Dean T. Yamaguchi

Th17 cells in inflammation and autoimmunity

T helper 17 (Th17), a distinct subset of CD4(+) T cells with IL-17 as their major cytokine, orchestrate the pathogenesis of inflammatory and autoimmune diseases. Dysregulated Th17 cells contribute to inflammatory and autoimmune diseases. Candidate biologics are in development for targeting IL-17, IL-17 receptors or IL-17 pathways. Several drugs that impact the IL-17 pathway are already in clinical trials for the treatment of autoimmune diseases. In this review we provide evidence for the role of Th17 cells in immune-mediated diseases. An understanding of the role of Th17 in these conditions will provide important insights and unravel novel targets for therapeutic intervention.

Anti-hyperglycemic activity of zinc plus cyclo (his-pro) in genetically diabetic Goto-Kakizaki and aged rats

We previously reported that treatment of streptozotocin-Induced diabetic rats with zinc plus cyclo (his-pro) (CHP) decreased fed blood glucose levels and water intake. The present study was conducted to examine the dose-dependent, acute, and chronic treatment effects of CHP on oral glucose tolerance (OGT), fed blood glucose levels, water intake, and plasma Insulin levels in young and aged Sprague-Dawley (S-D) rats, nondiabetic Wistar rats, and genetically diabetic Goto-Kakizaki (G-K) rats. Acute gastric gavage of 10 mg zinc plus 1.0 mg CHP/kg body weight significantly improved OGT in 4- and 13-month-old nondiabetic S-D rats and in 2-month-old diabetic G-K rats. Young S-D and G-K rats returned to pretreatment OGT values 1 week after acute gavage of zinc plus CHP (ZC), but improved OGT values persisted for at least 1 week after gavage in aged S-D rats. OGT values and fed blood glucose decreased to the greatest extent among other treatments when G-K rats were given free access to drinking water containing 1.0 to 1.5 mg CHP/L plus 10 mg zinc/L for 2 weeks. Although food and water intake showed a tendency to decrease, no statistically significant differences were observed in young G-K rats. Plasma insulin levels and blood glucose levels in both normal and diabetic G-K rats decreased with 2-week treatment with ZC. To test the direct effects of ZC on muscle tissue, we observed the effect of various doses of ZC on normal and G-K rat muscle slices. The optimal level of CHP alone for maximal muscle glucose uptake in muscle slices from normal rats was 10 μg/mL and 5.0 μg/mL in G-K rats, and ZC stimulated glucose uptake. However, no statistically significant difference was demonstrated between normal and G-K rat tissues in this study. These results indicate that oral intake of an optimal dose of ZC stimulates blood glucose metabolism, probably by stimulating muscle glucose utilization.

TNAP, TrAP, ecto-purinergic signaling, and bone remodeling.

Bone remodeling is a process of continuous resorption and formation/mineralization carried out by osteoclasts and osteoblasts, which, along with osteocytes, comprise the bone multicellular unit (BMU). A key component of the BMU is the bone remodeling compartment (BRC), isolated from the marrow by a canopy of osteoblast‐like lining cells. Although much progress has been made regarding the cytokine‐dependent and hormonal regulation of bone remodeling, less attention has been placed on the role of extracellular pH (pHe). Osteoclastic bone resorption occurs at acidic pHe. Furthermore, osteoclasts can be regarded as epithelial‐like cells, due to their polarized structure and ability to form a seal against bone, isolating the lacunar space. The major ecto‐phosphatases of osteoclasts and osteoblasts, acid and alkaline phosphatases, both have ATPase activity with pH optima several units different from neutrality. Furthermore, osteoclasts and osteoblasts express plasma membrane purinergic P2 receptors that, upon activation by ATP, accelerate bone osteoclast resorption and impair osteoblast mineralization. We hypothesize that these ecto‐phosphatases help regulate [ATP]e and localized pHe at the sites of bone resorption and mineralization by pH‐dependent ATP hydrolysis coupled with P2Y‐dependent regulation of osteoclast and osteoblast function. Furthermore, osteoclast cellular HCO  3− , formed as a product of lacunar V‐ATPase H+ secretion, is secreted into the BRC, which could elevate BRC pHe, in turn affecting osteoblast function. We will review the existing data addressing regulation of BRC pHe, present a hypothesis regarding its regulation, and discuss the hypothesis in the context of the function of proteins that regulate pHe. J. Cell. Biochem. 105: 655–662, 2008.

Transforming growth factor‐β, osteogenin, and bone morphogenetic protein‐2 inhibit intercellular communication and alter cell proliferation in MC3T3‐E1 cells

Intercellular communication by gap junctions has been implicated to function in the control of cell growth and differentiation in osseous tissues—processes which are regulated, in part, by peptide growth factors, including transforming growth factor‐beta (TGF‐β) and the bone morphogenetic proteins (BMPs). Using the osteoblastic cell line MC3T3‐E1, we tested the hypothesis that the effects of TGF‐β and BMPs on cell proliferation may be correlated to changes in intercellular communication. In a series of proliferation assays, MC3T3‐E1 cells were cultured in the presence of bone morphogenetic protein‐2 (BMP‐2) or TGF‐β for up to 48 hr. Proliferation of cells during the linear log phase (days 2 to 4) was assessed by 3H‐thymidine (3H‐TdR) incorporation. After times ranging from 6 to 48 hr, BMP‐2 significantly inhibited uptake of 3H‐TdR at doses of 50–800 ng/ml. Similarly, TGF‐β inhibited uptake of 3H‐TdR at doses of 2–32 ng/ml. In a separate group of experiments, intercellular communication through gap junctions was demonstrated by cell‐cell transfer of the fluorescent tracer, lucifer yellow, after microinjection. One series of experiments showed that the gap junctional intercellular communication (GJIC) of cells, incubated for 48 hr in the presence of the higher dose of osteogenin (OG) (5.0 vs. 0.5 μg/ml) or higher dose of TGF‐β (2.0 vs. 0.2 ng/ml), was significantly inhibited compared to control. In another series of experiments, time and dose dependent effects of BMP‐2 and TGF‐β on GJIC were investigated. In the time course experiments (3, 6, 12, 24, and 48 hr), TGF‐β (2.0 ng/ml) demonstrated a statistically significant effect in inhibiting GJIC as early as 6 hr, while BMP‐2 (50 ng/ml) inhibited GJIC after 24 and 48 hr of treatment. The dose‐dependent effects of BMP‐2 and TGF‐β on cell couplings, determined at 48 hr, showed significant inhibitory effects with BMP‐2 at 25 and 50 ng/ml and with TGF‐β at 2 and 4 ng/ml. The cell count results and injection study performed at 12 hr, at a fixed cell density, confirmed that the inhibitory effect was not due to differences in cell density. The 50% effective inhibitory concentrations (EC50) calculated for BMP‐2 and TGF‐β at 48 hr, showed no dose correlation between proliferation and GJIC, suggesting that these two events are independent occurrences. Additionally, marked morphological change was observed in the cells treated with TGF‐β. The observation may suggest that TGF‐β may have effects upon cytoskeletal elements in osseous tissues.

Inhibition of gap junction intercellular communication by extremely low-frequency electromagnetic fields in osteoblast-like models is dependent on cell differentiation.

Electromagnetic fields have been used to augment the healing of fractures because of its ability to increase new bone formation. The mechanism of how electromagnetic fields can promote new bone formation is unknown, although the interaction of electromagnetic fields with components of the plasma membrane of cells has been hypothesized to occur in bone cells. Gap junctions occur among bone forming cells, the osteoblasts, and have been hypothesized to play a role in new bone formation. Thus it was investigated whether extremely low‐frequency (ELF) magnetic fields alter gap junction intercellular communication in the pre‐osteoblastic model, MC3T3‐E1, and the well‐differentiated osteoblastic model, ROS 17/2.8. ELF magnetic field exposure systems were designed to be used for an inverted microscope stage and for a tissue culture incubator. Using these systems, it was found that magnetic fields over a frequency range from 30 to 120 Hz and field intensities up to 12.5 G dose dependently decreased gap junction intercellular communication in MC3T3‐E1 cells during their proliferative phase of development. The total amount of connexin 43 protein and the distribution of connexin 43 gap junction protein between cytoplasmic and plasma membrane pools were unaltered by treatment with ELF magnetic fields. Cytosolic calcium ([Ca2+]i) which can inhibit gap junction communication, was not altered by magnetic field exposure. Identical exposure conditions did not affect gap junction communication in the ROS 17/2.8 cell line and when MC3T3‐E1 cells were more differentiated. Thus ELF magnetic fields may affect only less differentiated or pre‐osteoblasts and not fully differentiated osteoblasts. Consequently, electromagnetic fields may aid in the repair of bone by effects exerted only on osteoprogenitor or pre‐osteoblasts. J. Cell. Physiol. 190: 180–188, 2002.

Body weight reduction in rats by oral treatment with zinc plus cyclo-(His-Pro).

Background and purpose:  We have previously shown that treatment with zinc plus cyclo‐(His‐Pro) (CHP) significantly stimulated synthesis of the insulin degrading enzyme and lowered plasma insulin and blood glucose levels, alongside improving oral glucose tolerance in genetically type 2 diabetic Goto‐Kakizaki (G‐K) rats and in aged obese Sprague‐Dawley (S‐D) rats. Thus, we postulated that zinc plus CHP (ZC) treatment might also improve body weight control in these rats. We therefore determined the effects of ZC treatment on body weights in both genetically diabetic, mature G‐K rats and non‐diabetic, obese S‐D rats.

Expression and localization of estrogen receptor-β in annulus cells of the human intervertebral disc and the mitogenic effect of 17-β-estradiol in vitro

BackgroundRecent evidence suggests that estrogens exert effects in different tissues throughout the body, and that the estrogen receptor β (ERβ) may be important for the action of estrogen (17-β-estradiol) on the skeleton. The cellular localization of ERβ in the human intervertebral disc, however, has not yet been explored.MethodsHuman disc tissue and cultured human disc cells were used for immunocytochemical localization of ERβ. mRNA was isolated from cultured human disc cells, and RT-PCR amplification of ERβ was employed to document molecular expression of this receptor. Cultured human disc cells were tested to determine if 17-β-estradiol stimulated cell proliferation.ResultsIn this report data are presented which provide evidence for ERβ gene expression in human intervertebral disc cells in vivo and in vitro. Culture of annulus cells in the presence of 10-7 M 17-β-estradiol significantly increased cell proliferation.ConclusionsThese data provide new insight into the biology of cells in the annulus of the intervertebral disc.

Osteogenesis on nanoparticulate mineralized collagen scaffolds via autogenous activation of the canonical BMP receptor signaling pathway

Skeletal regenerative medicine frequently incorporates deliverable growth factors to stimulate osteogenesis. However, the cost and side effects secondary to supraphysiologic dosages of growth factors warrant investigation of alternative methods of stimulating osteogenesis for clinical utilization. In this work, we describe growth factor independent osteogenic induction of human mesenchymal stem cells (hMSCs) on a novel nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG). hMSCs demonstrated elevated osteogenic gene expression and mineralization on MC-GAG with minimal to no effect upon addition of BMP-2 when compared to non-mineralized scaffolds (Col-GAG). To investigate the intracellular pathways responsible for the increase in osteogenesis, we examined the canonical and non-canonical pathways downstream from BMP receptor activation. Constitutive Smad1/5 phosphorylation with nuclear translocation occurred on MC-GAG independent of BMP-2, whereas Smad1/5 phosphorylation depended on BMP-2 stimulation on Col-GAG. When non-canonical BMPR signaling molecules were examined, ERK1/2 phosphorylation was found to be decreased in MC-GAG but elevated in Col-GAG. No differences in Smad2/3 or p38 activation were detected. Collectively, these results demonstrated that MC-GAG scaffolds induce osteogenesis without exogenous BMP-2 addition via endogenous activation of the canonical BMP receptor signaling pathway.

Cellular immunosenescence : an overview

Recent studies on space flights suggest that certain T cell immunologic activities are vulnerable to microgravitation. It would be desirable to know the extent to which these changes can be prevented or reversed. Since the changes observed are analogous to the effects of aging on immunity, a brief overview is presented of our current knowledge of age-related changes in immune cells and of the various interventional methods which have been used successfully in preventing the decline with age and in elevating the levels of immune functions of old individuals.

Angiogenic CXC Chemokine Expression During Differentiation of Human Mesenchymal Stem Cells Towards the Osteoblastic Lineage

The potential role of ELR+ CXC chemokines in early events in bone repair was studied using human mesenchymal stem cells (hMSCs). Inflammation, which occurs in the initial phase of tissue healing in general, is critical to bone repair. Release of cytokines from infiltrating immune cells and injured bone can lead to recruitment of MSCs to the region of repair. CXC chemokines bearing the Glu‐Leu‐Arg (ELR) motif are also released by inflammatory cells and serve as angiogenic factors stimulating chemotaxis and proliferation of endothelial cells. hMSCs, induced to differentiate with osteogenic medium (OGM) containing ascorbate, β‐glycerophosphate (β‐GP), and dexamethasone (DEX), showed an increase in mRNA and protein secretion of the ELR+ CXC chemokines CXCL8 and CXCL1. CXCL8 mRNA half‐life studies reveal an increase in mRNA stability upon OGM stimulation. Increased expression and secretion is a result of DEX in OGM and is dose‐dependent. Inhibition of the glucocorticoid receptor with mifepristone only partially inhibits DEX‐stimulated CXCL8 expression indicating both glucocorticoid receptor dependent and independent pathways. Treatment with signal transduction inhibitors demonstrate that this expression is due to activation of the ERK and p38 mitogen‐activated protein kinase (MAPK) pathways and is mediated through the Gαi‐coupled receptors. Angiogenesis assays demonstrate that OGM‐stimulated conditioned media containing secreted CXCL8 and CXCL1 can induce angiogenesis of human microvascular endothelial cells in an in vitro Matrigel assay. J. Cell. Biochem. 103: 812–824, 2008.