The prostaglandin E2 receptor PTGER2 and prostaglandin F2α receptor PTGFR mediate oviductal glycoprotein 1 expression in bovine oviductal epithelial cells.

Oviductal glycoprotein 1 (OVGP1), an oviductin, is involved in the maintenance of sperm viability and motility and contributes to sperm capacitation in the oviduct. In this study, the regulatory effects exerted by prostaglandin E2 (PGE2) and F2α (PGF2α) on OVGP1 expression via their corresponding receptors in bovine oviductal epithelial cells (BOECs) were investigated. BOECs were cultured in vitro, and their expression of receptors of PGE2 (PTGER1, PTGER2, PTGER3, and PTGER4) and PGF2α (PTGFR) was measured using RT-qPCR. Ca2+ concentration was determined with a fluorescence-based method and cAMP was quantified by enzyme-linked immunosorbent assays to verify activation of PTGER2 and PTGFR by their corresponding agonists in these cells. OVGP1 mRNA and protein expression was measured using RT-qPCR and western blotting, respectively, following PTGER2 and PTGFR agonist-induced activation. PTGER1, PTGER2, PTGER4, and PTGFR were found to be present in BOECs; however, PTGER3 expression was not detected. OVGP1 expression was significantly promoted by 10-6 M butaprost (a PTGER2 agonist) and decreased by 10-6 M fluprostenol (a PTGFR agonist). In addition, 3 µM H-89 (a PKA inhibitor) and 3 µM U0126 (an ERK inhibitor) effectively inhibited PGE2-induced upregulation of OVGP1, and 5 µM chelerythrine chloride (a PKC inhibitor) and 3 µM U0126 negated OVGP1 downregulation by PGF2α. In conclusion, this study demonstrates that OVGP1 expression in BOECs is enhanced by PGE2 via PTGER2-cAMP-PKA signaling, and reduced by PGF2α through the PTGFR-Ca2+-PKC pathway.

Effects of Melatonin, Aluminum Oxide, and Polymethylsiloxane Complex on the Expression of LYVE-1 in the Liver of Mice with Obesity and Type 2 Diabetes Mellitus.

The effects of melatonin, aluminum oxide, and polymethylsiloxane complex on the expression of LYVE-1 (lymphatic vessel endothelial hyaluronan receptor) in the liver were studied in db/db mice with experimental obesity and type 2 diabetes mellitus. The complex or placebo was administered daily by gavage from week 8 to week 16 of life. The animals receiving the complex exhibited enhanced, in comparison with the placebo group, immunohistochemical LYVE-1+ staining of endothelial cells in sinusoids. Enhanced expression of LYVE-1 was associated with less pronounced dilatation of interlobular arteries, veins, and lymphatic vessels. Thee findings suggest a protective effect of the complex towards structural changes in the liver of mice with obesity and type 2 diabetes.

Immune regulation by CD52-expressing CD4 T cells.

T-cell regulation by CD52-expressing CD4 T cells appears to operate by two different and possibly synergistic mechanisms. The first is by its release from the cell surface of CD4 T cells that express high levels of CD52 that then binds to the inhibitory sialic acid-binding immunoglobulin-like lectins-10 (Siglec-10) receptor to attenuate effector T-cell activation by impairing phosphorylation of T-cell receptor associated lck and zap-70. The second mechanism appears to be by crosslinkage of the CD52 molecules by an as yet unidentified endogenous ligand that is mimicked by a bivalent anti-CD52 antibody that results in their expansion.

Hyaluronidase treatment of acute lymphedema in a mouse tail model.

The purpose of this study was to investigate the impact of hyaluronidase (HAase) on lymphedema using an acute mouse tail lymphedema model. Six-week-old mice served to produce acute lymphedema and were then either treated with HAase injection or used as operative controls. An additional group of unmanipulated normal mice was used for comparison. Tail volumes were measured for 23 days and histological changes examined. Western blot analysis was conducted to quantify lymphatic vessel endothelial hyaluronan receptor (LYVE)-1, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, podoplanin, CD 44, and vascular endothelial growth factor receptor3 (VEGFR3) expression levels. The operative control group showed an increase in thickness of the dermis and subdermis, microlymphatic dilatation, and an increase in neutrophils. In contrast, the HAase treated group exhibited alleviation of inflammation evidenced by a decline in microlymphatic dilatation and neutrophils and an overall increase in microlymphatic vessels. Western blot analysis demonstrated that TNF-alpha and TGF-beta1 expression declined but CD44 expression increased in the HAase treated group. Levels of LYVE1, podoplanin, and VEGFR3 also increased significantly in the HAase group. Our results indicate that HAase treatment in the acute mouse tail model reduced lymphedema volume possibly through degradation of HA trafficking, which reduced inflammation and fibrosis in tissues and stimulated lymphangiogenesis.

In vivo modulation of T cell responses and protective immunity by TCR antagonism during infection.

Infectious agents are known to express altered peptide ligands that antagonize T cells in vitro; however, direct evidence of TCR antagonism during infection is still lacking, and its importance in the context of infection remains to be established. In this study, we used a murine model of infection with recombinant Listeria monocytogenes and addressed three issues that are critical for assessing the role of TCR antagonism in the modulation of the immune response. First, we demonstrated that the antagonist peptide efficiently inhibited the ability of the agonist to prime naive TCR-transgenic T cells in vivo. Second, we showed clonal memory T cells were antagonized during recall responses, resulting in loss of protective immunity. Lastly, we observed that even in the context of a polyclonal response, TCR antagonism greatly inhibits the agonist-specific response, leading to altered hierarchy of immunodominance and reduced T cell memory and protective immunity. These results provide direct evidence of clonal TCR antagonism of naive and memory CD8 T cells during infection and demonstrate the effect of TCR antagonism on protective immunity. Thus, agonist/antagonist interactions may play an important role in determining the immunodominance and repertoire of T cell targets, and evaluation of immune responses and vaccine strategies may require examination of not only agonists but also antagonists and their interactions during an infection.

Production of recombinant plant gum with tobacco cell culture in bioreactor and gum characterization.

Many plant gums, such as gum arabic, contain hydroxyproline-rich glycoproteins (HRGPs), which are also abundant components of the plant cell extracellular matrix. Here we expressed in transgenic BY2 Nicotiana tabacum (tobacco) cells, a synthetic gene encoding a novel HRGP-based gum, designated gum arabic-8 or (GA)(8). (GA)(8) encoded eight repeats of the consensus polypeptide sequence of gum arabic glycoprotein (GAGP): Gly-Pro-His-Ser-Pro-Pro-Pro-Pro-Leu-Ser-Pro-Ser-Pro-Thr-Pro-Thr-Pro-Pro-Leu, in which most of the Pro residues were posttranslationally modified to hydroxyproline (Hyp). (GA)(8) was expressed as a green fluorescent protein (GFP) fusion protein targeted to the culture medium, (GA)(8)GFP. The culture of the transgenic cells in a 5-L bioreactor showed that the production of (GA)(8)GFP was cell growth-associated. The extracellular yield of (GA)(8)GFP was 116.8 mg/L after 14 days of culture and accounted for 87% of the total fusion protein expressed. (GA)(8)GFP was purified from the culture medium by a combination of hydrophobic interaction, gel permeation, and reversed phase chromatography. Biochemical characterization indicated that the amino acid composition of the (GA)(8) module, after removal of GFP by proteolysis, was virtually identical to that of predicted by the GAGP consensus sequence and that carbohydrate, which occurred as arabinogalactan polysaccharides and small oligoarabinosides O-linked through the Hyp residues, accounted for 84% of the molecules' dry weight. Functional assays showed that (GA)(8) exhibited low viscosity in aqueous solution similar to native GAGP. However, neither GFP alone nor the (GA)(8) module could emulsify orange oil. However, the fusion protein (GA)(8)GFP possessed 1.28-fold better emulsification properties than native GAGP. This work demonstrates the feasibility and potential of a synthetic gene approach to the de novo design of novel glycoprotein-based gums and emulsifiers.

Regulation of osteoclastogenesis and RANK expression by TGF-beta1.

Transforming growth factor-beta (TGF-beta) has been shown to both inhibit and to stimulate bone resorption and osteoclastogenesis. This may be due, in part, to differential effects on bone marrow stromal cells that support osteoclastogenesis vs. direct effects on osteoclastic precursor cells. In the present study, we used the murine monocytic cell line, RAW 264.7, to define direct effects of TGF-beta on pre-osteoclastic cells. In the presence of macrophage-colony stimulating factor (M-CSF) (20 ng/ml) and receptor activator of NF-kappaB ligand (RANK-L) (50 ng/ml), TGF-beta1 (0.01-5 ng/ml) dose-dependently stimulated (by up to 120-fold) osteoclast formation (assessed by the presence of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and expression of calcitonin and vitronectin receptors). In addition, TGF-beta1 also increased steady state RANK mRNA levels in a time- (by up to 3.5-fold at 48 h) and dose-dependent manner (by up to 2.2-fold at 10 ng/ml). TGF-beta1 induction of RANK mRNA levels was present both in undifferentiated RAW cells as well as in cells that had been induced to differentiate into osteoclasts by a 7-day treatment with M-CSF and RANK-L. Using a fluorescence-labeled RANK-L probe, we also demonstrated by flow cytometry that TGF-beta1 resulted in a significant increase in the percentage of RANK+ RAW cells (P < 0.05), as well as an increase in the fluorescence intensity per cell (P < 0.05), the latter consistent with an increase in RANK protein expression per cell. These data thus indicate that TGF-beta directly stimulates osteoclastic differentiation, and this is accompanied by increased RANK mRNA and protein expression.

Threshold signaling of human Th0 cells in activation and anergy: modulation of effector function by altered TCR ligand.

Molecular interactions between TCR and its natural ligand, in the presence of costimulatory signals, elicit T cell effector functions, whereas subtle changes in the structure of antigenic peptides may induce only selected T cell effector function including anergy. In this study, we have investigated the immunological activity of an altered TCR ligand (p 2, 28-40A34,36) derived from the immunodominant T cell epitope of the group 2 allergen of house dust mite, in which residues at positions 34 and 36 were substituted by alanine. Elevated IFN-gamma synthesis was induced by equimolar concentrations of the analogue compared with native peptide (p 2, 28-40) and was paralleled by increased down-regulation of cell surface CD3. IL-5 and IL-10 production exhibit the same sensitivity to both peptides, implying that the induction of T cell effector functions are not all proportional to TCR occupancy. Both native peptide and the analogue bound to MHC class II (DRB1*1101) molecules with similar affinities. Furthermore, p 2, 28-40A34,36 induced T cell anergy at lower concentrations than native peptide. During the induction of anergy, TGF-beta production was comparable for both peptides, whereas IL-10 secretion was markedly increased but more so in response to p 2, 28-40A34,36. Membrane expression of costimulatory ligands CD80 and CD86 was similar for native peptide and p 2, 28-40A34,36 and increased in activation, whereas only CD86 was elevated during anergy. The modulation of T cell effector function with altered TCR ligands may have practical applications in reprogramming allergic inflammatory responses through the induction of T cell anergy and/or the promotion of Th1 cytokines.

Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL.

Osteoclasts, the multinucleated cells that resorb bone, develop from hematopoietic cells of monocyte/macrophage lineage. Osteoclast-like cells (OCLs) are formed by coculturing spleen cells with osteoblasts or bone marrow stromal cells in the presence of bone-resorbing factors. The cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors is essential for OCL formation. Recently, we purified and molecularly cloned osteoclastogenesis-inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF is a secreted member of the tumor necrosis factor receptor family and inhibits osteoclastogenesis by interrupting the cell-to-cell interaction. Here we report the expression cloning of a ligand for OPG/OCIF from a complementary DNA library of mouse stromal cells. The protein was found to be a member of the membrane-associated tumor necrosis factor ligand family and induced OCL formation from osteoclast progenitors. A genetically engineered soluble form containing the extracellular domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing factors. We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.

Heregulin induces in vivo proliferation and differentiation of mammary epithelium into secretory lobuloalveoli.

Mammary gland development and differentiation is mediated through the combined activities of systemic hormones and locally synthesized growth factors. To determine the in vivo response of mammary epithelium to heregulin (HRG), we implanted Elvax pellets containing HRG alpha or HRG beta within the mammary glands of prepubescent female mice in the presence or absence of exogenous estradiol and progesterone (E/ P). Mice treated in the same way with transforming growth factor alpha (TGF-alpha) were included as a positive control. Each growth factor treatment induced epithelial ductal branching in the presence or absence of E/P. In the absence of E/P, HRG beta did not effect terminal end bud formation, mammary epitheilum branching, or ductal migration. In contrast, TGF-alpha and HRG alpha induced ductal branching and HRG alpha induced ductal migration in the absence of E/P. The overall mammary response to growth factors was potentiated by the concomitant presence of E/P. In every case, the in vivo mammary epithelial responses to HRG alpha were more robust than TGF-alpha. Limited lobuloalveolar development was also observed in growth factor-treated mammary glands when E/P was present. Histological examination of growth factor-induced lobuloalveoli revealed secretory products within the lumen of HRG alpha and HRG beta lobuloalveoli. TGF-alpha-induced lobuloalveoli lacked similar secretory products.

Agonist-induced expression of tissue inhibitor of metalloproteinases and metalloproteinases by human macrophages is regulated by endogenous prostaglandin E2 synthesis.

The regulatory effect of endogenously synthesized eicosanoid metabolites on the expression of tissue inhibitor of metalloproteinases (TIMP), interstitial collagenase, and 92-kDa gelatinase by human macrophages was examined. TIMP and metalloproteinase production were stimulated with three agonists that produce distinct patterns of eicosanoid synthesis: lipopolysaccharide (10 micrograms/ml), denatured collagen (10 micrograms/ml), or zymosan (1 mg/ml). Indomethacin (3 micrograms/ml) or MK886 (3 microM), a specific inhibitor of 5-lipoxygenase, was used to examine the role of endogenous metabolites of arachidonic acid. Regardless of the agonist used, TIMP production by macrophages was inhibited 65% by indomethacin, synthesis of interstitial collagenase was reduced 70%, and expression of 92-kDa gelatinase was decreased 40%. In contrast, inhibition of leukotriene synthesis had no effect on metalloproteinase or TIMP production. The agonist-stimulated increase in TIMP and collagenase production was directly correlated to the cumulative prostaglandin E2 level induced by the agonist used. However, if response to an agonist was poor, the exogenous addition of prostaglandin E2 could not increase TIMP or collagenase production more than twofold, indicating an important permissive effect of the agonist on the regulation of each protein's expression. The mechanism of indomethacin inhibition of TIMP and collagenase production was studied by labeling the cells with [35S]-methionine and performing immunoprecipitation using specific antiserum. Indomethacin markedly inhibited the lipopolysaccharide-induced biosynthesis of both TIMP and collagenase. Northern analysis revealed parallel suppression of TIMP and collagenase steady-state mRNA levels by indomethacin, indicating pretranslational control. The regulation of inflammatory-cell TIMP and interstitial collagenase expression by prostaglandin E2 suggests that therapy inhibiting the cellular response to prostaglandins may be useful in cutaneous and systemic disease states involving macrophage-mediated connective-tissue destruction.