scholarly journals A method for isolating RNA from canine bone

BioTechniques ◽  
2020 ◽  
Vol 68 (6) ◽  
pp. 311-317
Author(s):  
Rebecca Nance ◽  
Payal Agarwal ◽  
Maninder Sandey ◽  
Dmytro Starenki ◽  
Jey Koehler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Connective Tissue ◽  
Bone Cells ◽  
Transcriptomic Analysis ◽  
Unique Challenge ◽  
Downstream Application ◽  
Transcriptomic Sequencing ◽  
Mineralized Matrix

Extracting sufficient quantity and quality RNA from bone is essential for downstream application, such as transcriptomic sequencing, to evaluate gene expression. Isolation of RNA from bone presents a unique challenge owing to the hypocellular, brittle and mineralized matrix, which makes homogenizing the tissue difficult and provides little RNA to work with. Removal of contaminating tissue, such as bone marrow and connective tissue, is essential for isolating RNA that is unique to osteoblasts, osteoclasts and osteocytes. This study established a method to effectively isolate RNA from normal canine bone cells using the phalanges, without contamination from other tissue types, for downstream transcriptomic analysis.

EXPRESSION OF FACTOR XIII SUBUNIT A IN DIFFERENT TYPES OF HUMAN MACROPHAGES

1987 ◽  
Author(s):  
R Adany ◽  
A Kiss ◽  
J Kappelmayer ◽  
R J Ablin ◽  
L Muszbek
Keyword(s):  
Bone Marrow ◽  
Connective Tissue ◽  
Cell Line ◽  
Factor Xiii ◽  
Bone Cells ◽  
Human Peripheral Blood ◽  
Coagulation Factor ◽  
Macrophage Cell ◽  
Subunit A ◽  
Different Types

In addition to plasma the presence of subunit a of blood coagulation Factor XIII (FXIIl) has been verified in platelets and megakariocytes. Most recently, we demonstrated that human peripheral blood monocytes also contain FXIII subunit a. The present study was designed 1/ to determine the stage in the maturation sequence of bone marrow monocytopoesis in which FXIII appears 2/ to establish if FXIII is retained during differentiation into macrophages 3/ to assess how general is the presence of FXIII subunit a in different types of macrophages. FXIII subunit a was immunomorphologically detected in bone marrow smears, in cytospin preparations of cells from serous cavities (pleural, peritoneal, pericardial and synovial spaces), and paraformaldehyde-fixed paraffin-embedded or frozen sections of different organs where classical types of macrophages have been described earlier (liver, lung, thymus, skin, connective tissue, prostate and developing bone) . Cells containing FXIII subunit a were intensively characterized by immunofluorescent and enzymecytochemical techniques in double and treble labeling systems. Its presence was clearly demonstrated in promonocytes of bone marrow, and in all probability, it is present in monoblasts, as well. FXIII was also found in macrophages from different serous cavities and in embryonic osteoclasts. Cells containing FXIII subunit a of connective tissue were found to be tissue histiocytes, and not fibroblasts as previously thought. Kupffer cells of the liver and Langerhans cells of the epidermis were negative supporting theories that these cells are not members of monocyte-derived macrophage cell population. Immunomorphological detection of FXIII subunit a seems to be a useful marker for labeling the continuum of monocyte/macrophage cell line from the earliest ftrais in the bone marrow to the mature forms of macrophages and might be a valuable tool in the cytological diagnosis of malignant disorders of this cell line.

scholarly journals TGF-β Mediates Suppression of Adipogenesis by Estradiol through Connective Tissue Growth Factor Induction

Endocrinology ◽  
2012 ◽  
Vol 153 (1) ◽  
pp. 254-263 ◽  
Author(s):  
Ashok Kumar ◽  
Ming Ruan ◽  
Kari Clifton ◽  
Farhan Syed ◽  
Sundeep Khosla ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Connective Tissue ◽  
Connective Tissue Growth Factor ◽  
Adipocyte Differentiation ◽  
Bone Cells ◽  
Mesenchymal Cell ◽  
Tissue Growth ◽  
Estradiol Treatment ◽  
Marrow Cavity

In the bone marrow cavity, adipocyte numbers increase, whereas osteoblast progenitor numbers decrease with aging. Because adipocytes and osteoblasts share a common progenitor, it is possible that this shift is due to an increase in adipocyte-lineage cells at the expense of osteoblast-lineage commitment. Estrogens inhibit adipocyte differentiation, and in both men and women, circulating estrogens correlate with bone loss with aging. In bone cells, estrogens stimulate expression of TGF-β and suppress mesenchymal cell adipogenesis. Using a tripotential mesenchymal cell line, we have examined whether estradiol suppression of adipocyte differentiation is due to stimulation of TGF-β and the mechanism by which TGF-β suppresses adipogenesis. We observed that estradiol-mediated suppression of adipogenic gene expression required at least 48 h treatment. TGF-β expression increased within 24 h of estradiol treatment, and TGF-β inhibition reversed estradiol influences on adipogenesis and adipocyte gene expression. Connective tissue growth factor (CTGF) mediates TGF-β suppression of adipogenesis in mouse 3T3-L1 cells. CTGF expression was induced within 24 h of TGF-β treatment, whereas estradiol-mediated induction required 48 h treatment. Moreover, estradiol-mediated induction of CTGF was abrogated by TGF-β inhibition. These data support that estradiol effects on adipogenesis involves TGF-β induction, which then induces CTGF to suppress adipogenesis.

Increased matrix gene expression by glucose in rat neural connective tissue cells in culture

Diabetes ◽  
1991 ◽  
Vol 40 (5) ◽  
pp. 605-611 ◽  
Author(s):  
P. Muona ◽  
J. Peltonen ◽  
S. Jaakkola ◽  
J. Uitto
Keyword(s):  
Gene Expression ◽  
Connective Tissue ◽  
Cells In Culture ◽  
Matrix Gene ◽  
Tissue Cells

scholarly journals Inhibition of glypican-1 expression induces an activated fibroblast phenotype in a human bone marrow-derived stromal cell-line

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sukhneeraj P. Kaur ◽  
Arti Verma ◽  
Hee. K. Lee ◽  
Lillie M. Barnett ◽  
Payaningal R. Somanath ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Bone Marrow ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Stromal Cell ◽  
Prostate Cancer Cell ◽  
Bone Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Prostate Cancer Cells

AbstractCancer-associated fibroblasts (CAFs) are the most abundant stromal cell type in the tumor microenvironment. CAFs orchestrate tumor-stromal interactions, and contribute to cancer cell growth, metastasis, extracellular matrix (ECM) remodeling, angiogenesis, immunomodulation, and chemoresistance. However, CAFs have not been successfully targeted for the treatment of cancer. The current study elucidates the significance of glypican-1 (GPC-1), a heparan sulfate proteoglycan, in regulating the activation of human bone marrow-derived stromal cells (BSCs) of fibroblast lineage (HS-5). GPC-1 inhibition changed HS-5 cellular and nuclear morphology, and increased cell migration and contractility. GPC-1 inhibition also increased pro-inflammatory signaling and CAF marker expression. GPC-1 induced an activated fibroblast phenotype when HS-5 cells were exposed to prostate cancer cell conditioned media (CCM). Further, treatment of human bone-derived prostate cancer cells (PC-3) with CCM from HS-5 cells exhibiting GPC-1 loss increased prostate cancer cell aggressiveness. Finally, GPC-1 was expressed in mouse tibia bone cells and present during bone loss induced by mouse prostate cancer cells in a murine prostate cancer bone model. These data demonstrate that GPC-1 partially regulates the intrinsic and extrinsic phenotype of human BSCs and transformation into activated fibroblasts, identify novel functions of GPC-1, and suggest that GPC-1 expression in BSCs exerts inhibitory paracrine effects on the prostate cancer cells. This supports the hypothesis that GPC-1 may be a novel pharmacological target for developing anti-CAF therapeutics to control cancer.

389 Combining transcriptomic- and tissue-based immune biomarkers to evaluate GB1275, a CD11b modulator, as a single agent or with pembrolizumab in patients with advanced solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A414-A414
Author(s):  
Wells Messersmith ◽  
Drew Rasco ◽  
Johann De Bono ◽  
Andrea Wang-Gillam ◽  
Wungki Park ◽  
...  
Keyword(s):  
Gene Expression ◽  
Solid Tumors ◽  
Peripheral Blood ◽  
Post Treatment ◽  
Transcriptomic Analysis ◽  
Gene Set Enrichment Analysis ◽  
Cancer Center ◽  
Advanced Solid Tumors ◽  
Core Tissue ◽  
Dose Dependent

BackgroundGB1275 is a first-in-class CD11b modulator in development as monotherapy and in combination with pembrolizumab or chemotherapy for the treatment of advanced solid tumors. Nonclinical data show that GB1275 reduced influx of tumor-associated myeloid-derived suppressor cells (MDSCs) and macrophages (TAMs), and repolarized M2 immuno-suppressive TAMs towards an M1 phenotype. We hypothesize that GB1275 administration can alleviate myeloid cell-mediated immunosuppressive effects and improve cancer treatment outcomes. A phase 1 trial evaluating GB1275 as monotherapy and in combination with pembrolizumab in specified advanced tumors in ongoing (NCT04060342).MethodsBlood gene expression variations as well as core tissue biopsies pre- and post-treatment were assessed following GB1275 monotherapy and combination with pembrolizumab. After obtaining informed consent, peripheral blood for MDSCs was collected from 21 patients pre- and two weeks post-treatment; core tissue biopsies were collected from 13 patients pre- and post-treatment. The frequency of MDSCs in whole blood was measured using the Serametrix MDSC FACS Assay. Gene expression transcriptome profiles were generated using NovaSeq platform. CD8 staining was performed at Neogenomics, and tumor infiltrating lymphocyte (TIL) quantification was performed by an independent pathologist.ResultsPreliminary statistical analysis of MDSC immunophenotyping pre- and post- treatment is consistent with the proposed mechanism of GB1275, showing modulation of peripheral blood MDSCs in some patients. Preliminary gene expression analysis in the blood showed dose-dependent clusters following treatment with GB1275 alone. Moreover, the transcriptomic analysis revealed two unique expression patterns for patients treated with GB1275 monotherapy or in combination with pembrolizumab. Gene Set Enrichment Analysis showed that the CD11b pathway is downregulated in patients treated with GB1275. Analyses of TIL count revealed an increase in lymphocyte trafficking into the tumor after treatment with GB1275 alone or in combination with pembrolizumab. CD8 expression and transcriptomic analysis are underway and will be presented.ConclusionsGB1275 alone or in combination with pembrolizumab demonstrates biological activity, which may be dose dependent. The observed increase in TILs after treatment is supportive of the mechanism of action of GB1275. Further biomarker analyses in blood and tissues are ongoing and will be correlated with clinical activity in a larger number of patients.Ethics ApprovalThis ongoing study is being conducted in accordance with the the Declaration of Helsinki and Council for International Organizations of Medical Sciences (CIOMS) International Ethical Guidelines. The study was approved by the Ethics Boards of University of Colorado Hospital, Washington University School of Medicine - Siteman Cancer Center, Memorial Sloan Kettering Cancer Center, The Sarah Cannon Research Institute/Tennessee Oncology, South Texas Accelerated Research Therapeutics, and The Royal Marsden NHS Foundation Trust.

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