Investigating cytokines in tear fluid and saliva may offer valuable information for understanding the pathogenesis of primary Sjögren's syndrome (pSS). Cytokine profiles in both tear fluid and saliva ...of pSS patients, non-Sjögren's syndrome (non-SS) subjects with sicca symptoms, and healthy controls without sicca complaints were analysed. Furthermore, relationships associating the severity of clinical ocular and oral manifestations with the upregulated cytokines were assessed. In tear fluid, pSS patients showed elevated levels of IL-1ra, IL-2, IL-4, IL-8, IL-12p70, IL-17A, IFN-γ, IP-10, MIP-1b, and Rantes compared to non-SS subjects and healthy controls. The increased cytokine levels (except IP-10) correlated significantly with reduced tear production, less stable tear film, and greater ocular surface damage. In saliva, pSS patients had a higher IP-10 level, which correlated with higher candida score; and an elevated MIP-1a level, which correlated significantly with lower unstimulated and stimulated whole saliva secretion rates. The upregulated cytokines identified in tear fluid and saliva of pSS patients show a clear interplay between innate and adaptive immune responses that may contribute to disease pathogenesis. The increase of IP-10 and MIP in both tears and saliva further emphasises the essential role of macrophages and innate immunity in pSS.
Epidemiological evidence suggests existing comorbidity between postmenopausal osteoporosis (OP) and cardiovascular disease (CVD), but identification of possible shared genes is lacking. The skeletal ...global transcriptomes were analyzed in trans-iliac bone biopsies (n = 84) from clinically well-characterized postmenopausal women (50 to 86 years) without clinical CVD using microchips and RNA sequencing. One thousand transcripts highly correlated with areal bone mineral density (aBMD) were further analyzed using bioinformatics, and common genes overlapping with CVD and associated biological mechanisms, pathways and functions were identified. Fifty genes (45 mRNAs, 5 miRNAs) were discovered with established roles in oxidative stress, inflammatory response, endothelial function, fibrosis, dyslipidemia and osteoblastogenesis/calcification. These pleiotropic genes with possible CVD comorbidity functions were also present in transcriptomes of microvascular endothelial cells and cardiomyocytes and were differentially expressed between healthy and osteoporotic women with fragility fractures. The results were supported by a genetic pleiotropy-informed conditional False Discovery Rate approach identifying any overlap in single nucleotide polymorphisms (SNPs) within several genes encoding aBMD- and CVD-associated transcripts. The study provides transcriptional and genomic evidence for genes of importance for both BMD regulation and CVD risk in a large collection of postmenopausal bone biopsies. Most of the transcripts identified in the CVD risk categories have no previously recognized roles in OP pathogenesis and provide novel avenues for exploring the mechanistic basis for the biological association between CVD and OP.
Seeking to improve the access to regenerative medicine, this study investigated the structural and transcriptional effects of storage temperature on human oral mucosal epithelial cells (OMECs).
Cells ...were stored at four different temperatures (4°C, 12°C, 24°C and 37°C) for two weeks. Then, the morphology, cell viability and differential gene expression were examined using light and scanning electron microscopy, trypan blue exclusion test and TaqMan gene expression array cards, respectively.
Cells stored at 4°C had the most similar morphology to non-stored controls with the highest viability rate (58%), whereas the 37°C group was most dissimilar with no living cells. The genes involved in stress-induced growth arrest (GADD45B) and cell proliferation inhibition (TGFB2) were upregulated at 12°C and 24°C. Upregulation was also observed in multifunctional genes responsible for morphology, growth, adhesion and motility such as EFEMP1 (12°C) and EPHA4 (4°C-24°C). Among genes used as differentiation markers, PPARA and TP53 (along with its associated gene CDKN1A) were downregulated in all temperature conditions, whereas KRT1 and KRT10 were either unchanged (4°C) or downregulated (24°C and 12°C; and 24°C, respectively), except for upregulation at 12°C for KRT1.
Cells stored at 12°C and 24°C were stressed, although the expression levels of some adhesion-, growth- and apoptosis-related genes were favourable. Collectively, this study suggests that 4°C is the optimal storage temperature for maintenance of structure, viability and function of OMECs after two weeks.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA methylation affects expression of associated genes and may contribute to the missing genetic effects from genome-wide association studies of osteoporosis. To improve insight into the mechanisms ...of postmenopausal osteoporosis, we combined transcript profiling with DNA methylation analyses in bone. RNA and DNA were isolated from 84 bone biopsies of postmenopausal donors varying markedly in bone mineral density (BMD). In all, 2529 CpGs in the top 100 genes most significantly associated with BMD were analyzed. The methylation levels at 63 CpGs differed significantly between healthy and osteoporotic women at 10% false discovery rate (FDR). Five of these CpGs at 5% FDR could explain 14% of BMD variation. To test whether blood DNA methylation reflect the situation in bone (as shown for other tissues), an independent cohort was selected and BMD association was demonstrated in blood for 13 of the 63 CpGs. Four transcripts representing inhibitors of bone metabolism-MEPE, SOST, WIF1, and DKK1-showed correlation to a high number of methylated CpGs, at 5% FDR. Our results link DNA methylation to the genetic influence modifying the skeleton, and the data suggest a complex interaction between CpG methylation and gene regulation. This is the first study in the hitherto largest number of postmenopausal women to demonstrate a strong association among bone CpG methylation, transcript levels, and BMD/fracture. This new insight may have implications for evaluation of osteoporosis stage and susceptibility.
Transplantation of cultured epidermal cell sheets (CES) can be life-saving for patients with large area burns. CES have also been successfully used to regenerate eye and urethral epithelia in animal ...models. Short-term storage aims to extend the transplantation window, offers flexibility in timing surgery and allows testing of CES quality, phenotype and sterility. This study investigated extended CES storage and explored the effect of additional re-incubation recovery time following storage. The proliferative quality of stored confluent versus pre-confluent CES was also investigated using functional testing. CES were stored at 12°C and results compared to non-stored control CES. Investigation of timepoints during 15 days storage revealed that viability began to deteriorate by day 11 and was associated with increased lactate in the storage medium. The percentage of apoptotic cells also significantly increased by day 11. Flow cytometry analysis of integrin β1 expression and cell size indicated best retention of stem cells at 7 days of storage. Functional testing of pre-confluent and confluent cells following 7 days storage showed that pre-confluent cells responded well to 1-day re-incubation after storage; they became highly prolific, increasing in number by ~67%. Conversely, proliferation in stored confluent cells declined by ~50% with 1-day re-incubation. Pre-confluent stored CES also had far superior stem cell colony forming efficiency (CFE) performance compared to the confluent group. Re-incubation improved CFE in both groups, but the pre-confluent group again out-performed the confluent group with significantly more colonies. In conclusion, a maximum storage period of 7 days is recommended. Use of pre-confluent cells and one day recovery incubation greatly improves viability, colony-forming ability and proliferation of cells stored for 7 days at 12°C. Thus, these recommendations should be considered under culture and storage of high-quality CES for clinical use.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mechanical loading exerts a profound influence on bone density and architecture, but the exact mechanism is unknown. Our study shows that expression of the neurological transcriptional factor zinc ...finger of the cerebellum 1 (
) is markedly increased in trabecular bone biopsies in the lumbar spine compared with the iliac crest, skeletal sites of high and low mechanical stress, respectively. Human trabecular bone transcriptome analyses revealed a strong association between
mRNA levels and gene transcripts characteristically associated with osteoblasts, osteocytes and osteoclasts. This supposition is supported by higher
expression in iliac bone biopsies from postmenopausal women with osteoporosis compared with age-matched control subjects, as well as strongly significant inverse correlation between
mRNA levels and BMI-adjusted bone mineral density (BMD) (Z-score).
promoter methylation was decreased in mechanically loaded vertebral bone compared to unloaded normal iliac bone, and its mRNA levels correlated inversely with
promoter methylation, thus linking mechanical stress to epigenetic control of gene expression. The findings were corroborated in cultures of rat osteoblast progenitors and osteoblast-like cells. This study demonstrates for the first time how skeletal epigenetic changes that are affected by mechanical forces give rise to marked alteration in bone cell transcriptional activity and translate to human bone pathophysiology.
We previously demonstrated that the silk protein sericin promotes pigmentation of retinal pigment epithelium (RPE) by activating the NF-κB pathway. Among numerous agents, NF-κB can be activated by ...hydrogen peroxide. In the present study, we explored possible associations between reactive oxygen species and sericin-induced melanogenesis in RPE. The proteome of human fetal RPE cultured for seven days with or without 1% sericin was analyzed using ingenuity pathway analysis (IPA). The proteomic data was verified by immunofluorescence and immunoblotting. Light microscopy and scanning electron microscopy were used to assess morphology. Dihydroethidium (DHE) and dihydrorhodamine (DHR) assays were used to measure superoxide and hydrogen peroxide species. Expression levels of proteins related to inflammation, differentiation, cell survival and cell adhesion were higher in cells cultured in Dulbecco's Modified Eagle Medium (DMEM) with 1% sericin, whereas cells cultured in DMEM alone showed higher expression levels of proteins associated with Bruch's membrane and cytoskeleton. Despite upregulation of inflammatory proteins, sericin co-cultured RPE yielded significantly higher cell viability compared to cells cultured without sericin. Addition of sericin to culture media significantly increased hydrogen peroxide-levels without significantly affecting superoxide-levels. We suggest that sericin-induced melanogenesis in cultured RPE is associated with elevated levels of superoxide dismutase, hydrogen peroxide and inflammatory proteins.
MicroRNAs (miRNAs) are small non-coding RNA molecules that post-transcriptionally regulate the translation of messenger RNAs. Given the crucial role of miRNAs in gene expression, genetic variants ...within miRNA-related sequences may affect miRNA function and contribute to disease risk. Osteoporosis is characterized by reduced bone mass, and bone mineral density (BMD) is a major diagnostic proxy to assess osteoporosis risk. Here, we aimed to identify miRNAs that are involved in BMD using data from recent genome-wide association studies (GWAS) on femoral neck, lumbar spine and forearm BMD. Of 242 miRNA-variants available in the GWAS data, we found rs11614913:C > T in the precursor
to be significantly associated with femoral neck-BMD (
-value = 9.9 × 10
, β = -0.038) and lumbar spine-BMD (
-value = 3.2 × 10
, β = -0.061). Furthermore, our sensitivity analyses using the Rotterdam study data showed a sex-specific association of rs11614913 with BMD only in women. Subsequently, we highlighted a number of
target genes, expressed in bone and associated with BMD, that may mediate the miRNA function in BMD. Collectively, our results suggest that
may contribute to variations in BMD level. Further biological investigations will give more insights into the mechanisms by which
control expression of BMD-related genes.
Background:
Clinical evidence suggests that body muscle mass is positively associated with bone mass, of significance for the elderly population at risk of osteoporosis (OP). Furthermore, muscle and ...bone interact mechanically and functionally, via local interactions as well as remotely via secreted components. Thus, it was of interest to compare muscle transcriptomes in postmenopausal OP and healthy women, and study effects of strength training on the muscle transcriptome, muscle stress proteins and bone mineral density (BMD).
Methods:
Skeletal muscle histological and genetic properties were compared in postmenopausal healthy (n = 18) and OP (n = 17) women before and after heavy-load strength training for 13–15 weeks. The cohorts were of similar age and body mass index without interfering diseases, medication or difference in lifestyle factors. Muscle biopsies obtained before and after intervention were studied histologically, and stress proteins and transcriptomes analyzed.
Results:
The OP women showed distinct muscle transcription profiles when compared with healthy women and had higher levels of the stress proteins HSP70 and α-β-crystalline. A set of 12 muscle transcripts, including ACSS3, FZD4, GNAI1 and IGF1, were differentially expressed before and after intervention (false discovery rate ⩽0.10, p ⩽0.001), and their corresponding bone transcripts were associated with BMD. Experimental data underline and describe the functionality of these genes in bone biology. OP women had 8% (p <0.01) higher proportion of type I fibres, but muscle fibre cross-sectional area did not differ. Muscle strength increased in both groups (p <0.01).
Conclusions:
Postmenopausal healthy and OP women have distinct muscle transcriptomes messenger ribonucleic acids (mRNAs) and microRNAs that are modulated by strength training, translating into key protein alterations and muscle fibre changes. The function of common skeletal muscle and bone genes in postmenopausal OP is suggestive of a shared disease trait.
Lipocalin 2 (LCN2) is an adipokine involved in many physiological functions, including bone metabolism. We previously demonstrated its implication in mouse models of mechanical unloading-induced ...osteoporosis and in a cohort of bed rest volunteers. We therefore aimed at studying its involvement in postmenopausal osteoporosis.
We measured serum LCN2 and correlated its levels to Dickkopf WNT Signaling Pathway Inhibitor 1 (DKK1), Tartrate Resistant Acid Phosphatase 5B (TRAcP5B), sclerostin, urinary N-terminal telopeptide of type I collagen (NTX), serum C-terminal telopeptide of type I collagen (CTX), parathyroid hormone and vitamin K by ELISA performed in a cohort of younger (50–65 years) and older (66–90 years) osteoporotic women in comparison to healthy subjects. A cohort of male healthy and osteoarthritic patients was also included. Sobel mediation analysis was used to test indirect associations among age, LCN2 and DKK1 or NTX.
LCN2 levels were unchanged in osteoporotic and in osteoarthritis patients when compared to healthy subjects and did not correlate with BMD. However, serum LCN2 correlated with age in healthy women (R = 0.44; P = 0.003) and men (R = 0.5; P = 0.001) and serum concentrations of DKK1 (R = 0.47; P = 0.003) and urinary NTX (R = 0.34; P = 0.04). Sobel mediation analysis showed that LCN2 mediates an indirect relationship between age and DKK1 (P = 0.02), but not with NTX, in healthy subjects.
Taken together, the results suggest a hitherto unknown association between LCN2, DKK1 and age in healthy individuals, but not in postmenopausal osteoporotic women.
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