Search

Cystectomy is the removal of all or part of the urinary bladder. It has been observed that there is significant regrowth of the bladder after partial cystectomy and this has been proposed to be through regeneration of the organ. Regrowth of tissue in mammals has been proposed to involve compensatory mechanisms that share many characteristics of true regeneration, like the growth of specialized structures such as blood vessels or nerves. However, the overall structure of the normal organ is not achieved. Here we tested if bladder growth after subtotal cystectomy (STC, removal of 50% of the bladder) was compensatory or regenerative. To do this we subjected adult female mouse bladders to STC and assessed regrowth using several established cellular parameters including histological, gene expression, cytokine accumulation and cell proliferation studies. Bladder function was analyzed using cystometry and the voiding stain on paper (VSOP) technique. We found that STC bladders were able to increase their ability to hold urine with the majority of volume restoration occurring within the first two weeks. Regenerating bladders had thinner walls with less mean muscle thickness, and they showed increased collagen deposition at the incision as well as throughout the bladder wall suggesting that fibrosis was occurring. Cell populations differed in their response to injury with urothelial regeneration complete by day 7, but stromal and detrusor muscle still incomplete after 8wks. Cells incorporated EdU when administered at the time of surgery and tracing of EdU positive cells over time indicated that many newborn cells originate at the incision and move mediolaterally. Basal urothelial cells and bladder mesenchymal stem cells but not smooth muscle cells significantly incorporated EdU after STC. Since anti-inflammatory cytokines play a role in regeneration, we analyzed expressed cytokines and found that no anti-inflammatory cytokines were present in the bladder 1wk after STC. Our findings suggest that bladder regrowth after cystectomy is compensatory and functions to increase the volume that the bladder can hold. This finding sets the stage for understanding how the bladder responds to cystectomy and how this can be improved in patients after suffering bladder injury. [ABSTRACT FROM AUTHOR]

Background: Obesity is a well-known risk factor of breast cancer in post-menopausal women that also correlates with a diminished therapeutic response. The influence of adipocytes and their secretome, i.e. adipokines, on the efficacy of hormone therapy has yet to be elucidated. Methods: We investigated, ex vivo, whether mature adipocytes, differentiated from adipose stem cells of normal-weight (MA20) or obese (MA30) women, and their secretions, were able to counteract the effects of tamoxifen (Tx) which is known to decrease neoplastic cell proliferation. Results: In a tridimensional model and in a model of co-culture, the anti-proliferative effect of Tx on MCF-7 cancer cells was counteracted by MA30. These two models highlighted two different specific gene expression profiles for genes encoding cytokines or involved in angiogenesis based on the adipocyte microenvironment and the treatment. Thus it notably showed altered expression of genes such as TNFα that correlated with IL-6. In addition, leptin, IL-6 and TNFα, at concentrations reflecting plasma concentrations in obese patients, decreased the anti-proliferative efficacy of 4-hydroxytamoxifen (a major active metabolite of Tx). Conclusions: These findings bring insights on adipocytes and mammary cancer cell interactions in Tx therapy, particularly in overweight/obese people. Indeed, patient’ adipokine status would give valuable information for developing individual strategies and avoid resistance to treatment. [ABSTRACT FROM AUTHOR]

Interleukin-31 (IL-31) is a type 2 helper T-cell-derived cytokine that has recently been shown to cause severe inflammation and tissue remodeling in multiple chronic diseases of the skin and lungs. IL-31 is upregulated in allergic and inflammatory diseases, including atopic dermatitis, asthma, cutaneous T-cell lymphomas, and allergic rhinitis, as well as autoimmune diseases such as systemic erythematosus. Overexpression of IL-31 in T cells causes severe inflammation, with histological features similar to skin lesions of patients with atopic dermatitis. However, the molecular mechanisms involved in IL31-driven pathological remodeling in skin diseases remain largely unknown. Here, we studied the role of IL-31 in skin damage as a result of intradermal administration of recombinant IL-31 into mice. Notably, IL-31 was sufficient to increase epidermal basal-cell proliferation and thickening of the epidermal skin layer. Our findings demonstrate a progressive increase in transepidermal water loss with chronic administration of IL-31 into the skin. Further, analysis of the skin transcriptome indicates a significant increase in the transcripts involved in epidermal-cell proliferation, epidermal thickening, and mechanical integrity. In summary, our findings demonstrate an important role for IL-31 signaling in epidermal cell proliferation and thickening that together may lead to impaired skin-barrier function in pathological remodeling of the skin. [ABSTRACT FROM AUTHOR]

Airway smooth muscle (ASM) cells play a critical role in the pathophysiology of asthma due to their hypercontractility and their ability to proliferate and secrete inflammatory mediators. microRNAs (miRNAs) are gene regulators that control many signaling pathways and thus serve as potential therapeutic alternatives for many diseases. We have previously shown that miR-708 and miR-140-3p regulate the MAPK and PI3K signaling pathways in human ASM (HASM) cells following TNF-α exposure. In this study, we investigated the regulatory effect of these miRNAs on other asthma-related genes. Microarray analysis using the Illumina platform was performed with total RNA extracted from miR-708 (or control miR)-transfected HASM cells. Inhibition of candidate inflammation-associated gene expression was further validated by qPCR and ELISA. The most significant biologic functions for the differentially expressed gene set included decreased inflammatory response, cytokine expression and signaling. qPCR revealed inhibition of expression of CCL11, CXCL10, CCL2 and CXCL8, while the release of CCL11 was inhibited in miR-708-transfected cells. Transfection of cells with miR-140-3p resulted in inhibition of expression of CCL11, CXCL12, CXCL10, CCL5 and CXCL8 and of TNF-α-induced CXCL12 release. In addition, expression of RARRES2, CD44 and ADAM33, genes known to contribute to the pathophysiology of asthma, were found to be inhibited in miR-708-transfected cells. These results demonstrate that miR-708 and miR-140-3p exert distinct effects on inflammation-associated gene expression and biological function of ASM cells. Targeting these miRNA networks may provide a novel therapeutic mechanism to down-regulate airway inflammation and ASM proliferation in asthma. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity. [ABSTRACT FROM AUTHOR]

Cytokine-like protein 1 (Cytl1), originally described as a protein expressed in CD34+ cells, was recently identified as a functional secreted protein involved in chondrogenesis and cartilage development. However, our knowledge of Cytl1 is still limited. Here, we determined the Cytl1 expression pattern regulated by ovarian hormones at both the mRNA and protein levels. We found that the endometrial expression of Cytl1 in mice was low before or on the first day of gestation, significantly increased during embryo implantation, and then decreased at the end of implantation. We investigated the effects of Cytl1 on endometrial cell proliferation, and the effects on the secretion of leukemia inhibitory factor (LIF) and heparin-binding epidermal growth factor (HB-EGF). We also explored the effect of Cytl1 on endometrial adhesion properties in cell-cell adhesion assays. Our findings demonstrated that Cytl1 is an ovarian hormone-dependent protein expressed in the endometrium that enhances the proliferation of HEC-1-A and RL95-2 cells, stimulates endometrial secretion of LIF and HB-EGF, and enhances the adhesion of HEC-1-A and RL95-2 cells to JAR spheroids. This study suggests that Cytl1 plays an active role in the regulation of embryo implantation. [ABSTRACT FROM AUTHOR]

Proangiogenic enzyme thymidine phosphorylase (TP) is a promising target for anticancer therapy, yet its action in non-small cell lung carcinoma (NSCLC) is not fully understood. To elucidate its role in NSCLC tumor growth, NCI-H292 lung mucoepidermoid carcinoma cells and endothelial cells were engineered to overexpress TP by viral vector transduction. NSCLC cells with altered expression of transcription factor Nrf2 or its target gene heme oxygenase-1 (HO-1) were used to study the regulation of TP and the findings from pre-clinical models were related to gene expression data from clinical NSCLC specimens. Overexpression of Nrf2 or HO-1 resulted in upregulation of TP in NCI-H292 cells, an effect mimicked by treatment with an antioxidant N-acetylcysteine and partially reversed by HO-1 knockdown. Overexpression of TP attenuated cell proliferation and migration in vitro, but simultaneously enhanced angiogenic potential of cancer cells supplemented with thymidine. The latter was also observed for SK-MES-1 squamous cell carcinoma and NCI-H460 large cell carcinoma cells. TP-overexpressing NCI-H292 tumors in vivo exhibited better oxygenation and higher expression of IL-8, IL-1β and IL-6. TP overexpression in endothelial cells augmented their angiogenic properties which was associated with enhanced generation of HO-1 and VEGF. Correlation of TP with the expression of HO-1 and inflammatory cytokines was confirmed in clinical samples of NSCLC. Altogether, the increased expression of IL-1β and IL-6 together with proangiogenic effects of TP-expressing NSCLC on endothelium can contribute to tumor growth, implying TP as a target for antiangiogenesis in NSCLC. [ABSTRACT FROM AUTHOR]

Human periodontal ligament cells (hPDLCs) possess stem cell properties, which play a key role in periodontal regeneration. Physical stimulation at appropriate intensities such as low-intensity pulsed ultrasound (LIPUS) enhances cell proliferation and osteogenic differentiation of mesechymal stem cells. However, the impacts of LIPUS on osteogenic differentiation of hPDLCs in vitro and its molecular mechanism are unknown. This study was undertaken to investigate the effects of LIPUS on osteogenic differentiation of hPDLCs. HPDLCs were isolated from premolars of adolescents for orthodontic reasons, and exposed to LIPUS at different intensities to determine an optimal LIPUS treatment dosage. Dynamic changes of alkaline phosphatase (ALP) activities in the cultured cells and supernatants, and osteocalcin production in the supernatants after treatment were analyzed. Runx2 and integrin β1 mRNA levels were assessed by reverse transcription polymerase chain reaction analysis after LIPUS stimulation. Blocking antibody against integrinβ1 was used to assess the effects of integrinβ1 inhibitor on LIPUS-induced ALP activity, osteocalcin production as well as calcium deposition. Our data showed that LIPUS at the intensity of 90 mW/cm2 with 20 min/day was more effective. The ALP activities in lysates and supernatants of LIPUS-treated cells started to increase at days 3 and 7, respectively, and peaked at day 11. LIPUS treatment significantly augmented the production of osteocalcin after day 5. LIPUS caused a significant increase in the mRNA expression of Runx2 and integrin β1, while a significant decline when the integrinβ1 inhibitor was used. Moreover, ALP activity, osteocalcin production as well as calcium nodules of cells treated with both daily LIPUS stimulation and integrinβ1 antibody were less than those in the LIPUS-treated group. In conclusion, LIPUS promotes osteogenic differentiation of hPDLCs, which is associated with upregulation of Runx2 and integrin β1, which may thus provide therapeutic benefits in periodontal tissue regeneration. [ABSTRACT FROM AUTHOR]

Skeletal muscle satellite cell function is largely dictated by the surrounding environment following injury. Immune cell infiltration dominates the extracellular space in the injured area, resulting in increased cytokine concentrations. While increased pro-inflammatory cytokine expression has been previously established in the first 3 days following injury, less is known about the time course of cytokine expression and the specific mechanisms of cytokine induced myoblast function. Therefore, the expression of IL-1β and IL-6 at several time points following injury, and their effects on myoblast proliferation, were examined. In order to do this, skeletal muscle was injured using barium chloride in mice and tissue was collected 1, 5, 10, and 28 days following injury. Mechanisms of cytokine induced proliferation were determined in cell culture using both primary and C2C12 myoblasts. It was found that there is a ∼20-fold increase in IL-1β (p≤0.05) and IL-6 (p = 0.06) expression 5 days following injury. IL-1β increased proliferation of both primary and C2C12 cells ∼25%. IL-1β stimulation also resulted in increased NF-κB activity, likely contributing to the increased proliferation. These data demonstrate for the first time that IL-1β alone can increase the mitogenic activity of primary skeletal muscle satellite cells and offer insight into the mechanisms dictating satellite cell function following injury. [ABSTRACT FROM AUTHOR]

During systemic inflammation different neutrophil subsets are mobilized to the peripheral blood. These neutrophil subsets can be distinguished from normal circulating neutrophils (CD16bright/CD62Lbright), based on either an immature CD16dim/CD62Lbright or a CD16bright/CD62Ldim phenotype. Interestingly, the latter neutrophil subset is known to suppress lymphocyte proliferation ex vivo, but how neutrophils become suppressive is unknown. We performed transcriptome analysis on the different neutrophil subsets to identify changes in mRNA expression that are relevant for their functions. Neutrophil subsets were isolated by fluorescence-activated cell sorting from blood of healthy volunteers that were administered a single dose of lipopolysaccharide (2 ng/kg i.v.) and the transcriptome was determined by microarray analysis. Interestingly, the CD16bright/CD62Ldim suppressive neutrophils showed an interferon-induced transcriptome profile. More importantly, IFN-γ, but not IFN-α or IFN-β stimulated neutrophils, acquired the capacity to suppress lymphocyte proliferation through the expression of programmed death ligand 1 (PD-L1). These data demonstrate that IFN-γ-induced expression of PD-L1 on neutrophils enables suppression of lymphocyte proliferation. Specific stimulation of neutrophils present at the inflammatory sites might therefore have a pivotal role in regulating lymphocyte-mediated inflammation and autoimmune disease. [ABSTRACT FROM AUTHOR]

This study aimed to clarify whether Gram-positive (G+) and Gram-negative (G−) bacteria affect antigen-presenting cells differently and thereby influence the immunogenicity of proteins they express. Lactobacilli, lactococci and Escherichia coli strains were transformed with plasmids conferring intracellular ovalbumin (OVA) production. Murine splenic antigen presenting cells (APCs) were pulsed with washed and UV-inactivated OVA-producing bacteria, control bacteria, or soluble OVA. The ability of the APCs to activate OVA-specific DO11.10 CD4+ T cells was assessed by measurments of T cell proliferation and cytokine (IFN-γ, IL-13, IL-17, IL-10) production. OVA expressed within E. coli was strongly immunogenic, since 500 times higher concentrations of soluble OVA were needed to achieve a similar level of OVA-specific T cell proliferation. Furthermore, T cells responding to soluble OVA produced mainly IL-13, while T cells responding to E. coli-expressed OVA produced high levels of both IFN-γ and IL-13. Compared to E. coli, G+ lactobacilli and lactococci were poor inducers of OVA-specific T cell proliferation and cytokine production, despite efficient intracellular expression and production of OVA and despite being efficiently phagocytosed. These results demonstrate a pronounced difference in immunogenicity of intracellular antigens in G+ and G− bacteria and may be relevant for the use of bacterial carriers in vaccine development. [ABSTRACT FROM AUTHOR]

Interleukin-26 (IL-26) is one of the cytokines secreted by Th17 cells whose role in human tumors remains unknown. Here, we investigated the expression and potential role of IL-26 in human gastric cancer (GC). The expression of IL-26 and related molecules such as IL-20R1, STAT1 and STAT3 was examined by real-time PCR and immunohistochemisty. The effects of IL-26 on cell proliferation and cisplatin-induced apoptosis were analyzed by BrdU cooperation assay and PI-Annexin V co-staining, respectively. Lentiviral mediated siRNA was used to explore its mechanism of action, and IL-26 related signaling was analyzed by western blotting. Human GC tissues showed increased levels of IL-26 and its related molecules and activation of STAT3 signaling, whereas STAT1 activation did not differ significantly between GC and normal gastric tissues. Moreover, IL-26 was primarily produced by Th17 and NK cells. IL-26 promoted the proliferation and survival of MKN45 and SGC-7901 gastric cancer cells in a dose-dependent manner. Furthermore, IL-20R2 and IL-10R1, which are two essential receptors for IL-26 signaling, were expressed in both cell lines. IL-26 activated STAT1 and STAT3 signaling; however, the upregulation of the expression of Bcl-2, Bcl-xl and c-myc indicated that the effect of IL-26 is mediated by STAT3 activation. Knockdown of STAT1 and STAT3 expression suggested that the proliferative and anti-apoptotic effects of IL-26 are mediated by the modulation of STAT1/STAT3 activation. In summary, elevated levels of IL-26 in human GC promote proliferation and survival by modulating STAT1/STAT3 signaling. [ABSTRACT FROM AUTHOR]

Activin, a member of the transforming growth factor-β superfamily, promotes the growth of preantral follicles and the proliferation of granulosa cells. However, little is known about the role of microRNAs in activin-mediated granulosa cell proliferation. Here, we reported a dose- and time-dependent suppression of microRNA-181a (miR-181a) expression by activin A in mouse granulosa cells (mGC). Overexpression of miR-181a in mGC suppressed activin receptor IIA (acvr2a) expression by binding to its 3′-untranslated region (3′-UTR), resulting in down-regulation of cyclin D2 and proliferating cell nuclear antigen expression, leading to inhibition of the cellular proliferation, while overexpression of acvr2a attenuated the suppressive effect of miR-181a on mGC proliferation. Consistent with the inhibition of acvr2a expression, miR-181a prevented the phosphorylation of the activin intracellular signal transducer, mothers against decapentaplegic homolog 2 (Smad2), leading to the inactivation of activin signaling pathway. Interestingly, we found that miR-181a expression decreased in ovaries of mice at age of 8, 12, and 21 days, as compared with that in ovaries of 3-day old mice, and its level was reduced in preantral and antral follicles of mice compared with that in primary ones. Moreover, the level of miR-181a in the blood of patients with premature ovarian failure was significantly increased compared with that in normal females. This study identifies an interplay between miR-181a and acvr2a, and reveals an important role of miR-181a in regulating granulosa cell proliferation and ovarian follicle development. [ABSTRACT FROM AUTHOR]

Decoding the complexity of multicellular organisms requires analytical procedures to overcome the limitations of averaged measurements of cell populations, which obscure inherent cell-cell heterogeneity and restrict the ability to distinguish between the responses of individual cells within a sample. For example, defining the timing, magnitude and the coordination of cytokine responses in single cells is critical for understanding the development of effective immunity. While approaches to measure gene expression from single cells have been reported, the absolute performance of these techniques has been difficult to assess, which likely has limited their wider application. We describe a straightforward method for simultaneously measuring the expression of multiple genes in a multitude of single-cell samples using flow cytometry, parallel cDNA synthesis, and quantification by real-time PCR. We thoroughly assess the performance of the technique using mRNA and DNA standards and cell samples, and demonstrate a detection sensitivity of ∼30 mRNA molecules per cell, and a fractional error of 15%. Using this method, we expose unexpected heterogeneity in the expression of 5 immune-related genes in sets of single macrophages activated by different microbial stimuli. Further, our analyses reveal that the expression of one 'pro-inflammatory' cytokine is not predictive of the expression of another 'pro-inflammatory' cytokine within the same cell. These findings demonstrate that single-cell approaches are essential for studying coordinated gene expression in cell populations, and this generic and easy-to-use quantitative method is applicable in other areas in biology aimed at understanding the regulation of cellular responses. [ABSTRACT FROM AUTHOR]