Expression Of RANTES Research Articles

Spinal SET7/9 may contribute to the maintenance of cancer-induced bone pain in mice.

Cancer-induced bone pain (CIBP) profoundly influences patients' quality of life. Exploring the mechanisms by which CIBP occurs is essential for developing efficacious therapies. Various studies have shown that proinflammatory factors were involved in CIBP. SET domain containing lysine methyltransferase 7/9 (SET7/9) may modulate the expression of NF-κB-dependent proinflammatory genes in vitro. However, whether SET7/9 may participate in the maintenance of CIBP remains unknown. In this study, NCTC 2472 cells were inoculated into the intramedullary space of the femur to establish a mouse model of CIBP. Upregulation of spinal SET7/9 expression was related to pain behaviours in tumour-inoculated mice. Intrathecal cyproheptadine (10 or 20nmol) attenuated response to painful stimuli in a dose-dependent manner. Moreover, there was a concomitant decrease in spinal SET7/9 and RANTES expression. The antinociceptive effects of cyproheptadine were abolished by pre-intrathecal administration of SET 7/9 (0.2μg) for 30minutes before intrathecal cyproheptadine (20nmol) administration. These results indicated that spinal SET7/9 may contribute to the maintenance of CIBP in mice. Hence, targeting of spinal SET7/9 might be a useful alternative therapy for the treatment of CIBP.

MyD88 contribution to ocular surface homeostasis.

The cornea must maintain homeostasis, enabling rapid response to injury and microbial insult, to protect the eye from insult and infection. Toll-like receptors (TLRs) are critical to this innate immune response through the recognition and response to pathogens. Myeloid differentiation primary response (MyD88) is a key signaling molecule necessary for Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R)-mediated immune defense and has been shown to be necessary for corneal defense during infection. Here, we examined the intrinsic role of TLR signaling in ocular surface tissues by determining baseline levels of inflammatory mediators, the response to mechanical stimuli, and corneal infection in MyD88-deficient mice (MyD88-/-). In addition, cytokine, chemokine, and matrix metalloproteinase (MMP) expression was determined in ocular surface cells exposed to a panel of TLR agonists. Compared to wild-type (WT) animals, MyD88-/- mice expressed lower MMP-9 levels in the cornea and conjunctiva. Corneal IL-1α, TNFα, and conjunctival IL-1α, IL-2, IL-6, and IL-9 levels were also significantly reduced. Additionally, CXCL1 and RANTES expression was lower in both MyD88-/- tissues compared to WT and IL-1R-/- mice. Interestingly, MyD88-/- mice had lower corneal sensitivities (1.01±0.31 gm/mm2) than both WT (0.59±0.16 gm/mm2) and IL-1R-/- (0.52±0.08 gm/mm2). Following Pseudomonas aeruginosa challenge, MyD88-/- mice had better clinical scores (0.5±0.0) compared to IL-1R-/- (1.5±0.6) and WT (2.3±0.3) animals, but had significantly more corneal bacterial isolates. However, no signs of infection were detected in inoculated uninjured corneas from either MyD88 or IL-1R-deficient mice. This work furthers our understanding of the importance of TLR signaling in corneal defense and immune homeostasis, showing that a lack of MyD88 may compromise the baseline innate response to insult.

RANTES levels in peripheral blood, CSF and contused brain tissue as a marker for outcome in traumatic brain injury (TBI) patients

BackgroundTraumatic brain injury (TBI) causes activation of several neurochemical and physiological cascades, leading to neurological impairment. We aimed to investigate the level of novel chemokine RANTES in plasma, cerebrospinal fluid (CSF) and contused brain tissue in traumatic brain injury patients and to correlate the expression of this chemokine with the severity of head injury and neurological outcome.MethodsThis longitudinal case control study was performed on 70 TBI patients over a period of 30 months. Glasgow coma scale (GCS) and Glasgow outcome score were used to assess the severity of head injury and clinical outcome. Level of RANTES was quantified in plasma (n = 60), CSF (N = 10) and contused brain tissue (n = 5). Alterations in the plasma levels on 1st and 5th day following TBI were assessed. Patients were categorized as severe (GCS < 8) (SHI), moderate and mild Head injury (GCS > 8–14). 15 healthy volunteers were taken as the control group.ResultsThe median plasma RANTES levels were 971.3 (88.40–1931.1); 999.2 (31.2–2054.9); 471.8 (370.9–631.9) for SHI, MHI and healthy control respectively and showed statistically significant variation (p = 0.005). There was no statistical difference in the mean 1st and 5th day RANTES levels for the SHI group. However, admission RANTES levels were significantly higher in patients who died than those who survived (p = 0.04). Also, RANTES levels were significantly higher in plasma as compared to contused brain tissue and CSF (p = 0.0001).ConclusionThis is the first study of its kind which shows that there is significant correlation of admission RANTES levels and early mortality. Another interesting finding was the significant upregulated in the expression of RANTES in plasma, compared to CSF and contused brain tissue following severe TBI.

Differences in RANTES and IL-6 levels among chronic rhinosinusitis patients with predominant gram-negative and gram-positive infection

BackgroundBacteria are suspected players in the pathogenesis of chronic rhinosinusitis (CRS), yet their exact role is not understood. We investigated the effect of planktonic and biofilm of staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) on the mucosa of CRS patients with gram-positive and gram-negative infections by measuring the levels of IL-6 and RANTES, a chemokine with activity on eosinophils and T lymphocytes.MethodsEthmoid mucosa of six CRS patients with gram-positive bacteria on culture and five with gram-negative bacteria were compared to ethmoid mucosa of 8 control patients. The tissue explants were stimulated with SA and PA extracts in planktonic and biofilm form for 6 hours, then RANTES levels were measured by ELISA.ResultsCompared to the control group, CRS patients with gram-negative predominance demonstrated a significantly higher level of RANTES expression in response to all forms of bacterial stimuli (P-value <0.05). Patients with gram-positive predominance showed a higher level of RANTES compere to control group, however, this difference was not significant (P-value >0.05).ConclusionsThe mucosa of CRS patients with gram-negative infections has a heightened innate immune response compared to controls and patients with gram-positive infections. It is possible that this response leads to the pathological eosinophilic inflammation seen in CRS.

559P Differential association of TGF-β expression with MCP-1 and RANTES expression in primary tumor depending on stages of breast cancer patients from Eastern India

559P Differential association of TGF-β expression with MCP-1 and RANTES expression in primary tumor depending on stages of breast cancer patients from Eastern India

559P Differential association of TGF-β expression with MCP-1 and RANTES expression in primary tumor depending on stages of breast cancer patients from Eastern India

559P Differential association of TGF-β expression with MCP-1 and RANTES expression in primary tumor depending on stages of breast cancer patients from Eastern India

Non-canonical NFκB activation promotes chemokine expression in podocytes

TNF-like weak inducer of apoptosis (TWEAK) receptor Fn14 is expressed by podocytes and Fn14 deficiency protects from experimental proteinuric kidney disease. However, the downstream effectors of TWEAK/Fn14 in podocytes are poorly characterized. We have explored TWEAK activation of non-canonical NFκB signaling in cultured podocytes. In cultured podocytes, TWEAK increased the expression of the chemokines CCL21, CCL19 and RANTES in a time-dependent manner. The inhibitor of canonical NFκB activation parthenolide inhibited the CCL19 and the early RANTES responses, but not the CCL21 or late RANTES responses. In this regard, TWEAK induced non-canonical NFκB activation in podocytes, characterized by NFκB2/p100 processing to NFκB2/p52 and nuclear migration of RelB/p52. Silencing by a specific siRNA of NIK, the upstream kinase of the non-canonical NFκB pathway, prevented CCL21 upregulation but did not modulate CCL19 or RANTES expression in response to TWEAK, thus establishing CCL21 as a non-canonical NFκB target in podocytes. Increased kidney Fn14 and CCL21 expression was also observed in rat proteinuric kidney disease induced by puromycin, and was localized to podocytes. In conclusion, TWEAK activates the non-canonical NFκB pathway in podocytes, leading to upregulation of CCL21 expression. The non-canonical NFκB pathway should be explored as a potential therapeutic target in proteinuric kidney disease.

K45A mutation of RIPK1 results in poor necroptosis and cytokine signaling in macrophages, which impacts inflammatory responses in vivo.

Receptor interacting protein kinase 1 (RIPK1) participates in several cell signaling complexes that promote cell activation and cell death. Stimulation of RIPK1 in the absence of caspase signaling induces regulated necrosis (necroptosis), which promotes an inflammatory response. Understanding of the mechanisms through which RIPK1 promotes inflammation has been unclear. Herein we have evaluated the impact of a K45A mutation of RIPK1 on necroptosis of macrophages and the activation of inflammatory response. We show that K45A mutation of RIPK1 results in attenuated necroptosis of macrophages in response to stimulation with LPS, TNFα and IFNβ in the absence of caspase signaling. Impairment in necroptosis correlated with poor phosphorylation of RIPK1, RIPK3 and reduced trimerization of MLKL. Furthermore, K45A mutation of RIPK1 resulted in poor STAT1 phosphorylation (at S727) and expression of RANTES and MIP-1α following TNF-R engagement in the absence of caspase activation. Our results further indicate that in the absence of stimulation by pathogen-associated molecular patterns (PAMPs), cellular inhibitors of apoptotic proteins (cIAPs) prevent the K45-dependent auto-phosphorylation of RIPK1, leading to resistance against necroptosis. Finally, RIPK1(K45A) mice displayed attenuated inflammatory response in vivo as they were significantly resistant against endotoxin shock, but highly susceptible against a challenge with Salmonella typhimurium. This correlated with reduced expression of IL-1β and ROS, and poor processing of caspase 8 by RIPK1(K45A) macrophages. Overall, these results indicate that K45 mediated kinase activity of RIPK1 is not only important for necroptosis but it also has a key role in promoting cytokine signaling and host response to inflammatory stimuli.

Determination on the Expression of Activated RANTES G 28 in HIV-1 Infection and in Co Infection with Hepatitis B Virus and Hepatitis C Virus and Its Effect on Leukocytes

HumanImmunodeficiencyVirus�(HIV),�coinfectedwithhepatitisBvirusandhepa- titisCviruschallengestheimmunesystem.�RegulatedonactivationnormalTcellexpressed� andsecreted�(RANTES)�isoneofthenaturalligandsforthechemokine,�usesreceptorwhich� potentlysuppressesviralinfection.�WeexaminedpossibleactivationofRANTESG�28�inHIV- 1�infectionandincoinfectionwithHBVandHCVanditseffectonleukocytes.�Leukocytes� fromHIV-1infectedHIV-1/HBVandHCV/HIVco-infectedindividualswereassessedbyflow� cytometryandAbacus�5�automated.�RANTESG�28�wasdeterminedbyPolymeraseChainRe- action.�RANTESG�28�demonstratedpositivecorrelationwithHIV-1,�HIV/HBV,�HIV-1/HCV� coinfectionandleukocytes.�Theassociationpresenteddifferenceincoefficientandpredication� ofitsoccurrence.�HighexpressionofRANTESG�28�wasindicatedwithstatedtheleukocytes.� Differenceinsignificancewasindicated.�Thereisneedofmoreresearchtostudyandunder- standthefunctionsofRANTESG�28�withreferencetohumansystemanditsroleinimmunity.

The Role of Microenvironment Expression of Pd-1 Ligands and Chemokines in Clinical Outcome of Hodgkin'S Lymphoma

ABSTRACT Aim: Classical Hodgkin's lymphoma (cHL) is characterized by only few malignant cells and an abundance of inflammatory cells. cHL infiltrating cells produce cytokines and growth factors that provide essential stimulatory signals for survival and proliferation of Hodgkin and Reed–Sternberg (HRS) cells. Moreover, clinical behaviour of cHL may be directly regulated by the cross-talk between HRS cells and other cells in their microenvironment. The aim of our study was to estimate the role of microenvironment expression of PD-1 ligands and chemokines in clinical outcome of cHL. Methods: The case group comprised 49 patients with cHL (stage IIA: 23, IIB: 3, III-IV: 23). The patients received chemotherapy regimens (ABVD or BEACOPP 14/esc) and radiotherapy by indications. cHL specimens were obtained from lymph node biopsies of patients at diagnosis. PD-L1, PD-L2, MIP-1a, RANTES mRNA expression levels were analyzed in fresh tissue specimens using real-time RT-PCR. Results: We observed that 26.5 % (13/49) of cHL cases were PD-L1 negative, 16.3 % (8/49) - RANTES negative and 14.3 % (7/49) – both PD-L1 and RANTES negative. MIP-1a and RANTES expression levels were slightly higher in mixed cellularity cHL (p = 0.15), whereas PD-L1 – in nodular sclerosis cHL and advanced stages of disease (p = 0.19). PD-L2 expression level was independent of histological cHL variant or disease stage. All cases with the absence of both PD-L1 and RANTES markers had a complete response to the therapy and long-term remission. For the patients with increasing of RANTES/MIP-1a rate, a trend for a higher risk of relapse was observed (P = 0.09). ROC analysis revealed that PD-L1 expression level in tumor is an important marker which is associated with clinical outcome of cHL patients (Se = 87.5 %; Sp = 64.5 %; AUC = 0.75; p = 0.002). High PD-L1 expression was associated with reduced progression-free survival (PFS) in cHL patients. The 2-year PFS rate for cHL patients with high PD-L1 expression was 47 % compared to 95 % for low or absent of PD-L1 expression (p = 0.04). Conclusions: Our results suggest that tumor microenvironment play an important role in clinical behavior of cHL. PD-L1 expression level can be used as a marker of prognosis in patients with cHL and represent an attractive target for a cHL immunotherapy in patients with poor outcome. Disclosure: All authors have declared no conflicts of interest.

La atorvastatina inhibe la progresión de la lesión aterosclerótica inducida por el factor inductor débil de apoptosis similar al factor de necrosis tumoral en ratones deficientes en apolipoproteína E

AimInteraction of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) with its receptor Fn14 accelerates atherosclerotic plaque development in ApoE deficient mice (ApoE KO). In this work, an analysis has been made on the effect of an HMG-CoA reductase inhibitor, atorvastatin, on atherosclerotic plaque development accelerated by TWEAK in ApoE KO mice. Materials and methodsEight week-old ApoE KO mice were fed with a high cholesterol diet for 4 weeks. The animals were then randomized into 3 groups: mice injected i.p. with saline, recombinant TWEAK (10μg/kg/twice a week), or recombinant TWEAK plus atorvastatin (1mg/kg/day) for 4 weeks. The lesion size, cellular composition, lipid and collagen content were analyzed, as well as inflammatory response in atherosclerotic plaques present in aortic root of mice. ResultsTWEAK treated mice showed an increase in atherosclerotic plaque size, as well as in collagen/lipid ratio compared with control mice. In addition, macrophage content, MCP-1 and RANTES expression, and NF-κB activation were augmented in atherosclerotic plaques present in aortic root of TWEAK treated mice compared with control mice. Treatment with atorvastatin prevented all these changes induced by TWEAK in atherosclerotic lesions. Atorvastatin treatment also decreased Fn14 expression in the atherosclerotic plaques of ApoE KO mice. ConclusionsAtorvastatin prevents the pro-atherogenic effects induced by TWEAK in ApoE KO mice, which could be related to the inhibition of Fn14 expression. The results of this study provide new information on the beneficial effects of statin treatment in cardiovascular diseases.

Immunosuppressive Mechanisms of Human Mesenchymal Stem Cells in Graft Vesus Host Disease Murine Models.

Background: Mesenchymal stem cells (MSCs) are proven to have immunosuppressive functions in vitro and in vivo via various mechanisms. We aim to demonstrate these mechanisms by treating GVHD murine models with human MSCs. Methods: Balb/c host mice were subjected to irradiation and received C57BL/6N donor T cell depleted bone marrow cells only (negative control) or with donor spleen CD4+ T lymphocyte (positive control). 12 mice were given 1 x 10ˆ6 human MSCs via tail vein (treatment group). 2 host mice received Dil-stained MSCs. Survival rate was monitored. At day 6 post HSCT, single cell suspensions, tissue lysates and paraffin sections were prepared from target tissues of 4-6 host mice for (1) flow cytometry to check for the presence of donor T lymphocytes in various tissues, (2) ELISA to check for levels of proinflammatory cytokines, chemokine ligands and T cell cycle regulatory pathway testing, (3) western blotting for expression of inducible nitric oxide synthase (iNOS) and (4) imaging by coherent anti-stokes raman spectroscopy (CARS). Tissues from 2 host mice who received Dil-stained MSCs were prepared for in vivo imaging studies at day 6 post HSCT. 2 mice sacrificed for histopathologic observation are given 3 doses of MSC and sacrificed at day 50 post HSCT. Results and conclusion: The immunosuppressive functions of MSCs were documented in our GVHD models by (1) Protection of GVHD mice from death probably due to decrease donor T lymphocytes in target tissues and levels of TNF-a, IFN-g and RANTES expression, (2) Increased levels of iNOS, an immunosuppressive soluble mediator, (3) Suppression of phosphorylation of STAT 5A/B proteins, indicative of T cell cycle arrest, (4) Less severe GVHD pathologic changes in the lungs, liver and colon; and (5) Documentation of the presence of MSCs in target tissues by observing increased fluorescence signals via in vivo imaging studies and differences in chemical bond vibratory signals using CARS.

Productive replication of Middle East respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response

The Middle East respiratory syndrome coronavirus (MERS-CoV) closely resembled severe acute respiratory syndrome coronavirus (SARS-CoV) in disease manifestation as rapidly progressive acute pneumonia with multi-organ dysfunction. Using monocyte-derived-dendritic cells (Mo-DCs), we discovered fundamental discrepancies in the outcome of MERS‐CoV‐ and SARS-CoV-infection. First, MERS-CoV productively infected Mo-DCs while SARS-CoV-infection was abortive. Second, MERS-CoV induced significantly higher levels of IFN-γ, IP-10, IL-12, and RANTES expression than SARS-CoV. Third, MERS-CoV-infection induced higher surface expression of MHC class II (HLA-DR) and the co-stimulatory molecule CD86 than SARS-CoV-infection. Overall, our data suggests that the dendritic cell can serve as an important target of viral replication and a vehicle for dissemination. MERS-CoV-infection in DCs results in the production of a rich combination of cytokines and chemokines, and modulates innate immune response differently from that of SARS-CoV-infection. Our findings may help to explain the apparent discrepancy in the pathogenicity between MERS-CoV and SARS-CoV.

1,25-(OH)2D3 inhibits the expression of RANTES in asthmatic rat airway smooth muscle cells

1,25-(OH)2D3 inhibits the expression of RANTES in asthmatic rat airway smooth muscle cells

Abstract 307: Deficiency of Lim-only Protein Fhl2 Accelerates Neointima Formation After Carotid Artery Ligation

Rationale The LIM-only protein FHL2, also known as DRAL or SLIM3, has a regulatory function in many physiological processes and is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells, but not in macrophages. FHL2 regulates SMC phenotype, but its function in vascular injury is unknown. Objective To assess the role of FHL2 in SMC-rich lesion formation after vascular injury and to elucidate the underlying mechanism. Methods &amp; Results Cultured aortic SMCs from FHL2-KO mice showed increased migration and increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. In agreement with this, overexpression of FHL2 in SMCs reduced CyclinD1 expression. In addition, FHL2-KO SMCs showed enhanced expression of pro-inflammatory cytokines in a NFkB dependent manner. Consistent with these findings, NFkB activity is higher in FHL2-KO SMCs. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice developed accelerated lesion formation compared with wild-type (WT)-mice. Furthermore, FHL2-KO mice displayed high number of macrophages in lesions and enhanced expression of RANTES and stromal derived factor-1α (SDF-1α) in ligated carotid arteries. SDF-1α expression was also increased in plasma of FHL2-KO mice. Finally, FHL2-KO mice showed enhanced Ki67 expression in lesions compared with wild-type (WT)-mice. Conclusion FHL2 deficiency in mice results in an exacerbated neointima formation by enhanced proliferation and migration of SMCs, possibly regulated via ERK1/2 and NFkB pathway.

Abstract 379: Evaluating tissue expression of RANTES with subtype specific breast cancer biomarkers found elevated in plasma.

Abstract Background: A previous analysis of 23 plasma proteins found that levels increased in cancer patients compared to benign-breast-disease controls when the cancer was analyzed based on tumor subtypes. Circulating levels of RANTES were significantly increased in cancer patients and were commonly associated with two or more of the following proteins; amphiregulin, EGF, HB-EGF, TGFa, PDGF, and VEGF in a tumor subtype specific pattern. However, it is unknown if expression of these proteins correlate in the tumor tissue of breast cancer subtypes thus contributing to the elevated expression seen in plasma. In this study, we evaluate the expression of 5 of plasma biomarkers in a 70-subject breast cancer tissue microarray (TMA) to determine if these proteins are increased differentially by tumor subtypes. Design: A BioChain TMA containing 70 duplicated cores covering the common types of breast cancer, 5 normal, and 5 benign cases are assayed using GBI Labs triple stain kits to evaluate subtype specific expression of amphiregulin and HB-EGF or PDGF and VEGF with RANTES. Imaging and quantitative assessment of the distribution and intensity of each stain was evaluated using a multispectral imaging (MSI) approach, automated morphologic analysis software and compared to a visual screen. Results: Results indicate that an algorithm can be develoed to accurately recognize tumor versus stromal tissue within each core. Furthermore, as compared to visual evaluation, the MSI approach has the added capability to separate the color of the three immunostains from the triple stain for precise quantification of each protein without crosstalk. The automated scoring algorithm compared well against visual assessment. Conclusion: The knowledge gained from correlating these subtype-specific patterns of tissue antigens with circulating levels in plasma may provide important information about the contribution of specific markers of breast cancers. Application of MSI to the triple staining in the TMA screen provided a superior quantitative method for evaluation of multiple chromogens linked proteins within a tumor core compared to visual observations/grading of the complex multicolor immunostaining in this study. Citation Format: James Mansfield, Chuanbo Xu, Rachel Gonzalez. Evaluating tissue expression of RANTES with subtype specific breast cancer biomarkers found elevated in plasma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 379. doi:10.1158/1538-7445.AM2013-379

The expression and functionality of stromal caveolin 1 in human adenomyosis

What is the expression pattern and functionality of caveolin 1 (CAV1) in the endometrium of patients with adenomyosis? The stromal CAV1 expression is down-regulated that leads to the release of a variety of molecules that either enhance the metastatic capacity of endometrial cells or contribute to adenomyosis-associated dysmenorrhea. Adenomyosis is characterized by invasion of endometrium into the uterine myometrium. CAV1 has been linked to tumor progression and clinical outcome in a variety of human malignancies; however, its role in adenomyosis development and adenomyosis-associated dysmenorrhea is still poorly recognized. We retrospectively analyzed the expression levels of CAV1 and RANTES protein using immunohistochemistry in 65 patients who were pathologically diagnosed with adenomyosis and 12 control women without related pathology, who were subjected to surgery between 2009 and 2010. Endometrial tissues from six additional normal females without related pathology were collected from 2011 to 2012; these tissues were subjected to subsequent primary cell culture experiments. The expression of CAV1 and RANTES was examined by immunohistochemistry in ectopic endometrium and paired eutopic endometrium of 65 adenomyosis patients and 12 control patients. Primary endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs) were isolated from 6 additional control females without related pathology. The expression of CAV1 in ESCs was either (i) inhibited by siRNA transfection and methyl-β-cyclodextrin (MβCD) treatment or (ii) increased by pcDNA3.1/CAV1 transfection. The impact of each treatment on the proliferation, migration and invasion of both ESCs and EECs was evaluated by methylthiazolydiphenyl-tetrazolium assay, colony formation assay, Transwell migration and invasion assay. Furthermore, ESC treatment with MβCD and siCAV1 was assessed for the effect on the expression of a panel of inflammatory cytokines. The levels of two pain mediators, nitric oxide (NO) and prostaglandin E2 (PGE2), were assessed in CAV-1-depleted and control ESCs, whereas immunoblotting was performed to characterize signaling pathways downstream to loss of stromal CAV1 in endometrium. The correlation between dysmenorrhea severity and stromal CAV1 and RANTES expression was further examined using 'Pearson's' correlation analysis. Stromal CAV1 expression in ectopic endometrium of adenomyosis patients was significantly lower than that of paired eutopic endometrium or normal controls as analyzed by immunohistochemistry (P < 0.001). Although no significant difference was observed in the proliferation of CAV1-depleted ESCs when compared with control group, EECs cultured with conditioned media from CAV1-depleted ESCs demonstrated a significantly elevated proliferation rate when compared with those treated with control ESC-conditioned media. Moreover, both CAV1-depleted ESCs and EECs cultured with conditioned media from CAV1-depleted ESCs showed enhanced migration and invasion capacity when compared with control group (P < 0.05). In contrast, incubation with conditioned media of ESCs with enforced CAV1 expression led to decreased proliferation capacity of EECs. Furthermore, the expression of RANTES in ESCs treated with MβCD and siCAV1 was significantly increased. Stromal RANTES expression in the ectopic endometrium of adenomyosis patients was significantly higher than that of paired eutopic endometrium or normal controls as analyzed by immunohistochemistry (P = 0.0026). Stromal CAV1 expression in eutopic endometrium was significantly lower in women with more severe dysmenorrhea (P < 0.05) and was negatively correlated with dysmenorrhea severity in adenomyosis patients (r(2) = 0.1549; P = 0.012, 'Pearson's' χ(2) test), whereas stromal RANTES expression in eutopic endometrium was significantly higher in women with more severe dysmenorrhea (P < 0.05) and was positively correlated with dysmenorrhea severity in adenomyosis patients (r(2) = 0.1646; P = 0.0094, 'Pearson's' χ(2) test). Silencing of CAV1 in ESCs led to increased release of NO and PGE2 when compared with control and was associated with enhanced activity of ERK-FAK signaling pathway. This study assessed the functional role of stromal CAV1 and RANTES in a small number of human adenomyosis samples by immunohistochemistry and in primary human ESCs by functional studies. In future investigations, a larger sample size should be adopted and the functional role of stromal CAV1 should be further characterized in animal models. Loss of stromal CAV1 expression may play a critical role in the pathogenesis of adenomyosis and is correlated with adenomyosis-related dysmenorrhea. National Basic Research Program of China and Ph.D. Programs Foundation of Ministry of Education of China. None.

Role of Leptin Signaling in the Pathogenesis of Angiotensin II–Mediated Atrial Fibrosis and Fibrillation

We examined the hypothesis that leptin signaling contributes to the atrial fibrosis and atrial fibrillation (AF) evoked by angiotensin II (AngII). Eight-week-old male CL57/B6 (CNT) and leptin-deficient ob/ob mice (Ob) were subcutaneously infused with AngII (2.0 mg/kg per day). Two weeks later, transesophageal burst pacing and an electrophysiological study using isolated perfused hearts were performed. Left-atrial tissues were collected to determine interstitial fibrosis by Masson trichrome staining, and the expressions of mRNAs related to inflammatory profibrotic signals were assessed. Left-atrial fibroblasts were isolated from adult Sprague-Dawley and Zucker rats. The effects of leptin (100 ng/mL) or AngII (100 nmol/L) treatment were evaluated. In CNT-AngII mice, leptin expression in the left atrium was upregulated (P<0.01). Transesophageal burst pacing induced atrial fibrillation in 88% (7/8) of CNT-AngII mice, but not in Ob-AngII mice (0/8; P<0.01). In isolated perfused hearts, atrial fibrillation was induced only in CNT-AngII mice (4/6; 67%). Interatrial conduction time was prolonged in CNT-AngII mice (P<0.01), but not in Ob-AngII mice. The upregulation of collagen 1, collagen 3, transforming growth factor-β1, α-smooth muscle actin, MCP-1, F4/80, and RANTES mRNA, which was seen in CNT-AngII mice, was attenuated in Ob-AngII mice. In cultured Sprague-Dawley rat atrial fibroblasts, AngII treatment increased leptin expression (P<0.01). Addition of leptin increased transforming growth factor-β1, α-smooth muscle actin, MCP-1, and RANTES expressions in Sprague-Dawley rat atrial fibroblasts, but not in Zucker rat atrial fibroblasts. Our results demonstrate for the first time that leptin signaling is essential for the development of atrial fibrosis and atrial fibrillation evoked by AngII.

Functional Screen for Secreted Proteins by Monoclonal Antibody Library and Identification of Mac-2 Binding Protein (Mac-2BP) as a Potential Therapeutic Target and Biomarker for Lung Cancer

Identification of secreted proteins of lung cancer could provide new candidates of serum biomarkers for cancer diagnosis or targets for therapeutic intervention. In this study, we developed a novel strategy that combined functional monoclonal antibody library screening technique and mass spectrometry to identify functional secreted proteins. BALB/c mice were immunized with cancer cells isolated from fresh human lung cancer tissues. The monoclonal antibody library containing 1160 mAbs was established with the mouse spleen cells, whose serum had most anti-proliferative effect on lung cancer cells. Monoclonal antibodies were subjected to an immunoreactive and functional screen and monoclonal antibodies that reacted strongly with secreted proteins in condition medium and lung cancer tissues with high inhibotion of cell proliferation were selected. Antigens that recognized by antibodies were obtained by immunoprecipitation and then identified by mass spectrometry. Mac-2-binding protein (Mac-2BP), the antigen of 13H3 antibody, was identified using this approach. Functional studies demonstrated that the 13H3 antibody suppressed lung cancer cell lines ANIP-973 and A549 proliferation in vitro and inhibit ANIP973 xenograft tumors growth in vivo by inducing cell-cycle arrest at G1 phase, with up-regulation of p27 and down-regulation of cyclin D1. Moreover, the serum level of Mac-2BP was significantly higher in lung cancer patients than healthy controls. At a cutoff value of 6 μg/ml, Mac-2BP might be a diagnostic biomarker of lung cancer, especially for SCLC. Mac-2BP concentrations of 6 μg/ml or higher was associated with poor overall survival in univariate analysis, and was an independent predictor in the multivariate COX analysis. Together, these results firstly demonstrated that Mac-2BP can be used as a therapeutic target and potential biomarker for lung cancer. Our strategy is feasible, which may facilitate the identification of novel secreted biomarkers of lung cancer.

STAT3-RANTES Autocrine Signaling Is Essential for Tamoxifen Resistance in Human Breast Cancer Cells

The acquisition of tamoxifen resistance is a major therapeutic problem in breast cancer. We developed a tamoxifen-resistant MCF-7 (TRM-7) cell line to elucidate the molecular mechanisms and factors associated with acquisition of such resistance. We showed that phosphorylation of STAT3 at tyrosine 705 (Y705) and RANTES expression are increased in response to tamoxifen in human breast cancer cells. On the basis of these results, we hypothesize that upregulated STAT3 phosphorylation and RANTES may be correlated with the development of drug resistance. Here, we showed that STAT3 and RANTES contribute to the maintenance of drug resistance. STAT3 phosphorylation is constitutively retained via a RANTES autocrine loop, which in turn upregulates anti-apoptotic signals in TRM-7 cells. STAT3-RANTES autocrine signaling affected expression of anti-apoptotic BCL-2 family genes and prevented TRM-7 cells from undergoing programmed cell death by inhibiting PARP and caspase-9 cleavage. Subsequently, blockade of STAT3 and RANTES in TRM-7 cells resulted in reduction of anti-apoptotic signals, which was rescued by exogenous RANTES treatment; drug resistance was also restored. Taken together, our results suggested that STAT3-RANTES autocrine signaling is essential for maintenance of drug resistance and inhibition of programmed cell death. These mechanisms of STAT3-RANTES autocrine signaling suggest a novel strategy for management of patients with tamoxifen-resistant tumors.