Misidentification and cross-contamination of cell lines are major problems of cell cultures that ... more Misidentification and cross-contamination of cell lines are major problems of cell cultures that can make scientific results and their reproducibility unreliable. This paper describes a PCR-based method for easily identifying or confirming the species of origin of cell lines by using a panel of oligonucleotides specific for the nine animal species most common in cell culture laboratories. A panel of 35 human and animal cell lines, whose species of origin were previously confirmed by isoenzyme assay, was studied with nine species-specific primer pairs that specifically anneal to DNA sequences codifying for human, cat, dog, mouse, rat, horse, rabbit, African Green monkey cytochrome c oxidase subunit I (cox I), and one primer pair specific for the cytochrome b gene of Chinese hamster. The amplified fragments were analyzed by electrophoresis in ethidium bromide-stained 2% agarose gels. The method is simple, rapid, highly sensitive, and useful for routinely monitoring the species identity of cell cultures.
We report the establishment in vitro of three-dimensional (3D) cultures of human osteoblasts (hOB... more We report the establishment in vitro of three-dimensional (3D) cultures of human osteoblasts (hOB) derived from normal adults and supported uniquely by the extracellular matrix (ECM) they deposit. Osteoblasts were cultured in 3D cultures in vitro for up to 120 days. The 3D cultures, examined at 25, 31, and 48 days, expressed protein markers of osteoblastic cells, namely osteonectin, collagen type I, fibronectin, osteopontin, bone sialoprotein, biglycan, and decorin. Sequentially, alkaline phosphatase (AP) and then Ca incorporation, mineralization of matrix (monitored by histochemistry and transmission electron microscopy), and finally osteocalcin expression, were detected in the 3D cultures. Ultrastructurally, morphology progressed from early to mature osteoblast and to osteocyte-like. Cells were embedded in a matrix with organized collagen type I fibers containing, increasingly with time of culture, needle-shaped crystals, often associated with matrix vesicles, characteristic of those in bone. During the culture (up to 120 days) there was an outgrowth of proliferating osteogenic cells from the 3D structure. Subcutaneous implantation in nude mice for 20 days of osteoblasts cultured in 3D culture for different lengths of time in vitro, showed progression of mineralization from the inner region of the implant outward, with peripheral cells being embedded in nonmineralized, collagen-rich matrix. The 3D implants were invaded by vessels derived from the host.
The pre-T cell receptor (pre-TCR) promotes the development of thymocytes with productive rearrang... more The pre-T cell receptor (pre-TCR) promotes the development of thymocytes with productive rearrangement at the TCR b chain locus by signaling in a ligand-independent fashion. The TCR b chain associates with the invariant pre-Ta (pTa) chain, which bears specific charged residues in the extracellular portion mediating pre-TCR self-oligomerization. In recombinase-deficient thymocytes, calnexin (CNX) associated with CD3 chains is inefficiently retained in the endoplasmic reticulum (ER) and weakly expressed in the plasma membrane. Deliberate cross-linking of CNX/CD3 complexes mimics pre-TCR signaling. Here, we show that, analogously to the pTa chain, surface CNX is palmitoylated and that CD3 prominently accumulated in lipid rafts upon cross-linking. Mutant CNX isoforms devoid of ER retention determined pre-TCR-like signaling and simulated b selection only when stably translocating CD3 to lipid rafts. Inclusion of the palmitoylated cytoplasmic tail from the pTa chain in recombinant CNX strikingly improved the pre-TCR-like signaling efficiency of CNX/ CD3 in rafts. This study indicates that lipid rafts in the plasma membrane represent proficient microdomains for the initiation of pre-TCR signaling, and supports the view that b selection by oligomerized pre-TCR is implemented by the pTa cytoplasmic tail.
Here we investigated the impact of CRT defi ciency on T cell function in immunodefi cient mice re... more Here we investigated the impact of CRT defi ciency on T cell function in immunodefi cient mice reconstituted with fetal liver crt −/− hemopoietic progenitors. These chimeric mice displayed severe immunopathological traits, which correlated with a lower threshold of T cell receptor (TCR) activation and exaggerated peripheral T cell response to antigen with enhanced secretion of infl ammatory cytokines. In crt −/− T cells TCR stimulation induced pulsatile cytosolic elevations of Ca 2+ concentration and protracted accumulation of nuclear factor of activated T cells in the nucleus as well as sustained activation of the mitogenactivated protein kinase pathways. These observations support the hypothesis that CRTdependent shaping of Ca 2+ signaling critically contributes to the modulation of the T cell adaptive immune response.
High Mobility Group 1 protein (HMGB1) is a chromatin component that, when leaked out by necrotic ... more High Mobility Group 1 protein (HMGB1) is a chromatin component that, when leaked out by necrotic cells, triggers in¯ammation. HMGB1 can also be secreted by activated monocytes and macrophages, and functions as a late mediator of in¯ammation. Secretion of a nuclear protein requires a tightly controlled relocation program. We show here that in all cells HMGB1 shuttles actively between the nucleus and cytoplasm. Monocytes and macrophages acetylate HMGB1 extensively upon activation with lipopolysaccharide; moreover, forced hyperacetylation of HMGB1 in resting macrophages causes its relocalization to the cytosol. Cytosolic HMGB1 is then concentrated by default into secretory lysosomes, and secreted when monocytic cells receive an appropriate second signal.
Misidentification and cross-contamination of cell lines are major problems of cell cultures that ... more Misidentification and cross-contamination of cell lines are major problems of cell cultures that can make scientific results and their reproducibility unreliable. This paper describes a PCR-based method for easily identifying or confirming the species of origin of cell lines by using a panel of oligonucleotides specific for the nine animal species most common in cell culture laboratories. A panel of 35 human and animal cell lines, whose species of origin were previously confirmed by isoenzyme assay, was studied with nine species-specific primer pairs that specifically anneal to DNA sequences codifying for human, cat, dog, mouse, rat, horse, rabbit, African Green monkey cytochrome c oxidase subunit I (cox I), and one primer pair specific for the cytochrome b gene of Chinese hamster. The amplified fragments were analyzed by electrophoresis in ethidium bromide-stained 2% agarose gels. The method is simple, rapid, highly sensitive, and useful for routinely monitoring the species identity of cell cultures.
We report the establishment in vitro of three-dimensional (3D) cultures of human osteoblasts (hOB... more We report the establishment in vitro of three-dimensional (3D) cultures of human osteoblasts (hOB) derived from normal adults and supported uniquely by the extracellular matrix (ECM) they deposit. Osteoblasts were cultured in 3D cultures in vitro for up to 120 days. The 3D cultures, examined at 25, 31, and 48 days, expressed protein markers of osteoblastic cells, namely osteonectin, collagen type I, fibronectin, osteopontin, bone sialoprotein, biglycan, and decorin. Sequentially, alkaline phosphatase (AP) and then Ca incorporation, mineralization of matrix (monitored by histochemistry and transmission electron microscopy), and finally osteocalcin expression, were detected in the 3D cultures. Ultrastructurally, morphology progressed from early to mature osteoblast and to osteocyte-like. Cells were embedded in a matrix with organized collagen type I fibers containing, increasingly with time of culture, needle-shaped crystals, often associated with matrix vesicles, characteristic of those in bone. During the culture (up to 120 days) there was an outgrowth of proliferating osteogenic cells from the 3D structure. Subcutaneous implantation in nude mice for 20 days of osteoblasts cultured in 3D culture for different lengths of time in vitro, showed progression of mineralization from the inner region of the implant outward, with peripheral cells being embedded in nonmineralized, collagen-rich matrix. The 3D implants were invaded by vessels derived from the host.
The pre-T cell receptor (pre-TCR) promotes the development of thymocytes with productive rearrang... more The pre-T cell receptor (pre-TCR) promotes the development of thymocytes with productive rearrangement at the TCR b chain locus by signaling in a ligand-independent fashion. The TCR b chain associates with the invariant pre-Ta (pTa) chain, which bears specific charged residues in the extracellular portion mediating pre-TCR self-oligomerization. In recombinase-deficient thymocytes, calnexin (CNX) associated with CD3 chains is inefficiently retained in the endoplasmic reticulum (ER) and weakly expressed in the plasma membrane. Deliberate cross-linking of CNX/CD3 complexes mimics pre-TCR signaling. Here, we show that, analogously to the pTa chain, surface CNX is palmitoylated and that CD3 prominently accumulated in lipid rafts upon cross-linking. Mutant CNX isoforms devoid of ER retention determined pre-TCR-like signaling and simulated b selection only when stably translocating CD3 to lipid rafts. Inclusion of the palmitoylated cytoplasmic tail from the pTa chain in recombinant CNX strikingly improved the pre-TCR-like signaling efficiency of CNX/ CD3 in rafts. This study indicates that lipid rafts in the plasma membrane represent proficient microdomains for the initiation of pre-TCR signaling, and supports the view that b selection by oligomerized pre-TCR is implemented by the pTa cytoplasmic tail.
Here we investigated the impact of CRT defi ciency on T cell function in immunodefi cient mice re... more Here we investigated the impact of CRT defi ciency on T cell function in immunodefi cient mice reconstituted with fetal liver crt −/− hemopoietic progenitors. These chimeric mice displayed severe immunopathological traits, which correlated with a lower threshold of T cell receptor (TCR) activation and exaggerated peripheral T cell response to antigen with enhanced secretion of infl ammatory cytokines. In crt −/− T cells TCR stimulation induced pulsatile cytosolic elevations of Ca 2+ concentration and protracted accumulation of nuclear factor of activated T cells in the nucleus as well as sustained activation of the mitogenactivated protein kinase pathways. These observations support the hypothesis that CRTdependent shaping of Ca 2+ signaling critically contributes to the modulation of the T cell adaptive immune response.
High Mobility Group 1 protein (HMGB1) is a chromatin component that, when leaked out by necrotic ... more High Mobility Group 1 protein (HMGB1) is a chromatin component that, when leaked out by necrotic cells, triggers in¯ammation. HMGB1 can also be secreted by activated monocytes and macrophages, and functions as a late mediator of in¯ammation. Secretion of a nuclear protein requires a tightly controlled relocation program. We show here that in all cells HMGB1 shuttles actively between the nucleus and cytoplasm. Monocytes and macrophages acetylate HMGB1 extensively upon activation with lipopolysaccharide; moreover, forced hyperacetylation of HMGB1 in resting macrophages causes its relocalization to the cytosol. Cytosolic HMGB1 is then concentrated by default into secretory lysosomes, and secreted when monocytic cells receive an appropriate second signal.
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Papers by Denise Ferrera