Comparison of ANA testing by indirect immunofluorescence or solid-phase assays in a low pre-test probability population for systemic autoimmune disease: the Camargo Cohort.

OBJECTIVES: Autoantibodies and, specifically antinuclear antibodies (ANA), are the hallmark of systemic autoimmune diseases (AID). In the last decades, there has been great technical development to detect these autoantibodies along with an increased request for this test by clinicians, while the overall pre-test probability has decreased. In this study, we compare the diagnostic performance of three different methods for ANA screening (indirect immunofluorescence [IIF], addressable laser bead immunoassay [ALBIA], and fluorescence enzyme immunoassay [FEIA]). METHODS: Serum samples at baseline visit from 2,997 participants from the Camargo Cohort, a population with an overall low pre-test probability for systemic AID, were analyzed with the three methods. Participants have a minimum follow-up of 10 years and the development of autoimmune diseases was collected from clinical records. RESULTS: The highest frequency of positive ANA was observed by IIF assay. However, ALBIA showed high sensitivity for AID. Likewise, solid phase assays (SPA) presented higher specificity than IIF for AID. ANA prevalence with any method was significantly higher in females and overall increased with age. Triple positivity for ANA was significantly related to the presence of anti-dsDNA-SSA/Ro60, Ro52, SSB/La, RNP, Scl-70, and centromere-specificities. No association was found for anti-Sm - RNP68, or ribosomal P - specificities. Noteworthy, triple positivity for ANA screening was associated with diagnosis of systemic AID both at baseline visit and follow-up. CONCLUSIONS: ANA detection by IIF may be better when the pre-test probability is high, whereas SPA techniques are more useful in populations with an overall low pre-test probability for systemic AID.

Crosstalk between caveolin-1/extracellular signal-regulated kinase (ERK) and ß-catenin survival pathways in osteocyte mechanotransduction.

Osteocyte viability is a critical determinant of bone strength and is promoted by both mechanical stimulation and activation of the Wnt signaling pathway. Earlier studies demonstrated that both stimuli promote survival of osteocytes by activating the ERKs. Here, we show that there is interaction between the caveolin-1/ERK and Wnt/ß-catenin signaling pathways in the transduction of mechanical cues into osteocyte survival. Thus, ERK nuclear translocation and anti-apoptosis induced by mechanical stimulation are abolished by the Wnt antagonist Dkk1 and the ß-catenin degradation stimulator Axin2. Conversely, GSK3ß phosphorylation and ß-catenin accumulation induced by mechanical stimulation are abolished by either pharmacologic inhibition of ERKs or silencing caveolin-1. In contrast, the canonical Wnt signaling inhibitor dominant-negative T cell factor does not alter ERK nuclear translocation or survival induced by mechanical stimulation. These findings demonstrate that ß-catenin accumulation is an essential component of the mechanotransduction machinery in osteocytes, albeit ß-catenin/T cell factor-mediated transcription is not required. The simultaneous requirement of ß-catenin for ERK activation and of ERK activation for ß-catenin accumulation suggests a bidirectional crosstalk between the caveolin-1/ERK and Wnt/ß-catenin pathways in mechanotransduction leading to osteocyte survival.

Prevalence and sociodemographic correlates of antinuclear antibody testing by indirect immunofluorescence or solid-phase assays in a Spanish population: the Camargo Cohort.

Autoantibodies are the hallmark of autoimmunity, and specifically, antinuclear antibodies (ANA) are one of the most relevant antibodies present in systemic autoimmune diseases (AID). In the present study, we evaluate the relationship between ANA and sociodemographic and biobehavioral factors in a population with a low pre-test probability for systemic AID. ANA were determined in serum samples at baseline visit from 2997 participants from the Camargo Cohort using indirect immunofluorescence assay, and two solid phase assays (SPA), addressable laser bead immunoassay, and fluorescence enzyme immunoassay. Sociodemographic and biobehavioral features of the subjects were obtained at baseline visit using a structured questionnaire. The prevalence of ANA positive results was significantly higher when indirect immunofluorescence assay was used as screening method in comparison with SPAs, being higher in females, older subjects, and those with higher C-reactive protein levels. Considering biobehavioral features, the prevalence was higher in those individuals with a sedentary lifestyle, and in ex- and non-alcohol users. Moreover, considering the relevance of the antibody load using ANA Screen, the prevalence of the antibody load also increased with age, especially in females. In conclusion, the prevalence of ANA varies depending on sociodemographic and biobehavioral features of the subjects, which could be relevant specifically in a population with a low pre-test probability for systemic AIDs.

Diffuse idiopathic skeletal hyperostosis (DISH) and trabecular bone score (TBS) in postmenopausal women: The Camargo cohort.

OBJECTIVES: The potential relationship between diffuse idiopathic skeletal hyperostosis (DISH) and bone microstructure has not been studied in women. We aimed to assess the association between the trabecular bone score (TBS) and DISH in postmenopausal women, as well as the role of other parameters related to bone metabolism, such as bone mineral density (BMD), calciotropic hormones, and bone remodeling markers. METHODS: Cross-sectional study, nested in a prospective population-based cohort (Camargo cohort). Clinical covariates, DISH, TBS, vitamin D, parathormone, BMD and serum bone turnover markers, were analyzed. RESULTS: We have included 1545 postmenopausal women (mean age, 62±9 years). Those with DISH (n = 152; 8.2%) were older and had a significantly higher prevalence of obesity, metabolic syndrome, hypertension, and type 2 diabetes mellitus (p<0.05). Moreover, they had lower TBS values (p = 0.0001) despite having a higher lumbar spine BMD (p<0.0001) and a higher prevalence of vertebral fractures than women without DISH (28.6% vs. 15.1%; p = 0.002). When analyzing DISH through Schlapbach grades, women without DISH had a median TBS value consistent with a normal trabecular structure while the values for women with DISH from grades 1 to 3 were consistent with a partially degraded trabecular structure. Women with vertebral fractures and DISH had a mean TBS corresponding to a degraded trabecular structure (1.219±0.1). After adjusting for confounders, the estimated TBS means were 1.272 (1.253-1.290) in the DISH group, and 1.334 (1.328-1.339) in the NDISH group (p<0.0001). CONCLUSION: An association between DISH and TBS has been shown in postmenopausal women, in which hyperostosis has been significantly and consistently related to trabecular degradation and, therefore, to deterioration in bone quality after adjusting for confounding variables.

CathepsinKCre mediated deletion of ßcatenin results in dramatic loss of bone mass by targeting both osteoclasts and osteoblastic cells.

It is well established that activation of Wnt/ßcatenin signaling in the osteoblast lineage leads to an increase in bone mass through a dual mechanism: increased osteoblastogenesis and decreased osteoclastogenesis. However, the effect of this pathway on the osteoclast lineage has been less explored. Here, we aimed to examine the effects of Wnt/ßcatenin signaling in mature osteoclasts by generating mice lacking ßcatenin in CathepsinK-expressing cells (Ctnnb1f/f;CtsKCre mice). These mice developed a severe low-bone-mass phenotype with onset in the second month and in correlation with an excessive number of osteoclasts, detected by TRAP staining and histomorphometric quantification. We found that WNT3A, through the canonical pathway, promoted osteoclast apoptosis and therefore attenuated the number of M-CSF and RANKL-derived osteoclasts in vitro. This reveals a cell-autonomous effect of Wnt/ßcatenin signaling in controlling the life span of mature osteoclasts. Furthermore, bone Opg expression in Ctnnb1f/f;CtsKCre mice was dramatically decreased pointing to an additional external activation of osteoclasts. Accordingly, expression of CathepsinK was detected in TRAP-negative cells of the inner periosteal layer also expressing Col1. Our results indicate that the bone phenotype of Ctnnb1f/f;CtsKCre animals combines a cell-autonomous effect in the mature osteoclast with indirect effects due to the additional targeting of osteoblastic cells.

The Effects of Androgens on Murine Cortical Bone Do Not Require AR or ERα Signaling in Osteoblasts and Osteoclasts.

In men, androgens are critical for the acquisition and maintenance of bone mass in both the cortical and cancellous bone compartment. Male mice with targeted deletion of the androgen receptor (AR) in mature osteoblasts or osteocytes have lower cancellous bone mass, but no cortical bone phenotype. We have investigated the possibility that the effects of androgens on the cortical compartment result from AR signaling in osteoprogenitors or cells of the osteoclast lineage; or via estrogen receptor alpha (ERα) signaling in either or both of these two cell types upon conversion of testosterone to estradiol. To this end, we generated mice with targeted deletion of an AR or an ERα allele in the mesenchymal (AR(f/y);Prx1-Cre or ERα(f/f);Osx1-Cre) or myeloid cell lineage (AR(f/y);LysM-Cre or ERα(f/f);LysM-Cre) and their descendants. Male AR(f/y);Prx1-Cre mice exhibited decreased bone volume and trabecular number, and increased osteoclast number in the cancellous compartment. Moreover, they did not undergo the loss of cancellous bone volume and trabecular number caused by orchidectomy (ORX) in their littermate controls. In contrast, AR(f/y);LysM-Cre, ERα(f/f);Osx1-Cre, or ERα(f/f);LysM-Cre mice had no cancellous bone phenotype at baseline and lost the same amount of cancellous bone as their controls following ORX. Most unexpectedly, adult males of all four models had no discernible cortical bone phenotype at baseline, and lost the same amount of cortical bone as their littermate controls after ORX. Recapitulation of the effects of ORX by AR deletion only in the AR(f/y);Prx1-Cre mice indicates that the effects of androgens on cancellous bone result from AR signaling in osteoblasts-not on osteoclasts or via aromatization. The effects of androgens on cortical bone mass, on the other hand, do not require AR or ERα signaling in any cell type across the osteoblast or osteoclast differentiation lineage. Therefore, androgens must exert their effects indirectly by actions on some other cell type(s) or tissue(s).

Estrogens, osteoarthritis and inflammation.

Estrogens participate in several biological processes through different molecular mechanisms. Their final actions consist of a combination of both direct and indirect effects on different organ and tissues. Estrogen may have pro- and anti-inflammatory properties depending on the situation and the involved tissue. In general, acute loss of estrogens increases the levels of reactive oxygen species and activates nuclear factor-κB and pro-inflammatory cytokine production, indicating their predominant anti-inflammatory properties. Furthermore, pro-inflammatory cytokine expression has been shown to be attenuated by estrogen replacement. Osteoarthritis and cardiovascular disease are two of the more prevalent diseases once menopause is established, which has suggested the link between estrogens and both processes. In addition, deletion of estrogen receptors in female mice results in cartilage damage, osteophytosis and changes in the subchondral bone of the joints suggesting that estrogens have a protective role on the maintenance of joint homeostasis. Furthermore, in spite of the negative effect of estrogen replacement reported in 2002 by the Women's Health Initiative study, several works published afterwards have explored the potential protective effect of estrogen supplementation in animal models and have postulated that these actions may justify a beneficial role of estrogens in different diseases where inflammation is the major feature. In this review, we will analyze the effects of estrogens on certain pathological situations such as osteoarthritis, some autoimmune diseases and coronary heart disease, especially in postmenopausal women.

Estrogen receptor-α signaling in osteoblast progenitors stimulates cortical bone accrual.

The detection of estrogen receptor-α (ERα) in osteoblasts and osteoclasts over 20 years ago suggested that direct effects of estrogens on both of these cell types are responsible for their beneficial effects on the skeleton, but the role of ERα in osteoblast lineage cells has remained elusive. In addition, estrogen activation of ERα in osteoclasts can only account for the protective effect of estrogens on the cancellous, but not the cortical, bone compartment that represents 80% of the entire skeleton. Here, we deleted ERα at different stages of differentiation in murine osteoblast lineage cells. We found that ERα in osteoblast progenitors expressing Osterix1 (Osx1) potentiates Wnt/ß-catenin signaling, thereby increasing proliferation and differentiation of periosteal cells. Further, this signaling pathway was required for optimal cortical bone accrual at the periosteum in mice. Notably, this function did not require estrogens. The osteoblast progenitor ERα mediated a protective effect of estrogens against endocortical, but not cancellous, bone resorption. ERα in mature osteoblasts or osteocytes did not influence cancellous or cortical bone mass. Hence, the ERα in both osteoblast progenitors and osteoclasts functions to optimize bone mass but at distinct bone compartments and in response to different cues.

The estrogen receptor-alpha in osteoclasts mediates the protective effects of estrogens on cancellous but not cortical bone.

Estrogens attenuate osteoclastogenesis and stimulate osteoclast apoptosis, but the molecular mechanism and contribution of these effects to the overall antiosteoporotic efficacy of estrogens remain controversial. We selectively deleted the estrogen receptor (ER)alpha from the monocyte/macrophage cell lineage in mice (ERalpha(LysM)(-/-)) and found a 2-fold increase in osteoclast progenitors in the marrow and the number of osteoclasts in cancellous bone, along with a decrease in cancellous bone mass. After loss of estrogens these mice failed to exhibit the expected increase in osteoclast progenitors, the number of osteoclasts in bone, and further loss of cancellous bone. However, they lost cortical bone indistinguishably from their littermate controls. Mature osteoclasts from ERalpha(LysM)(-/-) were resistant to the proapoptotic effect of 17beta-estradiol. Nonetheless, the effects of estrogens on osteoclasts were unhindered in mice bearing an ERalpha knock-in mutation that prevented binding to DNA. Moreover, a polymeric form of estrogen that is not capable of stimulating the nuclear-initiated actions of ERalpha was as effective as 17beta-estradiol in inducing osteoclast apoptosis in cells with the wild-type ERalpha. We conclude that estrogens attenuate osteoclast generation and life span via cell autonomous effects mediated by DNA-binding-independent actions of ERalpha. Elimination of these effects is sufficient for loss of bone in the cancellous compartment in which complete perforation of trabeculae by osteoclastic resorption precludes subsequent refilling of the cavities by the bone-forming osteoblasts. However, additional effects of estrogens on osteoblasts, osteocytes, and perhaps other cell types are required for their protective effects on the cortical compartment, which constitutes 80% of the skeleton.

Estrogens attenuate oxidative stress and the differentiation and apoptosis of osteoblasts by DNA-binding-independent actions of the ERalpha.

Estrogens diminish oxidative stress in bone and bone marrow, attenuate the generation of osteoblasts, and decrease the prevalence of mature osteoblast apoptosis. We have searched for the molecular mechanism of these effects using as tools a mouse model bearing an estrogen receptor alpha (ERalpha) knock-in mutation that prevents binding to DNA (ERalpha(NERKI/-)) and several osteoblast progenitor cell models expressing the wild-type ERalpha or the ERalpha(NERKI/-). We report that the ability of estrogens to diminish the generation of reactive oxygen species, stimulate the activity of glutathione reductase, and decrease the phosphorylation of p66(shc), as well as osteoblastogenesis and osteoblast number and apoptosis, were fully preserved in ERalpha(NERKI/-) mice, indicating that the DNA-binding function of the ERalpha is dispensable for all these effects. Consistent with the attenuation of osteoblastogenesis in this animal model, 17beta-estradiol attenuated bone morphogenetic protein 2 (BMP-2)-induced gene transcription and osteoblast commitment and differentiation in murine and human osteoblastic cell lines. Moreover, 17beta-estradiol attenuated BMP-2-induced differentiation of primary cultures of calvaria- or bone marrow-derived osteoblastic cells from ERalpha(NERKI/-) mice as effectively as in cells from wild-type littermates. The inhibitory effect of the hormone on BMP-2 signaling resulted from an ERalpha-mediated activation of ERKs and the phosphorylation of Smad1 at the linker region of the protein, which leads to proteasomal degradation. These results illustrate that the effects of estrogens on oxidative stress and the birth and death of osteoblasts do not require the binding of ERalpha to DNA response elements, but instead they result from the activation of cytoplasmic kinases.

FoxO-mediated defense against oxidative stress in osteoblasts is indispensable for skeletal homeostasis in mice.

Aging increases oxidative stress and osteoblast apoptosis and decreases bone mass, whereas forkhead box O (FoxO) transcription factors defend against oxidative stress by activating genes involved in free radical scavenging and apoptosis. Conditional deletion of FoxO1, FoxO3, and FoxO4 in 3-month-old mice resulted in an increase in oxidative stress in bone and osteoblast apoptosis and a decrease in the number of osteoblasts, the rate of bone formation, and bone mass at cancellous and cortical sites. The effect of the deletion on osteoblast apoptosis was cell autonomous and resulted from oxidative stress. Conversely, overexpression of a FoxO3 transgene in mature osteoblasts decreased oxidative stress and osteoblast apoptosis and increased osteoblast number, bone formation rate, and vertebral bone mass. We conclude that FoxO-dependent oxidative defense provides a mechanism to handle the oxygen free radicals constantly generated by the aerobic metabolism of osteoblasts and is thereby indispensable for bone mass homeostasis.

Oxidative stress antagonizes Wnt signaling in osteoblast precursors by diverting beta-catenin from T cell factor- to forkhead box O-mediated transcription.

We have elucidated that oxidative stress is a pivotal pathogenetic factor of age-related bone loss and strength in mice, leading to, among other changes, a decrease in osteoblast number and bone formation. To gain insight into the molecular mechanism by which oxidative stress exerts such adverse effects, we have tested the hypothesis that induction of the Forkhead box O (FoxO) transcription factors by reactive oxygen species may antagonize Wnt signaling, an essential stimulus for osteoblastogenesis. In support of this hypothesis, we report herein that the expression of FoxO target genes increases, whereas the expression of Wnt target genes decreases, with increasing age in C57BL/6 mice. Moreover, we show that in osteoblastic cell models, oxidative stress (exemplified by H(2)O(2)) promotes the association of FoxOs with beta-catenin, beta-catenin is required for the stimulation of FoxO target genes by H(2)O(2), and H(2)O(2) promotes FoxO-mediated transcription at the expense of Wnt-/T-cell factor-mediated transcription and osteoblast differentiation. Furthermore, beta-catenin overexpression is sufficient to prevent FoxO-mediated suppression of T-cell factor transcription. These results demonstrate that diversion of the limited pool of beta-catenin from T-cell factor- to FoxO-mediated transcription in osteoblastic cells may account, at least in part, for the attenuation of osteoblastogenesis and bone formation by the age-dependent increase in oxidative stress.

Skeletal involution by age-associated oxidative stress and its acceleration by loss of sex steroids.

Both aging and loss of sex steroids have adverse effects on skeletal homeostasis, but whether and how they may influence each others negative impact on bone remains unknown. We report herein that both female and male C57BL/6 mice progressively lost strength (as determined by load-to-failure measurements) and bone mineral density in the spine and femur between the ages of 4 and 31 months. These changes were temporally associated with decreased rate of remodeling as evidenced by decreased osteoblast and osteoclast numbers and decreased bone formation rate; as well as increased osteoblast and osteocyte apoptosis, increased reactive oxygen species levels, and decreased glutathione reductase activity and a corresponding increase in the phosphorylation of p53 and p66(shc), two key components of a signaling cascade that are activated by reactive oxygen species and influences apoptosis and lifespan. Exactly the same changes in oxidative stress were acutely reproduced by gonadectomy in 5-month-old females or males and reversed by estrogens or androgens in vivo as well as in vitro. We conclude that the oxidative stress that underlies physiologic organismal aging in mice may be a pivotal pathogenetic mechanism of the age-related bone loss and strength. Loss of estrogens or androgens accelerates the effects of aging on bone by decreasing defense against oxidative stress.

[Pyomyositis: retrospective review in a third-level hospital in the north of Spain]. / Piomiositis: una revisión retrospectiva en un hospital terciario del norte de España.

OBJECTIVE: To describe the clinical, radiological and microbiological features of a series of patients diagnosed with pyomyositis in a tertiary care university-affiliated center over a 12-year period. PATIENTS AND METHODS: The medical records of all patients diagnosed with pyomyositis between January 1992 and December 2003 were reviewed. The charts were retrieved from the hospital database. Data were extracted according to a standardized protocol and included clinical, radiological, laboratory and microbiological parameters. RESULTS: A total of 54 patients (mean age, 50 years, 61% men) had pyomyositis. The most frequent predisposing factors were diabetes mellitus (22%) and traumatic injury (20%), followed by neoplasms (9%). Primary pyomyositis was diagnosed in 25 patients (55%), and a contiguous source of infection was detected in the remainder, with skin infection being the most frequent (40%). The most common presentation was isolated inflammatory signs with or without other symptoms (94%). Isolated fever was documented in only one patient. Ultrasonography was the most common diagnostic procedure performed (32%), followed by CT scanning (18%). Forty-five patients underwent a drainage procedure combined with antibiotic therapy. Pyomyositis was monomicrobial in 20 cases, and polymicrobial in 12. The most frequent pathogen was Staphylococcus aureus followed by coagulase-negative staphylococci (6 cases). Sepsis developed in 4 patients, and recurrence was observed in 8 (15%). Mortality was 10% (5 patients). CONCLUSIONS: Pyomyositis is a relatively uncommon infection in temperate climates, and is often considered late in the diagnostic workup. Physicians should bear this disease in mind to avoid diagnostic delays and initiate prompt therapy, in order to improve the prognosis of these patients.

Piomiositis: una revisión retrospectiva en un hospital terciario del norte de España / Pyomyositis: Retrospective review in a third-level hospital in the north of Spain

Objetivo. Describir las características epidemiológicas y microbiológicas de los pacientes diagnosticados de piomiositis en un hospital universitario de tercer nivel durante 12 años. Pacientes y métodos. Revisión retrospectiva de las historias clínicas de los pacientes con piomiositis entre enero de 1992 y diciembre de 2003. Resultados. Se identificaron 54 pacientes. La media de edad fue de 50 años. El 61% de los casos eran varones. Los factores predisponentes más frecuentes fueron la diabetes (22%) y el traumatismo previo (20%), seguidos de las neoplasias (9%). La piomiositis primaria se diagnosticó en 25 pacientes (55%). En los demás se encontró un foco contiguo, el más frecuente de los cuales era la piel (40%). Los signos inflamatorios aislados o asociados a fiebre o leucocitosis fueron la forma habitual de presentación (94%). En cambio, la fiebre aislada se documentó sólo en un paciente. La ecografía fue el método diagnóstico más utilizado (32%) seguido de la tomografía computarizada (18%). Un total de 45 pacientes recibieron tratamiento combinado con antibióticos y drenaje. La piomiositis fue monomicrobiana en 20 casos y polimicrobiana en 12. El microorganismo aislado con más frecuencia fue Staphylococcus aureus, seguido de los estafilococos coagulasa negativos (6 casos). Un total de 4 pacientes presentaron sepsis y la piomiositis recurrió en ocho (15%). La tasa de mortalidad fue del 10% (5 pacientes). Conclusiones. La piomiositis es una entidad poco frecuente en climas templados y, por tanto, probablemente, infradiagnosticada. Un mayor conocimiento de esta enfermedad puede evitar un retraso en el diagnóstico y favorecer un tratamiento precoz que mejore el pronóstico de estos pacientes (AU)

Objective. To describe the clinical, radiological and microbiological features of a series of patients diagnosed with pyomyositis in a tertiary care university-affiliated center over a 12-year period. Patients and methods. The medical records of all patients diagnosed with pyomyositis between January 1992 and December 2003 were reviewed. The charts were retrieved from the hospital database. Data were extracted according to a standardized protocol and included clinical, radiological, laboratory and microbiological parameters. Results. A total of 54 patients (mean age, 50 years, 61% men) had pyomyositis. The most frequent predisposing factors were diabetes mellitus (22%) and traumatic injury (20%), followed by neoplasms (9%). Primary pyomyositis was diagnosed in 25 patients (55%), and a contiguous source of infection was detected in the remainder, with skin infection being the most frequent (40%). The most common presentation was isolated inflammatory signs with or without other symptoms (94%). Isolated fever was documented in only one patient. Ultrasonography was the most common diagnostic procedure performed (32%), followed by CT scanning (18%). Forty-five patients underwent a drainage procedure combined with antibiotic therapy. Pyomyositis was monomicrobial in 20 cases, and polymicrobial in 12. The most frequent pathogen was Staphylococcus aureus followed by coagulase-negative staphylococci (6 cases). Sepsis developed in 4 patients, and recurrence was observed in 8 (15%). Mortality was 10% (5 patients). Conclusions. Pyomyositis is a relatively uncommon infection in temperate climates, and is often considered late in the diagnostic workup. Physicians should bear this disease in mind to avoid diagnostic delays and initiate prompt therapy, in order to improve the prognosis of these patients (AU)