Intracellular Secretion Research Articles

Laboratory and Genetic Investigation of Mutations Accounting for Congenital Fibrinogen Disorders.

Congenital fibrinogen disorders are classified into two types of plasma fibrinogen defects: type I (quantitative fibrinogen deficiencies), that is, hypofibrinogenemia or afibrinogenemia, in which there are low or absent plasma fibrinogen antigen levels, respectively, and type II (qualitative fibrinogen deficiencies), that is, dysfibrinogenemia or hypodysfibrinogenemia, in which there are normal or reduced antigen levels associated with disproportionately low functional activity. These disorders are caused by mutations in the three fibrinogen-encoding genes FGA, FGB, and FGG. Afibrinogenemia is associated with mild to severe bleeding, whereas hypofibrinogenemia is often asymptomatic. For these quantitative disorders, the majority of mutations prevent protein production. However, in some cases, missense or late-truncating nonsense mutations allow synthesis of the mutant fibrinogen chain, but intracellular fibrinogen assembly and/or secretion are impaired. Qualitative fibrinogen disorders are associated with bleeding, thrombosis, or both thrombosis and bleeding, but many dysfibrinogenemias are asymptomatic. The majority of cases are caused by heterozygous missense mutations. Here, we review the laboratory and genetic diagnosis of fibrinogen gene anomalies with an updated discussion of causative mutations identified.

Modulation of intracellular expression of IFNγ and IL-2 in culture of splenic T lymphocytes by some flavonoid glycosides of Alchornea floribunda

Context Alchornea floribunda Müll. Arg. (Euphorbiaceae) leaves are widely used in ethnomedicine for the management of rheumatism, arthritis and toothache.Objective In this study, flavonoid glycosides isolated from Alchornea floribunda were screened for their effect on the intracellular expression of interferon-gamma (IFNγ) and interleukin-2 (IL-2) type-1 cytokines.Materials and methods Chromatographic purification of the ethyl acetate fraction of the methanol leaf extract led to the isolation of seven flavonoid glycosides (1–7). Their structures were elucidated by 1D and 2D nuclear magnetic resonance and mass spectrometry. Splenocytes were treated with graded concentrations of the compounds (6.25–25 μg/mL) and incubated for 24 h. Thereafter, their effect on the expression of IFNγ and IL-2 by CD4+ and CD8+ T-lymphocytes was evaluated using intracellular cytokine staining and FACS analysis.Results Compounds 1–7 (6.25–25 μg/mL) caused the up-regulation of activated CD8+ (57.85–72.45% versus 57.85% for untreated control) and, to a lesser extent, activated CD4+ (3.21–7.21% versus 2.75% for the untreated control) T-lymphocytes that were both largely interferon-gamma-releasing in treated mouse T lymphocytes relative to untreated control. FACS data analysis showed that stimulation with all the compounds increased the proportion of CD8+/IFNγ+ and CD4+/IFNγ+ T lymphocytes up to two-fold when compared with the cells in untreated control wells. Intracellular IL-2 secretion by treated T cells was not detected.Conclusion This recorded T-lymphocyte-specific immune-modulatory property may contribute to explain in part the dynamics associated with the ethnomedicine of Alchornea floribunda, and may find relevance as a necessary cellular immune response precursor to infection-associated disease management.

Abstract P2-06-10: Loss of COX5B inhibits proliferation and promotes senescence via mitochondrial dysfunction in breast cancer

Abstract Background: COX5B, a peripheral subunit of the cytochrome c oxidase complex, is reported to maintain the stability of this complex and impact on cell viability. Recently, several studies demonstrated that cox5b may involve in cancer progression. In our study, we also found that COX5B was upregulated both in breast cancer tissues and cells. However, the role and mechanism of COX5B in breast cancer remain unclear. Therefore, the aim of this study is to validate COX5B functions and its underlying mechanisms in breast cancer cells. Methods: SILAC (stable isotope labeling with amino acids in cell culture) assay was conducted in mammary epithelial cells and its paired breast tumor cells, and the results was validated by other two paired tissues. Forty tumors and twenty normal or benign samples were implied for COX5B expression by IHC. Online Kaplan Meier plotting tool was used to confirm the association between COX5B expression and clinical outcome. Three Cell lines (MDA-MB-231, MDA-MB-468 and MCF-7) were constructed by sh-RNA and the efficiency was confirmed by qPCR and western blot and then migration, proliferation and senescence characteristics were evaluated. Furthermore, microarray was implemented to detect potential mechanisms and the associated genes were assessed by qPCR. ELISA was carried out for IL8 secretion. Condition medium was collected from stable knock down cells and used as attractant in lower chamber of tranwell assay to examine microenvironment influence. Finally, the intracellular ATP glucose uptake and lactate secretion were evaluated, as well as mitochondrial membrane potential and ROS production. Results: We found that the level of COX5B was elevated in breast cancer and cell lines. Patients with high levels of COX5B were likely to have a longer disease-free survival than patients with low levels. Furthermore, loss of COX5B inhibited proliferation and induced the senescence of breast cancer cells, which was accompanied by increases in the secretion of IL-8 and other cytokines. Additionally, conditioned medium form COX5B knockdown cells increased the migration of breast cancer cells compared with conditioned medium from control cells. The mechanisms underlying these processes were associated with mitochondrial dysfunction due to increased ROS production and decreased MMP and metabolism disorder. Conclusion: Our results show that loss of COX5B inhibits proliferation and promotes senescence in breast cancer cells, which is associated with mitochondrial dysfunction. Indeed, loss of COX5B has different functions in target cells and the surrounding environments. These findings may provide a new perspective for the design of anti-cancer therapy combining with anti-IL8 treatment. Citation Format: Gao S-P, Sun H-F, Jiang H-L, Li L-D, Jin W. Loss of COX5B inhibits proliferation and promotes senescence via mitochondrial dysfunction in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-06-10.

Sodium Lauryl Sulfate Stimulates the Generation of Reactive Oxygen Species through Interactions with Cell Membranes.

Sodium lauryl sulfate (SLS), a representative anionic surfactant, is well-known to induce rough skin following single or multiple topical applications. The mechanism by which SLS induces rough skin is thought to result from the disruption of skin moisture function consisting of NMF and epidermal lipids. However, a recent study demonstrated that topically applied SLS easily penetrates into the living cell layers of the epidermis, which suggests that physiological alterations of keratinocytes might cause the SLS-induced rough skin. This study was conducted to clarify the effects of SLS on keratinocytes to demonstrate the contribution of SLS to the induction of rough skin. In addition, the potentials of other widely used anionic surfactants to induce rough skin were evaluated. HaCaT keratinocytes treated with SLS had increased levels of intracellular ROS and IL-1α secretion. Application of SLS on the surface of a reconstructed epidermal equivalent also showed the increased generation of ROS. Further, SLS-treated cells showed an increase of intracellular calpain activity associated with the increase of intracellular Ca2+ concentration. The increase of intracellular ROS was abolished by the addition of BAPTA-AM, a specific chelator of Ca2+. In addition, IL-1α also stimulated ROS generation by HaCaT keratinocytes. An ESR spin-labeling study demonstrated that SLS increased the fluidity of membranes of liposomes and cells. Together, those results indicate that SLS initially interacts with cell membranes, which results in the elevation of intracellular Ca2+ influx. Ca2+ stimulates the secretion of IL-1α due to the activation of calpain, and also increases ROS generation. IL-1α also stimulates ROS generation by HaCaT keratinocytes. We conclude from these results that the elevation of intracellular ROS levels is one of the causes of SLS-induced rough skin. Finally, among the other anionic surfactants tested, sodium lauryl phosphate has less potential to induce rough skin because of its lower generation of ROS.

Effect of Asiasarum Root Extract and Its Constituents on Interleukin-1β-Stimulated Matrix Metalloproteinase-1 Secretion from Human Gingival Fibroblasts.

Asiasarum root (roots and rhizome of Asiasarum sieboldii or A. heterotropoides var. mandshuricum) has been frequently used in traditional Chinese medicinal formulas for the management of oral malodor syndrome caused by periodontal disease. However, there are no scientific reports concerning these effects and the mechanism of action. The objective of this study was to examine the inhibitory effects of Asiasarum root and its constituents on oral malodor syndrome and periodontal disease. A 50% ethanolic extract of Asiasarum root (AR-ext) showed L-methionine γ-lyase (METase) inhibitory activity at a concentration of 200 µg/mL, and inhibited interleukin (IL)-1β-stimulated matrix metalloproteinase (MMP)-1 secretion from human gingival fibroblasts (HGFs) at a concentration of 10 and 50 µg/mL without cytotoxic effects. Activity-guided fractionation of the AR-ext suggested that METase inhibitory activity was attributable to a mixture of linoleic and oleic acid, because these unsaturated fatty acids showed weak METase inhibitory activities. Similar fractionation using MMP-1 secretion inhibitory activity led to the isolation of two unsaturated fatty acid amides, (2E,4E,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide (1) and (2E,4E,8Z,10Z)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide (2), as active constituents with inhibitory activity on MMP-1 secretion from HGFs. To elucidate the inhibition mechanism on MMP-1 secretion, the effect of 2 on mitogen-activated protein kinase (MAPK) phosphorylation was examined. Western blotting analysis revealed that 2 (10 µM) reduced the phosphorylation of p38 and c-Jun-N-terminal kinase. These results suggested that 2 suppresses intracellular MMP-1 expression and MMP-1 secretion from IL-1β-stimulated HGFs by down-regulation of MAPK phosphorylation.

Effects of Lipopolysaccharide on Human First Trimester Villous Cytotrophoblast Cell Function In Vitro.

It has been shown that adverse obstetrical outcomes such as pre-eclampsia and intrauterine growth retardation correlate with maternal infection. In this study, we investigated mechanisms involved in infection-associated abnormalities in cytotrophoblast function. Primary human first trimester cytotrophoblast cells were isolated and treated with lipopolysaccharide (LPS). Levels of the cytokines and chemokines were measured and cytotrophoblast invasion was investigated. In addition, first trimester decidual macrophages were isolated and treated with the conditioned medium from LPS-treated cytotrophoblast cells, and macrophage migration was assessed. Coculturing decidual macrophages with cytotrophoblast cells was conducted to investigate macrophage costimulatory molecule and receptor expression and intracellular cytokine production. We found that LPS exposure increased cytotrophoblast production of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, and chemokines IL-8, macrophage inflammatory protein (MIP)-1alpha, and CXCL12 in a dose-dependent manner. In addition, LPS decreased cytotrophoblast invasion, and its effect was Toll-like receptor 4 (TLR4)-dependent and partly TNF-alpha-dependent. Conditioned medium from LPS-stimulated cytotrophoblast cells increased decidual macrophage migration and this effect was partly TLR4-dependent. Furthermore, coculturing decidual macrophages with LPS-exposed cytotrophoblast cells up-regulated macrophage CD80 and CD86 expression and intracellular TNF-alpha and IL-12p40 production, while down-regulating macrophage CD206 and CD209 expression and intracellular IL-10 secretion. LPS-stimulated macrophages also inhibited cytotrophoblast invasion. In conclusion, our results indicate that LPS increases the production of a subset of proinflammatory cytokines and chemokines by human first trimester cytotrophoblast cells, decreases cytotrophoblast invasion, and alters the cross talk between cytotrophoblast cells and decidual macrophages.

Splenic B-cell marginal zone lymphoma with marked plasmocytic differentiation: tumor variant from Mott cells?

Splenic B-cell marginal zone lymphoma (SMZL) – a rare B-cell non-Hodgkin, s lymphoma, which is represented morphologically by mature lymphoid cells, corresponding to lymphocytes of secondary follicles marginal zone by immunological characteristics. Plasma cell differentiation can be in marginal zone lymphoma, but we described a single case of abundance of Mott cells as a tumor substrate in splenic B-cell marginal zone lymphoma. We present the first case of SMZL represented by Mott cells. This was the second case of Mott cells tumor described in Russia. This observation is the only case among the collected material of splenic lymphomas. The morphological pattern is characterized by marked proliferation of monoclonal lymphoid cells with plasma cell differentiation with presence of Mott cells and is evidence of intense intracellular secretion of immunoglobulins.

Astrocytes spatially restrict VEGF signaling by polarized secretion and incorporation of VEGF into the actively assembling extracellular matrix.

The spatial organization of vascular endothelial growth factor (VEGF) signaling is a key determinant of vascular patterning during development and tissue repair. How VEGF signaling becomes spatially restricted and the role of VEGF secreting astrocytes in this process remains poorly understood. Using a VEGF-GFP fusion protein and confocal time-lapse microscopy, we observed the intracellular routing, secretion and immobilization of VEGF in scratch-activated living astrocytes. We found VEGF to be directly transported to cell-extracellular matrix attachments where it is incorporated into fibronectin fibrils. VEGF accumulated at β1 integrin containing fibrillar adhesions and was translocated along the cell surface prior to internalization and degradation. We also found that only the astrocyte-derived, matrix-bound, and not soluble VEGF decreases β1 integrin turnover in fibrillar adhesions. We suggest that polarized VEGF release and ECM remodeling by VEGF secreting cells is key to control the local concentration and signaling of VEGF. Our findings highlight the importance of astrocytes in directing VEGF functions and identify these mechanisms as promising target for angiogenic approaches.

Oleacein enhances anti-inflammatory activity of human macrophages by increasing CD163 receptor expression

BackgroundOleacein (dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol; 3,4-DHPEA-EDA) have been proven to possess antioxidant and anti-inflammatory activity. PurposeIn this study, we examined whether oleacein could increase CD163 and IL-10 receptor expression as well as HO-1 intracellular secretion in human macrophages. MethodsEffect of oleacein (10 and 20 μmol/l) or oleacein together with complexes of haemoglobin (Hb) and haptoglobin 1-1 (Hp11) or haptoglobin 2-2 (Hp22) on expression of IL-10 and CD163 receptor was determined by Flow Cytometry. Expression of CD163mRNA was measured by real-time quantitative RT-PCR. Heme oxygenase 1 (HO-1) intracellular secretion in macrophages was investigated by enzyme-linked immunosorbent assay (ELISA). ResultsOleacein (OC) together with complexes HbHp11 or HbHp22 stimulated the expression of CD163 (30-100-fold), IL-10 (170-300-fold) and HO-1 secretion (60-130-fold) after 5 days of coincubation. The 2-fold (24 h), 4-fold (48 h) increase of CD163 mRNA level and its final (72 h) decrease was also observed. ConclusionOur results suggested that oleacein enhances anti-inflammatory activity of complexes haemoglobin with haptoglobin 1-1 and 2-2 and could play a potential role in the prevention of inflammatory disease related to atherosclerosis.

Biogenesis and cytotoxicity of APOL1 renal risk variant proteins in hepatocytes and hepatoma cells

Two APOL1 gene variants, which likely evolved to protect individuals from African sleeping sickness, are strongly associated with nondiabetic kidney disease in individuals with recent African ancestry. Consistent with its role in trypanosome killing, the pro-death APOL1 protein is toxic to most cells, but its mechanism of cell death is poorly understood and little is known regarding its intracellular trafficking and secretion. Because the liver appears to be the main source of circulating APOL1, we examined its secretory behavior and mechanism of toxicity in hepatoma cells and primary human hepatocytes. APOL1 is poorly secreted in vitro, even in the presence of chemical chaper-ones; however, it is efficiently secreted in wild-type transgenic mice, suggesting that APOL1 secretion has specialized requirements that cultured cells fail to support. In hepatoma cells, inducible expression of APOL1 and its risk variants promoted cell death, with the G1 variant displaying the highest degree of toxicity. To explore the basis for APOL1-mediated cell toxicity, endoplasmic reticulum stress, pyroptosis, autophagy, and apoptosis were examined. Our results suggest that autophagy represents the predominant mechanism of APOL1-mediated cell death. Overall, these results increase our understanding of the basic biology and trafficking behavior of circulating APOL1 from the liver.

Enhancement of VEGF-Mediated Angiogenesis by 2-N,6-O-Sulfated Chitosan-Coated Hierarchical PLGA Scaffolds.

Rapid and controlled vascularization within scaffolds remains one of the key limitations in tissue engineering applications. This study describes the fabrication and characterization of 2-N,6-O-sulfated chitosan (26SCS)-coated hierarchical scaffold composed of poly(lactic-co-glycolic acid) (PLGA) microspheres, as a desirable vehicle for vascular endothelial growth factor (VEGF) delivery and consequent angiogenic boosting in vitro. Owing to the hierarchical porous structure and high affinity between VEGF and 26SCS, the 26SCS-modified PLGA (S-PLGA) scaffold possesses excellent entrapment and sustained release of VEGF. Using human umbilical vein endothelial cells (HUVECs) as a cell model, the VEGF-loaded S-PLGA scaffold shows desirable cell viability and attachment. The bioactivity of released VEGF is validated by intracellular nitric oxide secretion and capillary tube formation, demonstrating the improved capacity of VEGF-mediated pro-angiogenesis ascribed to 26SCS incorporation. Such a strategy will afford an effective method to prepare a scaffold with promoted angiogenesis.

The effect of SERPINF1 in-frame mutations in osteogenesis imperfecta type VI

Osteogenesis imperfecta type VI is caused by mutations in SERPINF1, which codes for pigment-epithelium derived factor (PEDF). Most of the reported SERPINF1 mutations lead to premature termination codons, but three in-frame insertion or deletion mutations have also been reported. It is not clear how such in-frame mutations lead to OI type VI. In the present study we therefore investigated how SERPINF1 in-frame mutations affect the intracellular localization and secretion of PEDF. Skin fibroblasts affected by SERPINF1 in-frame mutations transcribed SERPINF1 at slightly reduced levels but secretion of PEDF was markedly diminished. Two deletions (p.F277del and the deletion of SERPINF1 exon 5) were associated with retention of PEDF in the endoplasmic reticulum and a stress response in osteoblastic cells. A recurrent in-frame duplication of three amino acids (p.Ala91_Ser93dup) appeared to lead to intracellular degradation but no retention in the endoplasmic reticulum or stress response. Immunofluorescence imaging in transiently transfected osteoblastic MC3T3-E1 cells suggested that PEDF affected by in-frame mutations was not transported along the secretory pathway. MC3T3-E1 osteoblasts stably overexpressing SERPINF1 with the p.Ala91_Ser93dup mutation had decreased collagen type I deposition and mineralization. Thus, the assessed homozygous in-frame deletions or insertions lead to retention or degradation within cellular compartments and thereby interfere with PEDF secretion.

734 Tango1 Regulates Collagen I Intracellular Trafficking and Secretion in Hepatic Stellate Cells

734 Tango1 Regulates Collagen I Intracellular Trafficking and Secretion in Hepatic Stellate Cells

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca2+]i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca2+]i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48h to a variety of stressors: cytokines (low-grade inflammation), 28mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca2+]i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca2+]i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca2+]i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3mM glucose) observed for FFAs and also for 28G. We also clamped [Ca2+]i using 30mM KCl+250μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca2+]i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca2+]i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion.

Number and brightness analysis of sFRP4 domains in live cells demonstrates vesicle association signal of the NLD domain and dynamic intracellular responses to Wnt3a

The Wnts are secreted, lipidated glycoproteins that play a role in cellular processes of differentiation, proliferation, migration, survival, polarity and stem cell self-renewal. The majority of Wnts biological effects are through binding to specific frizzled (Fzd) receptor complexes leading to activation of downstream pathways. Secreted frizzled-related proteins (sFRPs) were first identified as antagonists of Wnt signalling by binding directly to Wnts. They comprise two domains, a Fzd-like cysteine rich domain (CRD) and a netrin-like domain (NLD). Subsequently sFRPs have been shown to also interact with Fzd receptors and more diverse functions have been identified, including potentiation of Wnt signalling. Many aspects of the biology of this family remain to be elucidated. We used the number and brightness (N&B) method, a technique based on fluorescence fluctuation analysis, to characterise the intracellular aggregation and trafficking of sFRP4 domains. We expressed sFRP4 and its’ domains as EGFP fusions and then characterised the effect of endogenous Wnt3a by fluorescence confocal imaging. We observed vesicular trafficking of sFRP4 and that the NLD domain has a vesicular association signal. We found that sFRP4 and the CRD formed oligomeric aggregates in the perinuclear region while the NLD was distributed evenly throughout the cell with a larger proportion of aggregates. Most significantly we observed intracellular redistribution of sFRP4 in response to Wnt3a suggesting that Wnt3a can modulate intracellular localisation and secretion of sFRP4. Our results reveal a number of novel findings regarding sFRP4 which are likely to have relevance to this wider family.

Serglycin is part of the secretory repertoire of LPS-activated monocytes.

Monocytes play multiple roles in the immune system, and are active in both acute and chronic diseases. Patients exposed to bacterial infections depend on monocytes in defense reactions, but excessive immune reactions may also cause morbidity through systemic inflammatory responses. Few studies have addressed the importance of proteoglycans, and in particular, the hematopoietic serglycin, in such monocyte immune reactions. Adherent primary monocytes were cultured in absence and presence of LPS. Media were analyzed by ELISA for detection of serglycin. Lysed cell fractions were used to determine the mRNA level of serglycin. Monocytes were also cultured on chamber slides to investigate if serglycin could be detected intracellularly by immunocytochemistry. Monocytes secreted serglycin, and LPS-stimulation increased the secretion. Secretion of inflammatory cytokines increased to a larger extent than serglycin. mRNA levels of serglycin were also increased, suggesting both increased expression and secretion. Immunocytochemistry revealed the presence of serglycin in intracellular vesicles, many destined for secretion. Serglycin containing vesicles increased in number and size when the cells were exposed to LPS. Intracellular vesicle localization and secretion of the proteoglycan serglycin is shown for the first time in primary human monocytes. Monocyte activation by LPS increased the expression and secretion of serglycin, suggesting roles for serglycin in inflammatory processes.

Activation of decidual invariant natural killer T cells promotes lipopolysaccharide-induced preterm birth.

Invariant natural killer T (iNKT) cells are crucial for host defense against a variety of microbial pathogens, but the underlying mechanisms of iNKT cells activation by microbes are not fully explained. In this study, we investigated the molecular mechanisms of iNKT cell activation in lipopolysaccharide (LPS)-stimulated preterm birth using an adoptive transfer system and diverse neutralizing antibodies (Abs) and inhibitors. We found that adoptive transfer of decidual iNKT cells to LPS-stimulated iNKT cell deficient Jα18(-/-) mice that lack invariant Vα14Jα281T cell receptor (TCR) expression significantly decreased the time to delivery and increased the percentage of decidual iNKT cells. Neutralizing Abs against Toll-like receptor 4 (TLR-4), CD1d, interleukin (IL)-12 and IL-18, and inhibitors blocking the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase (ERK) significantly reduced in vivo percentages of decidual iNKT cells, their intracellular interferon (IFN)-γ production and surface CD69 expression. In vitro, in the presence of the same Abs and inhibitors used as in vivo, decidual iNKT cells co-cultured with LPS-pulsed dendritic cells (DCs) showed significantly decreased extracellular and intracellular IFN-γ secretion and surface CD69 expression. Our data demonstrate that the activation of decidual iNKT cells plays an important role in inflammation-induced preterm birth. Activation of decidual iNKT cells also requires TLR4-mediated NF-κB, MAPK p38 and ERK pathways, the proinflammatory cytokines IL-12 and IL-18, and endogenous glycolipid antigens presented by CD1d.

Recombinant BMP4 and BMP7 increase activin A production by up-regulating inhibin βA subunit and furin expression in human granulosa-lutein cells.

Granulosa cell-derived activins play important roles in the regulation of ovarian functions. To date, there is limited information pertaining to the intracellular regulation, assembly, and secretion of endogenous activin A in human granulosa cells. The aim of this study was to examine the effects of BMP4 and BMP7 on furin expression and activin A production as well as the underlying mechanisms of action in human granulosa cells. An established immortalized human granulosa cell line (SVOG) and primary granulosa-lutein cells were used as study models. Expression of inhibin subunits and furin as well as activin A accumulation were examined after exposure to recombinant human BMP4 or BMP7. A BMP type I receptor inhibitor (dorsomorphin), a furin inhibitor (Decanoyl-Arg-Val-Lys-Arg-chloromethylketone), and small interfering RNAs targeting SMAD4 and furin were used to verify the specificity of the effects and investigate potential mechanisms. The study was conducted in an academic center. Specific mRNA and protein levels were examined using real time qPCR and Western blot. Activin A levels were measured using enzyme immunoassay. Treatment with bone morphogenetic protein (BMP) 4 and BMP7 significantly increased furin mRNA and protein, inhibin βA mRNA, and activin A accumulation. Pre-treatment with dorsomorphin or SMAD4 knockdown reversed the stimulatory effects of BMP4 and BMP7 on furin and inhibin βA expression. In addition, furin knockdown or pre-treatment with a furin inhibitor attenuated the BMP4- and BMP7-induced accumulation of activin A. Recombinant BMP4 and BMP7 increase the production of bioactive mature activin A by up-regulating both the production and proteolytic processing of inhibin βA subunit in human granulosa cells. The enhancement of inhibin βA subunit processing is attributable to a SMAD-dependent up-regulation of its proprotein convertase, furin. These findings provide a potential mechanism by which theca cells can regulate neighboring granulosa cells in the ovary.

Dual Monitoring of Secretion and ATP Levels during Chondrogenesis Using Perfusion Culture-Combined Bioluminescence Monitoring System.

Skeletal pattern formation in limb development depends on prechondrogenic condensation which prefigures the cartilage template. However, although morphogens such as TGF-βs and BMPs have been known to play essential roles in skeletal patterning, how the morphogens induce prechondrogenic cells to aggregate and determine patterns of cartilage elements has remained unclear. Our previous study reported that ATP oscillations are induced during chondrogenesis. This result suggests the possibility that ATP oscillations lead to the oscillatory secretion of morphogens, due to the fact that secretion process requires ATP. To examine the correlation between ATP oscillations and secretion levels of morphogens, we have developed perfusion culture-combined bioluminescence monitoring system to simultaneously monitor intracellular ATP levels and secretion levels. Using this system, we found that secretory activity oscillates in phase with ATP oscillations and that secretion levels of TGF-β1 and BMP2 oscillate during chondrogenesis. The oscillatory secretion of the morphogens would contribute to amplifying the fluctuation of the morphogens, underlie the spatial patterning of morphogens, and consequently lead to skeletal pattern formation.

Secretory Carcinoma of the Breast: Report of Two Cases and Review of the Literature.

Secretory carcinoma of the breast is an extremely rare subtype of breast cancer characterized by intracellular or extracellular secretion and granular eosinophilic cytoplasm of the neoplastic cells. The disease which was considered to be predominant in younger age group has been recognized in adult population too and tends to show slow growth and indolent behavior. The disease occurs preferentially in females and only 27 cases have been reported amongst males. An optimal treatment for the disease subtype has been debated because of the paucity of data. We report two cases (one female and one male) of this rare disease that underwent treatment at our institution.