Dynamic expression of VDR and 1-­alpha-­hydroxylase in differentiated and re-­differentiated human articular chondrocytes

Abstract of a presentation at a conference of the International Cartilage Repair Society.Purpose: The goal was to investigate potential roles played by vitamin D in the regulation of joint cartilage biology. We studied the expression of two central elements of vitamin D metabolism, namely the vitamin D receptor and its converting enzyme 1­α­hydroxylase in human knee cartilage and chondrocytes. Methods and Materials: Expression of receptor and enzyme was determined by immunohistochemistry/immunofluorescence, reversetranscriptase PCR and western blot on differentiated, de­differentiated and re­differentiated chondrocytes. Cartilage was harvested from a macroscopically healthy looking area of the lateral femoral condyle during knee replacement surgery in 4 otherwise healthy patients aged 50­70. Suspension cultures of differentiated chondrocytes were established by short enzymatic digestion of cartilage using Collagenase XI and further incubation in non­adherent vessels. De­differentiated cells were the result of serial expansion of chondrocytes during 4 weeks after isolation in monolayers cultures. Chondrocyte re­differentiation was achieved by propagating cell pellets for 3 weeks in the presence of chondro­inductive morphogens. Results: Both protein and gene expression of vitamin D receptor appear to be very low or undetectable in native cartilage and/or differentiated chondrocytes. In contrast, receptor expression was upregulated in dedifferentiated cells after monolayer expansion, however, this upregulation was lost when cells regained chondrogenic phenotype in 3D pellets. The expression of 1­α­hydroxylase was observed on the superficial layer of chondrocytes in native cartilage, which correlated with weak but detectable outcomes by PCR and western blot on differentiated cultures. Similarly, levels of the enzyme were increased after cell expansion in monolayers and decreased in 3D pellet cultures. Conclusion: Our study uncover a previously unknown regulation of vitamin D receptor between differentiated and redifferentiated phenotypes in cartilage cells. Furthermore, this study is pioneering on investigating the expression of 1­α­hydroxylase in cartilage tissue and chondrocytes. Further work is needed to ascertain if receptor and enzyme expression is regulated in disease conditions or affected by inflammatory environments

Failure to Find a Conditioned Placebo Analgesic Response

Background: Associative learning has, in several studies, been modulated by the sex of the participant. Consistent with this, a recent review found that conditioned nocebo effects are stronger in females than in males.Purpose: It has been suggested that conditioned placebo responses are stronger in females, and this hypothesis was investigated in the present study. Cortisol and measures of negative emotions were taken to investigate if these processes could mediate any conditioned placebo effects.Methods: Cold pain was applied to the volar forearm. The Conditioned group received inert capsules prior to two presentations of less painful stimulations, to associate intake of the capsules with reduced pain. The pain control group received the same painful stimulation as the Conditioned group, but no capsules. The Capsule control group received the capsules in the same way as the Conditioned group, but no decrease in the painful stimulation. Participant sex was crossed across groups. It was hypothesized that in the Conditioned group, an expectation of reduced pain should be induced after administration of the capsules, and this should generate placebo analgesia, and mostly so in females.Results: The Conditioned group reported lower pain during conditioning, and rated the capsules as more effective painkillers than the capsule control group. However, placebo analgesia was not reliably observed in the Conditioned group.Conclusion: The placebo capsules were rated as effective painkillers, but this did not translate into a placebo analgesic effect. This could be due to violation of response expectancies, too few conditioning trials, and differences in pain ratings in the pre-test that could be due to previous experience with painkillers

Human articular chondrocytes express ChemR23 and chemerin; ChemR23 promotes inflammatory signalling upon binding the ligand chemerin21-157

Chemerin is a chemotactic peptide which directs leukocytes expressing the chemokine-like receptor ChemR23 towards sites of inflammation. ChemR23 is a G protein-coupled receptor which binds several different ligands, and it is also expressed by other cell types such as adipocytes. In addition to chemotaxis, recent reports suggest that ChemR23 is capable of mediating either inflammatory or anti-inflammatory effects, depending on the type of ligand it binds. In the present study, we aimed to clarify whether human chondrocytes express ChemR23 and chemerin, and whether chemerin/ChemR23 signalling could affect secretion of inflammatory mediators. Tissue sections were taken from human knee joints and labelled with antibodies towards chemerin and ChemR23. Chondrocytes from cartilage tissue were isolated, cultured and assessed for chemerin and ChemR23 expression by PCR and immunolabelling. Receptor activation and intracellular signalling were studied by assessment of phosphorylated mitogen activated protein kinases (MAPKs) and phosphorylated Akt after stimulating cells with recombinant chemerin21-157. Biological effects of chemerin21-157 were investigated by measuring secretion of pro-inflammatory cytokines and metalloproteases in cell supernatants. Both serially cultured human articular chondrocytes and resident cells in native cartilage expressed chemerin and ChemR23. Stimulating cells with chemerin21-157 resulted in phosphorylation of p44/p42 MAPKs (ERK 1/2) and Akt (Ser 473). Also, significantly enhanced levels of the pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), tumour necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and the matrix metalloproteases MMP-1, MMP-2, MMP-3, MMP-8 and MMP-13 were detected. These results demonstrate that human chondrocytes express both the receptor ChemR23 and the ligand chemerin. Chemerin21-157 stimulation engaged signal-transduction pathways that further promoted inflammatory signalling in chondrocytes, as judged by an enhanced secretion of cytokines and metalloproteases. Taken together, the previously reported chemotaxis and the present findings suggest that the receptor and its ligand may play pivotal roles in joint inflammation

Expression and function of Leukotriene B4 receptors in human articular chondrocytes

Leukotriene B4 (LTB4) is linked to osteoarthritis (OA) development however the expression of LTB4 receptors in cartilage cells and the physiological effects of LTB4 on cartilage tissue remain unknown. In this study we find that human articular chondrocytes express LTB4 receptors and that these receptors are functional, however, LTB4 does not seem to affect importantly some primary chondrocyte functions

The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis

Published version. Source at http://dx.doi.org/10.1186/s12885-016-2545-1 Background: Formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor mainly expressed by the cells of myeloid origin, where it mediates the innate immune response to bacterial formylated peptides. High expression of FPR1 has been detected in various cancers but the function of FPR1 in tumorigenesis is poorly understood. Methods: Expression of FPR1 in neuroblastoma cell lines and primary tumors was studied using RT-PCR, western blotting, immunofluorescence and immunohistochemistry. Calcium mobilization assays and western blots with phospho-specific antibodies were used to assess the functional activity of FPR1 in neuroblastoma. The tumorigenic capacity of FPR1 was assessed by xenografting of neuroblastoma cells expressing inducible FPR1 shRNA, FPR1 cDNA or control shRNA in nude mice. Results: FPR1 is expressed in neuroblastoma primary tumors and cell lines. High expression of FPR1 corresponds with high-risk disease and poor patient survival. Stimulation of FPR1 in neuroblastoma cells using fMLP, a selective FPR1 agonist, induced intracellular calcium mobilization and activation of MAPK/Erk, PI3K/Akt and P38-MAPK signal transduction pathways that were inhibited by using Cyclosporin H, a selective receptor antagonist for FPR1. shRNA knock-down of FPR1 in neuroblastoma cells conferred a delayed xenograft tumor development in nude mice, whereas an ectopic overexpression of FPR1 promoted augmented tumorigenesis in nude mice. Conclusion: Our data demonstrate that FPR1 is involved in neuroblastoma development and could represent a therapy option for the treatment of neuroblastoma

Hormone replacement therapy use and plasma levels of sex hormones in the Norwegian Women and Cancer Postgenome Cohort – a cross-sectional analysis

<p>Abstract</p> <p>Background</p> <p>Hormone replacement therapy use (HRT) is associated with increased breast cancer risk. Our primary objective was to explore hormone levels in plasma according to HRT use, body mass index (BMI) and menopausal status. A secondary objective was to validate self-reported questionnaire information on menstruation and HRT use in the Norwegian Women and Cancer postgenome cohort (NOWAC).</p> <p>Methods</p> <p>We conducted a cross-sectional study of sex hormone levels among 445 women aged 48–62 who answered an eight-page questionnaire in 2004 and agreed to donate a blood sample. The samples were drawn at the women's local general physician's offices in the spring of 2005 and sent by mail to NOWAC, Tromsø, together with a two-page questionnaire. Plasma levels of sex hormones and Sex Hormone Binding Globulin (SHBG) were measured by immunometry. 20 samples were excluded, leaving 425 hormone measurements.</p> <p>Results</p> <p>20% of postmenopausal women were HRT users. The plasma levels of estradiol (E₂) increased with an increased E₂dose, and use of systemic E₂-containing HRT suppressed the level of Follicle Stimulating Hormone (FSH). SHBG levels increased mainly among users of oral E₂preparations. Vaginal E₂application did not influence hormone levels. There was no difference in BMI between HRT users and non-users. Increased BMI was associated with increased E₂and decreased FSH and SHBG levels among non-users. Menopausal status defined by the two-page questionnaire showed 92% sensitivity (95% CI 89–96%) and 73% specificity (95% CI 64–82%), while the eight-page questionnaire showed 88% sensitivity (95% CI 84–92%) and 87% specificity (95% CI 80–94%). Current HRT use showed 100% specificity and 88% of the HRT-users had plasma E₂levels above the 95% CI of non-users.</p> <p>Conclusion</p> <p>Users of systemic E₂-containing HRT preparations have plasma E₂and FSH levels comparable to premenopausal women. BMI has an influence on hormone levels among non-users. NOWAC questionnaires provide valid information on current HRT use and menopausal status among Norwegian women who are between 48 and 62 years old.</p

Cortisol levels and cognitive profile in major depression: A comparison of currently and previously depressed patients

Source at https://doi.org/10.1016/j.psyneuen.2018.08.024.The association between depressive symptoms and elevated cortisol levels, and depression and cognitive functioning, has been less robust in outpatients with symptoms in the mild to moderate range. Furthermore, the association between elevated cortisol levels and cognitive functioning is unclear. In the present study, currently depressed (n = 37), previously depressed (n = 81) and never depressed controls (n = 50) were assessed on a range of neuropsychological measures. Salivary cortisol was measured in the morning and evening. Participants with current depression were non-hospitalized and had symptoms predominately in the mild to moderate range. Elevated salivary evening cortisol, but not morning cortisol, was significantly related to depressive symptoms. The difference in cortisol levels between the previously depressed group and the never depressed controls was not significant. The groups had significantly different cognitive profiles, with the currently depressed performing poorer on tasks related to working memory compared to the never depressed controls. Both the currently and previously depressed performed worse on attentional tasks. The findings indicate that outpatients with mild to moderate depression have elevated cortisol levels and limited mild cognitive impairments. Furthermore, mild impairments in attention may persist after remission, indicating that this could be a trait-marker in depression. The present study did not find support for a significant relationship between cortisol and cognitive functioning

No significant effect on bone mineral density by high doses of vitamin D3 given to overweight subjects for one year

<p>Abstract</p> <p>Background</p> <p>In meta-analyses supplementation with vitamin D appears to reduce incidence of fractures, and in cross-sectional studies there is a positive association between serum 25-hydroxyvitamin D (25(OH)D) levels and bone mineral density (BMD). However, the effect of supplementation with high doses of vitamin D on BMD is more uncertain and could in theory have both positive and negative effects.</p> <p>Methods</p> <p>The study was a one year, double blind placebo-controlled intervention trial performed at the University Hospital of North Norway. 421 subjects, 21 - 70 years old, were included and 312 completed the study. The subjects were randomized to vitamin D₃40.000 IU per week (DD group), vitamin D₃20.000 IU per week (DP group), or placebo (PP group). All subjects were given 500 mg calcium daily. Serum 25(OH)D, osteoprotegrin (OPG), receptoractivator of nuclear factor-kappaB ligand (RANKL), and BMD at the lumbar spine and the hip were measured before and at the end of the study.</p> <p>Results</p> <p>At baseline the mean serum 25(OH)D levels were 58 nmol/L (all subjects) and increased to 141 and 100 nmol/L in the DD and DP groups, respectively. After one year, no significant differences were found between the three groups regarding change in BMD, serum OPG or RANKL.</p> <p>Conclusions</p> <p>Supplementation with high doses of vitamin D for one year does not appear to have a negative effect on BMD in healthy subjects. In order to disclose a positive effect, subjects with low BMD and/or low serum 25(OH)D levels need to be studied.</p> <p>Trial registration</p> <p>The trial was registered at ClinicalTrials.gov (NCT00243256).</p

Dynamic expression of VDR and 1-­alpha-­hydroxylase in differentiated and re-­differentiated human articular chondrocytes

Purpose: The goal was to investigate potential roles played by vitamin D in the regulation of joint cartilage biology. We studied the expression of two central elements of vitamin D metabolism, namely the vitamin D receptor and its converting enzyme 1­α­hydroxylase in human knee cartilage and chondrocytes. Methods and Materials: Expression of receptor and enzyme was determined by immunohistochemistry/immunofluorescence, reversetranscriptase PCR and western blot on differentiated, de­differentiated and re­differentiated chondrocytes. Cartilage was harvested from a macroscopically healthy looking area of the lateral femoral condyle during knee replacement surgery in 4 otherwise healthy patients aged 50­70. Suspension cultures of differentiated chondrocytes were established by short enzymatic digestion of cartilage using Collagenase XI and further incubation in non­adherent vessels. De­differentiated cells were the result of serial expansion of chondrocytes during 4 weeks after isolation in monolayers cultures. Chondrocyte re­differentiation was achieved by propagating cell pellets for 3 weeks in the presence of chondro­inductive morphogens. Results: Both protein and gene expression of vitamin D receptor appear to be very low or undetectable in native cartilage and/or differentiated chondrocytes. In contrast, receptor expression was upregulated in dedifferentiated cells after monolayer expansion, however, this upregulation was lost when cells regained chondrogenic phenotype in 3D pellets. The expression of 1­α­hydroxylase was observed on the superficial layer of chondrocytes in native cartilage, which correlated with weak but detectable outcomes by PCR and western blot on differentiated cultures. Similarly, levels of the enzyme were increased after cell expansion in monolayers and decreased in 3D pellet cultures. Conclusion: Our study uncover a previously unknown regulation of vitamin D receptor between differentiated and redifferentiated phenotypes in cartilage cells. Furthermore, this study is pioneering on investigating the expression of 1­α­hydroxylase in cartilage tissue and chondrocytes. Further work is needed to ascertain if receptor and enzyme expression is regulated in disease conditions or affected by inflammatory environments

Co-expression of 1α-hydroxylase and vitamin D receptor in human articular chondrocytes

Background: The aim was to investigate whether resident chondrocytes in human articular cartilage and in subculture express vitamin D receptor (VDR) and the enzyme that hydroxylates the prohormone 25(OH)D3 to the active hormone 1α,25(OH)2D3, namely 1α-hydroxylase (CYP27B1). Any putative effects of vitamin D on chondrocytes were also explored. Methods: Cartilage from human osteoarthritic knee joints, cultured chondrocytes and cells grown in 3D spheroids were examined for the expression of VDR and 1α-hydroxylase by PCR, Western blots and immunolabelling. Receptor engagement was judged by visualizing nuclear translocation. The effects of 25(OH)D3 and 1α,25(OH)2D3 on chondrocyte functions were assessed in proliferation-, chondrogenesis- and cartilage signature-gene expression assays. The capability of chondrocytes to hydroxylate 25(OH)D3 was determined by measuring the concentration of metabolites. Finally, a putative regulation of receptor and enzyme expression by 1α,25(OH)2D3 or interleukin (IL)-1β, was investigated by Western blot. Results: Gene expression was positive for VDR in freshly isolated cells from native cartilage, cells subcultured in monolayers and in spheroids, whereas protein expression, otherwise judged low, was apparent in monolayers. Nuclear translocation of VDR occurred upon 1α,25(OH)2D3 treatment. Transcripts for 1α-hydroxylase were detected in freshly isolated cells, cultured cells and spheroids. Western blots and immunolabelling detected 1α-hydroxylase protein in all materials, while staining of tissue appeared confined to cells at the superficial layer. A dose-dependent 1α,25(OH)2D3 production was measured when the enzyme substrate was supplied to cell cultures. Western blots revealed that the VDR, but not 1α-hydroxylase, was induced by IL-1β treatment in adherent cells. Proliferation in monolayers was enhanced by both 25(OH)D3 and 1α,25(OH) 2D3, and both compounds had negative effects on chondrogenesis and cartilage-matrix genes. Conclusions: VDR expression in resident cartilage chondrocytes, generally considered differentiated cells, is elusive. A similar pattern applies for redifferentiated chondrocytes in spheroid cultures, whereas dedifferentiated cells, established in monolayers, stably express VDR. Both 25(OH)D3 and 1α,25(OH)2D3 are able to potentiate cell proliferation but have a negative impact in proteoglycan synthesis. Chondrocytes express 1α-hydroxylase and may contribute to the production of 1α,25(OH)2D3 into the joint environment. Effects of vitamin D could be unfavourable in the context of cartilage matrix synthesis