17 research outputs found
Role of tartrate-resistant acid phosphatase in bone remodeling
Tartrate-resistant acid phosphatase is a metalloenzyme that exists as two isoforms: the monomeric TRAP 5a and the proteolytically cleaved TRAP 5b, responsible for phosphatase activity, which is highly expressed in osteoclasts (OCs). TRAP 5b has been used as a serum marker of bone resorption, as it correlates with the absolute number of OCs and with resorption markers such as CTX-I. Despite being used as biomarker for bone metabolic diseases, little is known about the role of TRAP isoforms in OCs and thus bone remodeling (the process of bone degradation by osteoclasts, and bone formation by osteoblasts). Therefore, this thesis aimed to investigate the role of TRAP isoforms in bone remodeling.
To enable the investigation of TRAP 5a and 5b we (1) developed a sandwich TRAP 5a/5b ELISA for the quantification of human TRAP isoforms. This ELISA was then used for (2) evaluating the expression and secretion pattern of TRAP 5a and 5b in healthy individuals and during OC differentiation. Additionally, we used the ELISA to (3) investigated if TRAP protein levels correlate to osteoarthritis (OA) or rheu- matoid arthritis (RA). Here we correlated the phosphorylation status of the known TRAP in vivo substrate, osteopontin, to the TRAP isoforms. (4) Using a competitive inhibitor for TRAP 5b, we studied the role of TRAP in OCs differentiation. Lastly, we (5) developed a high throughput system to identify a subclone in a murine cell line that is a more homogeneous and stable OC precursors that could be used as a screening tool for OC biology studies.
A double TRAP 5a/5b sandwich ELISA was developed and designed as a two-step process. Using the ELISA, we showed that in vitro cultures of OCs secrete not only TRAP 5b but also TRAP 5a and that both isoforms were present intracellular estab- lishing that 5b can also be formed intracellular in OCs. Correlation between TRAP 5a and 5b indicated a dependence between TRAP 5a and formation of 5b. There was a positive correlation in both serum from healthy men, and media from in vitro OC cultures of not only 5b but also TRAP 5a with CTX-I further suggesting that TRAP 5a also originates partly from OCs. Measurement of TRAP 5a and 5b in synovial fluid from OA and RA patients revealed a correlation between low TRAP 5b/ TRAP 5a ratio and phosphorylated osteopontin. This suggested that synovial fluid from RA patients contained an insufficient amount of TRAP 5b increasing levels of phos- phorylated OPN leading to a higher OC activation and bone destruction. Inhibition of TRAP 5b using the competitive inhibitor, 5-phenylnicotinic acid, decreased the number of OCs formed and the expression of several OC markers. However, some OCs were able to fuse and resorb bone.
In this thesis we show that measurement of TRAP isoforms protein is an important tool in research and possibly also in diagnostic to understand the biological implications of TRAP 5a and 5b in OCs, which may lead to therapeutic targeting of certain isoforms for inflammatory and metabolic bone diseases. We further show that TRAP is involved in the bone remodeling process in OCs and defects in TRAP may cause alterations in OCs function and differentiation
Longitudinal study of cytokine expression, lipid profile and neuronal growth factors in human breast milk from term and preterm deliveries
Breast milk (BM) is considered as a reference for infant nutrition. The role of bioactive components, such as cytokines, hormones, growth factors (GFs) and fatty acids (FAs) is poorly known, but they might be implicated in immune response development. The aim of this study was to identify the lipid profile and the spectrum of cytokines and neuronal GF in BM samples and analyse the influence of gestational age and lactation time on these components. This study used a longitudinal prospective method for the characterization of cytokines, FAs and GFs global profiles in 120 BM samples from 40 healthy mothers (20 preterm and 20 term) collected as colostrum, transitional and mature milk. The cytokines were analysed by protein array (Ray Bio® Human Cytokine Array G6. Ray Biotech, Inc. Norcross, GA, USA) and the FAs were analysed by gas chromatography. The FA profile was similar between the term and the preterm BM samples. Omega-3-α-linoleic and docosahexaenoic acid (DHA) and omega-6-linoleic acid were the most abundant in the term and preterm samples during lactation. Omega-3 ETA and omega-3 EPA we observed exclusively in the preterm samples. The cytokine profile showed a different trend based on gestational age. A significantly higher expression of neurotrophic factors was found in the mature preterm milk samples as compared to the mature term samples. Our study is the first to identify the influence and interactions of perinatal factors on cytokine, GFs and FAs in human milk. © 2015 by the authors; licensee MDPI, Basel, Switzerland.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe
''Microbial mucosa l colon ic shifts associated to the developm ent of colorectal cancer Reveal the presence of diffe rent bacterlal and archaeal bioma rkers''
[EN] Study of microbiota composition through qPCR and pyrosequencing of feces and biopsies samples from
patients in differents stages of progression pattern of colorectal cancer disease.[ES] Estudio mendiante qPCR y pirosecuenciación del gen 16SrRNA de la ccomposición
microbiana de muestras de heces y biopsias de pacientes en diferentes estadías de progresión del cáncer de colon.Mira Pascual, L. (2014). Cancer de Colon & Microbiota. http://hdl.handle.net/10251/53408Archivo delegad
A Sub-Clone of RAW264.7-Cells Form Osteoclast-Like Cells Capable of Bone Resorption Faster than Parental RAW264.7 through Increased De Novo Expression and Nuclear Translocation of NFATc1
The murine macrophage cell line RAW264.7 is extensively used as a progenitor to study osteoclast (OC) differentiation. RAW264.7 is a heterogeneous cell line, containing sub-clones with different abilities to form OCs. The aim of this study was to identify characteristics within the heterogeneous RAW264.7 cells that define sub-clones with an augmented ability to form bone-resorbing OCs (H9), as well as sub-clones representing non-OCs (J8). RAW264.7 sub-clones were isolated by single cell cloning. Selection was based on TRAP/cathepsin K expression in sub-clone cultures without added RANKL. Sub-clones before and after differentiation with RANKL were assayed for multiple OC-characteristics. Sub-clone H9 cells presented a higher expression of OC-markers in cultures without added RANKL compared to the parental RAW264.7. After 6 days of RANKL stimulation, sub-clone H9 cells had equal expression levels of OC-markers with RAW264.7 and formed OCs able to demineralize hydroxyapatite. However, sub-clone H9 cells displayed rapid differentiation of OC already at Day 2 compared to Day 4 from parental RAW264.7, and when cultured on plastic and on bone they were more efficient in resorption. This rapid differentiation was likely due to high initial expression/nuclear translocation of OC master transcription factor, NFATc1. In contrast to H9, J8 cells expressed initially very low levels of OC-markers, and they did not respond to RANKL-stimulation by developing OC-characteristics/OC-marker expression. Hence, H9 is an additional clone suitable for experimental setup requiring rapid differentiation of large numbers of OCs
The adipokine tartrate-resistant acid phosphatase 5a in serum correlates to adipose tissue expansion in obesity
Longitudinal Study of Cytokine Expression, Lipid Profile and Neuronal Growth Factors in Human Breast Milk from Term and Preterm Deliveries
Breast milk (BM) is considered as a reference for infant nutrition. The role of bioactive components, such as cytokines, hormones, growth factors (GFs) and fatty acids (FAs) is poorly known, but they might be implicated in immune response development. The aim of this study was to identify the lipid profile and the spectrum of cytokines and neuronal GF in BM samples and analyse the influence of gestational age and lactation time on these components. This study used a longitudinal prospective method for the characterization of cytokines, FAs and GFs global profiles in 120 BM samples from 40 healthy mothers (20 preterm and 20 term) collected as colostrum, transitional and mature milk. The cytokines were analysed by protein array (Ray Bio® Human Cytokine Array G6. Ray Biotech, Inc. Norcross, GA, USA) and the FAs were analysed by gas chromatography. The FA profile was similar between the term and the preterm BM samples. Omega-3-α-linoleic and docosahexaenoic acid (DHA) and omega-6-linoleic acid were the most abundant in the term and preterm samples during lactation. Omega-3 ETA and omega-3 EPA we observed exclusively in the preterm samples. The cytokine profile showed a different trend based on gestational age. A significantly higher expression of neurotrophic factors was found in the mature preterm milk samples as compared to the mature term samples. Our study is the first to identify the influence and interactions of perinatal factors on cytokine, GFs and FAs in human milk
The adipokine tartrate-resistant acid phosphatase 5a in serum correlates to adipose tissue expansion in obesity
<p><b>Purpose:</b> Tartrate-resistant acid phosphatase (TRAP) exists as two isoforms, 5a and 5b. TRAP 5a is elevated in adipose tissue of obese women, interacts with pre-adipocytes and is linked to insulin-sensitive hyperplastic obesity when overexpressed in mice. The aim of this study was to investigate the relation between serum TRAP 5a, adiposity indices and metabolic syndrome risk markers in lean and obese women, using a newly developed TRAP 5a-specific ELISA.</p> <p><b>Materials and methods:</b> A TRAP 5a sandwich ELISA was optimized using TRAP 5a-specific monoclonal antibodies and tested in sera of healthy males. TRAP 5a levels were quantitated in sera from healthy males and lean and obese women.</p> <p><b>Results:</b> Serum TRAP 5a protein levels were lower in obese women in comparison with lean. In obese, but not in lean women, serum TRAP 5a correlated positively to % fat mass, BMI, waist- and hip circumference, waist-to-hip ratio and PAI, while no correlations to serum leptin, HOMA, glucose, insulin, FFA, HDL, TG, APO-A1 and APO-B were observed.</p> <p><b>Conclusions:</b> TRAP 5a serum levels correlated positively to anthropometric obesity parameters but not to metabolic syndrome risk factors, indicating that serum TRAP 5a is associated with pathological adipose tissue expansion.</p
Longitudinal Study of Cytokine Expression, Lipid Profile and Neuronal Growth Factors in Human Breast Milk from Term and Preterm Deliveries
Synovial tissue immunohistology.
<p>The antibody staining in each panel is shown as brown color. The generic TRAcP antibody (A, B, E, F), which stains both 5A and 5B isoforms, localized mostly in the lining layer (1) cells in a similar pattern between RA and OA, but a slightly more intensive staining of the sublining layer (2) cells and stroma could be seen in OA samples. The pattern of staining within deep stroma (3), which consists mostly of dense connective tissue and fat, was also similar between the diseases. TRAcP 5A stain (C, D, G, H) localized similarly to the generic antibody, and no differences were found between staining intensities of the sample groups. This indicates that most of TRAcP in the synovial tissue is likely in the 5A form, but differences in TRAcP 5B levels within the synovial tissue are possible between OA and RA. Both TRAcP antibodies were also localized in endothelial (asterisk) and lymphatic cells within the lymphatic follicle (arrow). OPN antibody stain (J, K) localized in the extracellular matrix, with only slight intracellular staining seen. OPN staining was most intense in the sublining layer (2), while the stainings of the lining layer (1) and deep stroma were lighter. OPN antibody did not localize in the endothelial cells (asterisk) but was found within the lymph node (arrow) stroma and some cells within it. No difference was found in the pattern or intensity of staining between RA and OA samples.</p
Summary of phospho-OPN and TRAcP isoform data.
<p>The TRAcP 5B/5A ratio (A) is lowered in RF+ RA and this correlates with the level of OPN’s phosphorylation (B), since TRAcP 5B is the primary phosphatase of OPN, its phosphorylation is decreased in RA. An image of phospho-OPN staining on western blot (C) is shown to highlight the differences, the photo was cropped to allow better visualization of the bands. In conclusion, we demonstrate that OPN is significantly more phosphorylated in RA than in OA synovia, and there are no significant differences between seropositive and seronegative groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182904#pone.0182904.g001" target="_blank">Fig 1A</a>). TRAcP 5B/5A ratio in synovial fluid is increased in OA, and there is a significant negative correlation between the measured ratios and phospho-OPN levels.</p