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PubMed2005

Co-culture of osteoblasts and chondrocytes modulates cellular differentiation in vitro.

Biological integration of cartilage grafts with subchondral bone remains a significant clinical challenge. We hypothesize that interaction between osteoblasts and chondrocytes is important in regenerating the osteochondral interface on tissue-engineered osteochondral grafts. We describe here a sequential co-culturing model which permits cell-cell contact and paracrine interaction between osteoblast and chondrocytes in 3-D culture. This model was used to determine the effects of co-culture on the phenotypic maintenance of osteoblasts and chondrocytes. It was found that while chondrocytes synthesized a type II collagen and glycosaminoglycan (GAG) matrix, GAG deposition was significantly lower in co-culture. Alkaline phosphatase activity was maintained in osteoblasts, but cell-mediated mineralization in co-culture was markedly lower compared to osteoblast controls. These results collectively suggest that interactions between osteoblasts and chondrocytes modulate cell phenotypes, and the importance of these interactions on osteochondral interface regeneration will be explored in future studies.

Animals↗
PubMed2005

Contrasting effects of insulin and cellular differentiation on expression of the novel insulin receptor substrate APS in skeletal muscle.

The novel insulin receptor substrate protein APS is highly expressed in insulin-sensitive tissues and plays an important role in insulin-mediated glucose uptake and GLUT4 translocation via the Cbl/CAP pathway. Tyrosine phosphorylation of APS leads to recruitment of c-Cbl and Crk, while overexpression of APS mutant inhibits GLUT4 translocation in response to insulin, but the regulation of APS expression in skeletal muscle has not been previously reported. L6 myoblasts were differentiated in 2% FBS and serum starved for 24h prior to stimulation for 24h with either insulin 1 microM (n=6), rosiglitazone 10 microM (n=6), resistin 500 nM (n=6) or the MAP kinase inhibitor PD098059 50 microM (n=6) for 30 min, followed by insulin 1 microM for 24h. Semi-quantitative real-time RT-PCR was used to determine the expression of APS mRNA relative to the control gene TF2D. APS expression was markedly upregulated by myoblast differentiation (0.55+/-0.08 versus 1.14+/-0.08, p=0.001), and this effect was augmented by addition of rosiglitazone 10 microM for 24h to the differentiated myotubes (1.50+/-0.09, p=0.025). Insulin caused a 3.1-fold decrease in APS mRNA expression (0.37+/-0.01 versus 1.14+/-0.08, p=0.001), an effect that was attenuated by the MAP kinase inhibitor PD098059 (0.80+/-0.03, p=0.001). Exposure to resistin produced a modest decrease (1.4-fold) in myotube expression of APS (0.8+/-0.09, p=0.025). In conclusion, this is the first study to show that exposure to insulin markedly reduces the expression of APS in skeletal muscle via a MAP kinase dependent pathway, whereas myocyte differentiation and rosiglitazone increase APS expression. Changes in APS expression may be important in the aetiology and therapeutic reversal of insulin resistance in skeletal muscle.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Developmental changes of keratin expression in chick embryo olfactory epithelium in relation to cellular differentiation and neurogenesis in vivo and in vitro.

Olfactory embryogenesis was studied using an anti-chick keratin antibody on chick embryo sections as well as in vitro. Olfactory placodes form at embryonic day 3 (ED3) in the anterior facial ectoderm and invaginate to form the nasal pits. At ED5, the epidermal ectoderm and respiratory epithelium show the same dense cytokeratin immunoreaction. In contrast, absence of keratin expression in the basal part of olfactory epithelial primordium, in the deeper nasal pit area, coincides with one of the critical first steps of olfactory neurogenesis. However, beginning with periphero-central olfactory synaptogenesis at ED8, a new basal cell population starts to express keratin in the olfactory epithelium. Keratin positive cells appear to correspond, by their epithelial localisation and morphology, to sustentacular and basal cells. This interpretation was confirmed in vitro with ED14 chick primary olfactory cultures where TrKA immunoreactivity was used as a marker of horizontal basal cells (HBCs). After ED15, late keratin expression was detected in forming Bowman's glands. The density of keratin expressing basal cells was measured between ED10 and ED20, and appeared highest in the median part of the olfactory epithelium, the area of most active olfactory neurogenesis and neuronal maturation. Thus, keratin expression corresponds to a specialisation of horizontal basal cells as active neuronal stem cells.

Animals↗
PubMed2005

Plasmodium chabaudi chabaudi (AS): differential cellular responses to infection in resistant and susceptible mice.

The infection with blood stages of Plasmodium chabaudi chabaudi (AS) was followed in BALB/c and DBA/2 mice. Both strains show a peak parasitemia by 7-9 days after infection, display splenic hypercellularity of T and B cells, thymic atrophy, nearly complete depletion of B cells in the bone marrow, and mount comparable polyclonal IgM and IgG responses in the serum. In contrast, these strains diverge in some aspects of the immune response and susceptibility to infection: while BALB/c survive, 70-80% of DBA/2 die within 2 weeks; BALB/c but not DBA/2 show marked increases in the levels of splenic gamma/delta and regulatory T cells, dendritic cells and macrophages and parasite-specific IgM and IgG levels; however, lower levels of TNF-alpha and IL-12 were observed. These results suggest the relevance of different cell populations that are known to participate/regulate specific antibody responses and cytokine production in the susceptibility to infection.

Animals↗
PubMed2006

Quantitative analysis of the effect of cell type and cellular differentiation on protective antigen binding to human target cells.

We quantitatively measured protective antigen (PA) binding to human cells targeted by anthrax lethal toxin (LT). Affinities were less than 50 nM for all cells, but differentiated cells (macrophages and neutrophils) had significantly increased PA binding and endothelial cells demonstrated the most binding. Combined with the function of such cells, this suggests that PA receptors interact with the extracellular matrix and that differentiation increases the number of PA-specific receptors, which supports previously observed differentiation-induced LT susceptibility. Our results quantifiably confirm that the generality of PA binding will complicate its use as a tumor targeting agent.

Animals↗
PubMed2003

Immunohistochemical observations of cellular differentiation and proliferation in endochondral bone formation from grafted periosteum: expression and localization of BMP-2 and -4 in the grafted periosteum.

PURPOSE: To clarify the involvement of bone morphogenetic proteins (BMPs) in the proliferation and differentiation of osteo/chondrogenic cells during the process of bone formation from grafted periosteum. MATERIAL AND METHODS: Tibial periosteum of young Japanese white rabbits was grafted into suprahyoid muscles and removed after 7, 9, 14 or 21 days. BMP-2, -4, proliferative cell nucleus antigen (PCNA) immunoreaction and Alcian blue staining in grafted periosteum was then sought microscopically. RESULTS: PCNA positive cells in the grafted periosteum expressed BMP-2 at 7 days. These cells differentiated into chondroblasts that expressed BMP-2 and Alcian blue at 9 days. After 14 days, cartilage formation was seen, and BMP-2 and -4 expressions were observed in mature and hypertrophic chondrocytes. Endochondral ossification was observed at 21 days and osteoblasts showed both BMP-2 and -4 expression. CONCLUSION: Both BMP-2 and -4 appear to play regulatory roles in the process of endochondral ossification from grafted periosteum, due to their involvement in the proliferation and differentiation into chondrogenic and osteogenic cells.

Alcian Blue↗
PubMed2006

Differential cellular expression of LIGHT and its receptors in early gestation human placentas.

LIGHT (homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes) is an apoptosis-inducing member of the tumor necrosis factor family of ligands. Messenger RNAs encoding LIGHT and its receptors, lymphotoxin-beta receptor (LTbetaR), decoy receptor-3 (DcR3) and herpes virus entry mediator (HVEM), are present in first trimester and term placentas. Proteins have been localized to specific cells in term but not earlier gestation placentas. Here, we have studied LIGHT and its receptors in early (6-7 weeks) and early-to-middle (8-13 weeks) gestation using immunohistology. Notable cell-specific, gestation-related features were identified. LIGHT and two of its receptors, a membrane-bound receptor that mediates apoptosis (LTbetaR) and a soluble receptor that interferes with LIGHT signaling (DcR3), were present in syncytiotrophoblast and cytotrophoblast cells in all samples but were detected in placental stromal cells only at week 8 and thereafter. HVEM, a membrane-bound receptor that protects against apoptosis, was expressed only on syncytiotrophoblast. These observations suggest that the LIGHT system may regulate early to middle stages of placental development via cell-specific, temporally programmed expression of the ligand and its receptors, and may also assist in preserving placental immune privilege.

Apoptosis↗
PubMed2005

Binding of pRB to the PHD protein RBP2 promotes cellular differentiation.

pRB can enforce a G1 block by repressing E2F-responsive promoters. It also coactivates certain non-E2F transcription factors and promotes differentiation. Some pRB variants activate transcription and promote differentiation despite impaired E2F binding and transcriptional repression capabilities. We identified RBP2 in a screen for proteins that bind to such pRB variants. RBP2 resembles other chromatin-associated transcriptional regulators and RBP2 binding tracked with pRB's ability to activate transcription and promote differentiation. RBP2 and pRB colocalize and pRB/RBP2 complexes were detected in chromatin isolated from differentiating cells. RBP2 siRNA phenocopied restoration of pRB function in coactivation and differentiation assays, suggesting that pRB prevents RBP2 from repressing genes required for differentiation. In addition, two bromodomain-containing proteins were identified as RBP2 targets that are transcriptionally activated by pRB in an RBP2-dependent manner. Our results suggest that promotion of differentiation by pRB involves neutralization of free RBP2 and transcriptional activation of RBP2 targets linked to euchromatin maintenance.

Animals↗
PubMed2001

Differential cellular expression of neurotrophins in cortical tubers of the tuberous sclerosis complex.

Neurotrophins and their receptors modulate cerebral cortical development. Tubers in the tuberous sclerosis complex (TSC) are characterized histologically by disorganized cortical cytoarchitecture and thus, we hypothesized that expression of neurotrophin mRNAs and proteins might be altered in tubers. Using in situ transcription and mRNA amplification to probe cDNA arrays, we found that neurotrophin-3 (NT3) and trkB mRNA expression were reduced whereas neurotrophin-4 (NT4) and trkC mRNA expression were increased in whole tuber sections. Alterations in mRNA abundance were defined in single microdissected dysplastic neurons (DNs) and giant cells (GCs). NT3 mRNA expression was reduced in GCs and trkB mRNA expression was reduced in DNs. NT4 mRNA expression was increased in DNs and trkC mRNA expression was increased in both DNs and GCs. In three patients, TSC2 locus mutations were confirmed and the mean tuberin mRNA expression levels was reduced across all nine cases. Consistent with these observations, NT3 mRNA expression was reduced but trkC mRNA expression was increased in vitro in human NTera2 neurons (NT2N) transfected with a tuberin antisense construct that reduced tuberin expression. Western analysis of tuber homogenates and computer-assisted densitometry of immunolabeled sections confirmed the neurotrophin mRNA expression data in whole sections and single neurotrophin immunoreactive cells. We conclude that alterations in NT4/trkB and NT3/trkC expression may contribute to tuber formation during brain development as downstream effects of the hamartin and tuberin pathway in TSC.

Adolescent↗
PubMed2003

Progesterone induces cellular differentiation in MDA-MB-231 breast cancer cells transfected with progesterone receptor complementary DNA.

Progesterone is an important regulator of growth and differentiation in breast tissues. In this study, the effect of progesterone on cell differentiation was evaluated in the estrogen receptor-negative and progesterone receptor (PR)-negative MDA-MB-231 cell line which was transfected with PR-complementary DNA. Morphological changes were analyzed at the ultrastructural level by scanning and transmission electron microscopy. Progesterone-treated PR-transfected cells exhibited a more protracted and well spread morphology with an increase in organelles such as mitochondria and rough endoplasmic reticulum as compared to the rounded form of control vehicle (0.1% ethanol)-treated PR-transfected cells. Vehicle and progesterone-treated MDA-MB-231 cells transfected with the pSG5 plasmid (transfection control cells) had similar rounded morphology as control vehicle-treated PR-transfected cells. Immunofluorescence staining revealed that expression of E-cadherin, a differentiation marker, was more prominent in progesterone-treated cells. Expression of keratin and vimentin but not beta-catenin was up-regulated in progesterone treated cells when evaluated by immunoblotting. As signal transducers and activators of transcription (STAT) molecules have been implicated in mammary differentiation, we analyzed the expression of Stat 1, 3, 5a, and 5b proteins and found a significant up-regulation of the Stat 5b protein in progesterone-treated cells. We have provided in vitro evidence of the close association of PR with differentiation in breast cancer. It is likely that the Stat 5b protein may play a major role in progesterone-induced differentiation in breast cancer cells.

Blotting, Western↗
PubMed1999

Divergent cellular differentiation pathways during the invasive stage of cutaneous malignant melanoma progression.

Melanocytic nevus cells in the dermis adopt many morphological features of Schwann cells. These differentiation-related changes typically are not observed in melanomas. However, nevus cells do not fully recapitulate a Schwann cell phenotype, because they lack expression of mature myelin-associated proteins. In this study, melanocytic nevi and malignant melanomas were examined by immunohistochemistry for expression of low-affinity nerve growth factor receptor (p75NGFR), neural cell adhesion molecule (CD56/N-CAM), and growth-associated phosphoprotein-43 (GAP-43). These three proteins define the earliest stages of Schwann cell development but are not expressed in myelinated Schwann cells or normal melanocytes. p75NGFR was expressed in 25 of 25 (100%) and CD56/N-CAM and GAP-43 in 23 of 25 (92%) nevi, predominantly in type C nevus cells and nevic corpuscles. Most (84%) of the nevi expressed all three proteins. In primary invasive and metastatic melanoma, expression of each of the three proteins was limited to </=20% of lesions but was not observed in any melanoma in situ (chi(2 )P < 0.0001). None of the melanomas expressed all three proteins (ANOVA P < 0.0001). These data confirm and extend earlier studies by showing that terminal differentiation of melanocytes in the dermis recapitulates some aspects observed in the earliest stages of Schwann cell development and that invasive melanomas follow a divergent pathway. Studying these early differentiation events may help to identify specific defects in the relevant signaling pathways and establish tenable targets for therapy of advanced-stage melanoma.

Biomarkers, Tumor↗
PubMed1996

Differential cellular distribution of glutathione--an endogenous antioxidant--in the guinea pig inner ear.

Electronmicroscopic immunogold cytochemistry was applied to investigate the localization of glutathione (GSH) in the guinea pig inner ear. GSH immunoreactivity was preferentially distributed in basal cells and intermediate cells of the stria vascularis, and in fibrocytes and capillary endothelial cells in the spiral ligament and vestibular endorgans. The present findings suggest that the synthesis of GSH, a peptide known to protect against ototoxic compounds, depends on restricted cell populations in the inner ear.

Animals↗
PubMed2003

The age-related resistance of rats to Plasmodium berghei infection is associated with differential cellular and humoral immune responses.

In this study, we investigated how the age of rats would affect the course of infection of and the immune response to Plasmodium berghei. Both young (4-week-old) and adult rats (8-week-old) can be infected with P. berghei ANKA strain, with significantly higher levels of infected red blood cells in young rats. While 100% of young rats succumbed to infection, adult rats were able to clear blood parasites and no mortality was observed. Analysis of cellular distribution and circulating cytokines demonstrated the persistence of CD4+/CD25+ T cells and high expression of circulating interleukin-10 (IL-10) during the progression of infection in young-susceptible rats, whereas high levels of CD8+ T cells and natural killer T cells are detected in adult-resistant rats. Analysis of antibody isotypes showed that adult rats produced significantly higher levels of interferon-gamma (IFN-gamma)-dependent IgG2c antibodies than young rats during infection. Further evaluation of the role of IL-10, IFN-gamma and of immune cells showed that only the adoptive transfer of spleen cells from adult-resistant rats was able to convert susceptibility of young-susceptible rats to a resistant phenotype. These observations suggest that cell-mediated mechanisms are crucial for the control of a primary infection with P. berghei in young rats.

Adoptive Transfer↗
PubMed2001

Differential cellular expression of tumor necrosis factor-alpha and Type I tumor necrosis factor receptor after transient global forebrain ischemia.

We examined the expression of tumor necrosis factor-alpha (TNF-alpha) and the Type I tumor necrosis factor receptor, (TNFR1), in relation to c-fos, a known regulator gene of immediate cellular responses, after an extended period of global ischemia. The number of TNF-alpha mRNA expressing cells peaked in most brain areas after 8 h of reperfusion. Significant increases in TNFR1 mRNA expression were evident in the cortex at 2 and 8 h of reperfusion and after 8 h of reperfusion in the CA3/CA4 region of the hippocampus. Transient neuronal c-fos mRNA expression preceded these responses. TNF-alpha immunoreactivity was seen in neurons>>>oligodendrocytes=perivascular cells=ependymal cells=vessel wall structures. After ischemia/reperfusion, increased TNF-alpha immunoreactivity was evident only in oligodendrocytes. TNFR1 immunoreactivity in sham brains manifested in bundles of cellular fibers of variable length and thickness. In post-ischemic brains, immunoreactivity in these cellular processes representing mainly astroglial extensions was suppressed at 2 h but recovered partially by 8 and 24 h of reperfusion. In contradiction, transient ischemia-induced TNFR1 immunoreactivity was observed in somas of large cortical neurons, in activated microglia/macrophages, perivascular and endothelial cells.Taken together, the increase in neuronal TNF-alpha mRNA appeared not to be followed by substantial translation to protein in the cerebral tissue after an extended period of global ischemia. However, there was increased neuronal TNFR1 immunostaining in conjunction with increased immunostaining for TNF-alpha in oligoglial elements, which suggests signaling to neurons by enhanced oligoglial TNF-alpha.

Animals↗
PubMed2002

Effect of cellular differentiation on 11beta-hydroxysteroid dehydrogenase activity in the intestine.

11beta-Hydroxysteroid dehydrogenase (11betaHSD) converts endogenous glucocorticoids to their biologically inactive 11-dehydro derivatives and is therefore able to determine, at least in part, the biological action of glucocorticoids. Type 1 11betaHSD has both oxidase and reductase activities interconverting corticosterone and 11-dehydrocorticosterone, whereas type 2 11betaHSD has only oxidase activity converting corticosterone to 11-dehydrocorticosterone. Since 11betaHSD expression is regulated during development and by hormones in a tissue-specific manner and since glucocorticoids play an important role in postnatal intestinal maturation, we investigated the role of corticosteroids and cytodifferentiation in the regulation of intestinal 11betaHSD. Using rat intestinal organ cultures and epithelial cell lines derived from rat small intestine (IEC-6, IEC-18) and from human colon adenocarcinoma (Caco-2, HT-29), we analyzed the effect of corticosteroids and cytodifferentiation on 11betaHSD. Screening of the clonal cell lines showed that Caco-2 cells expressed by far the greatest 11betaHSD2 oxidase activity, lower activity was observed in HT-29 cells, and lowest activity was seen in IEC cells. Treatment with dexamethasone (50 nM) increased the activity of 11betaHSD2 in IEC-6 cells (+59%) and HT-29 cells (+31%), whereas aldosterone (50 nM) stimulated 11betaHSD2 in IEC-6 cells only (+31%). Caco-2 cells and IEC-18 cells did not respond to corticosteroids. Growth of IEC-6 cells on Matrigel, treatment of HT-29 cells with butyrate, and postconfluency of Caco-2 cells increased not only the markers of cytodifferentiation, such as alkaline phosphatase and sucrase, but also the activity of 11betaHSD2 in all of these cell lines (IEC-6, +96%; HT-29, +139%; Caco-2, +95%). Addition of corticosteroids to these more differentiated cell cultures did not enhance 11betaHSD2 activity. In intestinal organ cultures of suckling rat small intestine, dexamethasone and aldosterone stimulated 11betaHSD by more than 300%. We conclude that corticosteroids markedly and differentially regulate intestinal 11betaHSD2 and that cytodifferentiation of intestinal epithelial cells is associated with upregulation of 11betaHSD2 activity that is independent of corticosteroids.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗