Presence Of M2 Research Articles

Aqueous extract of pulp of maqui-berry (Aristotelia chilensis) induces apoptosis in human endometrial carcinoma ishikawa cells via mitochondrial and endoplasmic reticulum stress pathways

In Chilean traditional medicine, Aristotelia chilensis (Mol) Stuntz is commonly utilized for a variety of conditions (particularly digestive problems). We investigated the effects of an aqueous extract from the fruit pulp of A. chilensis (Maqui berry: M2) on endoplasmic reticulum stress and mitochondrial pathway-induced early apoptosis in Ishikawa cells. Using the MTT assay, the aqueous extract was found to have an inhibitory impact on human endometrial cancer Ishikawa cells, with an IC50 of 5.36 μg/mL. The changes in Ishikawa cells under the effect of M2 were detected by flow cytometry with cycle arrest in S phase, reduced mitochondrial membrane potential, ROS and calcium ion aggregation. Western blotting and RT-qPCR were used to identify the intracellular mRNA and protein alterations. The presence of M2 resulted in the up-regulation of mitochondrial pathway-related proteins Bad, Bax, and Cyto C, down-regulation of Bcl-2, and up-regulation of endoplasmic reticulum stress-related proteins IRE1α, p-eif2α, ATF4, and CHOP. The findings imply that A. chilensis pulp may be used as a possible raw material for the innovative functional applications.

EFFECT OF M2 MACROPHAGE-DERIVED SOLUBLE FACTORS ON DIFFERENTIATION OF SH-SY5Y CELLS

Macrophages play a key role in triggering and regulation of neuroregeneration. The characteristic feature of macrophages is pronounced plasticity, which manifests itself in the ability of macrophages to change their functional phenotype depending on the micromilieu. Apoptotic cell clearance (efferocytosis) is an important inducer of a macrophage polarization to M2 phenotype under pathological settings. Previously, we have developed an original protocol for the generation of M2-like macrophages, polarized by efferocytosis under serum-deprived conditions (M2 (LS), Low Serum). The present study was aimed to assess a neuroregenerative potential of M2 (LS) macrophages. We studied their effect on the differentiation of SH-SY5Y cells in comparison with retinoic acid (RA). As the morphological criteria of differentiation we have assessed the relative content of differentiated cells, i.e., cells with a neurite length exceeding the cell body length, and the average neurite length on days 3, 7, and 13. The ratio of neuron-like (N-type) and epithelial-like (S-type) cells in cultures was also assessed. SH-SY5Y cells were characterized by a low level of spontaneous differentiation, both under standard conditions (10% FBS) and serum deprivation (1% FBS). Upon RA treatment, SH-SY5Y cells stopped proliferating and underwent neuronal differentiation. Cultivation of SH-SY5Y cells in the presence of M2 (LS) conditioned medium also led to a significant increase in the relative content of differentiated cells, the average length of neurite-like processes, as well as a change in the balance of S- and N-type cells towards a pronounced predominance of the latter. The morphological features of differentiation were significantly less pronounced at early stage (day 3) of differentiation as compared with the RA-induced changes and reached the level of positive control only at later stages (day 13) (p < 0.05). In contrast to retinoic acid, M2 (LS) conditioned medium induced neuronal differentiation of SH-SY5Y cells without suppressing their proliferative activity. The data obtained may indicate a high neuroregenerative potential of M2 macrophages in vitro, which is realized through soluble factors and manifests itself in promoting SH-SY5Y differentiation.

Tumor-Released Products Promote Bone Marrow-Derived Macrophage Survival and Proliferation

Macrophages play a central role within the tumor microenvironment, with relevant implications for tumor progression. The modulation of their phenotype is one of the mechanisms used by tumors to escape from effective immune responses. This study was designed to analyze the influence of soluble products released by tumors, here represented by the tumor-conditioned media of two tumor cell lines (3LL from Lewis lung carcinoma and MN/MCA from fibrosarcoma), on murine macrophage differentiation and polarization in vitro. Data revealed that tumor-conditioned media stimulated macrophage differentiation but influenced the expression levels of macrophage polarization markers, cytokine production, and microRNAs of relevance for macrophage biology. Interestingly, tumor-derived soluble products supported the survival and proliferation rate of bone marrow precursor cells, an effect observed even with mature macrophages in the presence of M2 but not M1 inducers. Despite presenting low concentrations of macrophage colony-stimulating factor (M-CSF), tumor-conditioned media alone also supported the proliferation of cells to a similar extent as exogenous M-CSF. This effect was only evident in cells positive for the expression of the M-CSF receptor (CD115) and occurred preferentially within the CD16+ subset. Blocking CD115 partially reversed the effect on proliferation. These results suggest that tumors release soluble products that not only promote macrophage development from bone marrow precursors but also stimulate the proliferation of cells with specific phenotypes that could support protumoral functions.

Necrosis Factor-α (TNF-α) and the Presence of Macrophage M2 and T Regulatory Cells in Nasopharyngeal Carcinoma.

To investigate the correlation between TLR3 and pro-inflammatory cytokines (TNFα, IL6) expression with the distribution of macrophage M2 and Treg on Epstein Barr virus-encoded RNAs (EBER+) nasopharyngeal carcinoma (NPC) tissues. A total of 23 FFPE NPC tissue samples were obtained from patients in Dr. Sardjito General Hospital, Yogyakarta, Indonesia in 2008-2010, which expressed EBER was collected. The expressions of TLR3, TNFα, and IL6 were examined using immunofluorescence assay. The distribution of macrophage M2 and Treg were examined by immunohistochemistry with anti-CD163 and -FOXP3 antibodies, respectively. The quantification of fluorescence intensity was analyzed by the RGB space method using ImageJ software. The M2 interpretation was done by the eyeballing method and the M2 scores were divided into 0 (negative), 1 (scant), 2 (focal), 3 (abundant). The average number of Treg FOXP3+ cells in five high power fields was counted. The relationship between variables were tested by the Spearman correlation test, and the coefficient correlation was used to see the correlation between variables. All EBER+ NPC specimens showed TLR3 expression intracellularly. The expression of TNFα could be observed in the cell membranes and secreted extracellularly, while IL6 was secreted to the extracellular area. The expression of TNFα was two times higher than IL6. Most specimens showed low M2 score (56.52%) and high Treg (52.17%). A positive correlation was found between TLR3 and IL6 (12.9%). TNFα was positively correlated with the M2 distribution of 13.7% and Treg distribution of 12.9%, while the rest were explained by other factors. TNFα has a positive correlation with M2 and Treg distribution,but mostly through a different mechanism other than EBER-TLR3 interaction. Possibly, other pro-inflammatory and anti-inflammatory cytokines are involved in the formation of the NPC microenvironment, especially related to the presence of M2 and Treg, which provide immunosuppressive effects in NPC tumors.<br />.

Mathematical investigation of innate immune responses to lung cancer: The role of macrophages with mixed phenotypes

Macrophages’ role in the evolution of solid tumours is a well accepted fact, with the M1-like macrophages having an anti-tumour role and the M2-like macrophages having a pro-tumour role. Despite the fact that some clinical studies on lung tumours have emphasised also the presence of macrophages with mixed M1 and M2 phenotypes in addition to macrophages with distinct phenotypes, the majority of studies still use the distinct M1-M2 classification to predict the evolution of tumours and patient survival. In this theoretical study we use a mathematical modelling and computational approach to investigate the role of macrophages with mixed phenotype on growth/control/elimination of lung tumours. We show that tumour control in the presence of M2→M1 re-polarising treatments is mainly the result of macrophages with mixed phenotypes (due to the assumption of short half-life of M1-like macrophages). We also show that the half-life of various macrophage phenotypes (distinct M1 or mixed M1/M2 phenotypes) impacts the outcome of various therapeutic strategies targeting tumour-associated macrophages. All these results suggest the need for a better experimental understanding of the kinetics of macrophages inside solid tumours.

Influence of polysiloxane microspheres on hydrophobicity, structure and mechanical properties of paper materials

The aim of the study was to investigate the impact of three types of polysiloxane microspheres on the barrier properties, structure and mechanical properties of paper. An influence of new silicon filler on properties of cellulose paper sheet was analyzed. Polysiloxane microspheres were used as an additive introduced into the network of cellulosic fibers in order to obtain new functional properties of the paper. The following types of microspheres were used in the research: M1 hydrophilic of average diameter 23.5 µm, M2 hydrophobic of average diameter 3.1 µm and M3 hydrophobic of average diameter 23.5 µm. The obtained handsheets were analyzed for changes in apparent density, roughness, tensile strength, bursting strength, and tear resistance. Wettability and resistance to liquid were characterized by contact angle measurement, penetration dynamics analysis and uniformity of liquid penetration measured using an extended liquid penetration analyser. It was found that the presence of M2 (small diameter) microspheres improved significantly the paper’s hydrophobicity without changing the mechanical properties. The addition of M1 and M3 (large diameter) microspheres decreased the mechanical properties of the paper samples and did not improve their hydrophobicity. However, M1 microspheres resulted in increased uniformity of liquid penetration through the paper structure. The presented studies also show that it is possible to obtain paper with high hydrophobic properties only through the filling application when polysiloxane microspheres are used for this purpose. The results also indicate that it is not necessary to hydrophobize the entire material structure in order to achieve its high hydrophobicity.Graphic abstract

MP01-16 IL-10 AND CXCL10 URINE QUANTIFICATION AS USEFUL BIOMARKERS TO PREDICT BCG RESPONSE IN BLADDER CANCER PATIENTS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (MP01)1 Apr 2020MP01-16 IL-10 AND CXCL10 URINE QUANTIFICATION AS USEFUL BIOMARKERS TO PREDICT BCG RESPONSE IN BLADDER CANCER PATIENTS Felix Guerrero-Ramos*, Pablo Abad-Lopez, Cristian Suarez-Cabrera, Marta Rodriguez-Izquierdo, Carmen Gomez-Cañizo, Federico de la Rosa-Kehrmann, Victor Garcia-Martinez, Alejandra Bernardini, Iris Lodewijk, Guillermo Paramio, Alfredo Rodriguez-Antolin, Jesus M Paramio, and Marta Dueñas Felix Guerrero-Ramos*Felix Guerrero-Ramos* More articles by this author , Pablo Abad-LopezPablo Abad-Lopez More articles by this author , Cristian Suarez-CabreraCristian Suarez-Cabrera More articles by this author , Marta Rodriguez-IzquierdoMarta Rodriguez-Izquierdo More articles by this author , Carmen Gomez-CañizoCarmen Gomez-Cañizo More articles by this author , Federico de la Rosa-KehrmannFederico de la Rosa-Kehrmann More articles by this author , Victor Garcia-MartinezVictor Garcia-Martinez More articles by this author , Alejandra BernardiniAlejandra Bernardini More articles by this author , Iris LodewijkIris Lodewijk More articles by this author , Guillermo ParamioGuillermo Paramio More articles by this author , Alfredo Rodriguez-AntolinAlfredo Rodriguez-Antolin More articles by this author , Jesus M ParamioJesus M Paramio More articles by this author , and Marta DueñasMarta Dueñas More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000815.016AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Identifying biomarkers to predict BCG response before beginning the treatment is an unsolved need in the clinical practice for patients with high risk non-muscle invasive bladder cancer. Nowadays, there is no method to predict BCG response in our patients. In this context, protumor activity of M2 macrophages could be mediating this treatment response, so evaluation of cytokines as IL-10 and CXCL10 in urine could be an indicator of their activity on patient’s urothelium. The main aim of this work is to evaluate if expression levels of IL-10 and CXCL10 in urine samples obtained before treatment are able to identify BGC responders and non-responders. We also aimed to find out the correlation of these levels in urine with the presence of M2 macrophages in tumor tissue samples obtained after transurethral resection of the bladder tumor (TURBT) and before the onset of BCG instillations. METHODS: Tumor tissue samples as well as urine samples were collected from a paired sample set (n= 20) from patients before BCG treatment at our Urology Department. Response to BCG instillations was defined as no recurrence after 2 years. Informed consent was obtained from all patients. The expression levels of IL-10 and CXCL10 were measured by ELISA and RT-qPCR. The percentage of M2 polarized macrophages was calculated by immunohistochemistry staining using CD163 as M2 surface marker. We also carried out nanostring profiling with IO360 panel to characterize immune cell population in tumor tissue. RESULTS: We found a good correlation between the levels of IL-10 and CXCL10 measured by ELISA in urine samples and the levels determined by RT-qPCR in urine and tumor tissue. In order to define if the presence of M2 macrophages before BCG instillation correlates with the BCG response rate, we quantified CD163-positive cells in tissue samples from responders and non-responders. We found that patients with higher count of CD163+ infiltrating cells show worse response to BCG. We found that IL-10 quantification is a good predictor for BCG response. CONCLUSIONS: The presence of M2 polarized macrophages in TURBT tissue as well as M2 markers in urine samples of bladder cancer patients appear to be predictive for BCG response. However, validation in a larger cohort is needed to confirm our data. Source of Funding: None. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e8-e8 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Felix Guerrero-Ramos* More articles by this author Pablo Abad-Lopez More articles by this author Cristian Suarez-Cabrera More articles by this author Marta Rodriguez-Izquierdo More articles by this author Carmen Gomez-Cañizo More articles by this author Federico de la Rosa-Kehrmann More articles by this author Victor Garcia-Martinez More articles by this author Alejandra Bernardini More articles by this author Iris Lodewijk More articles by this author Guillermo Paramio More articles by this author Alfredo Rodriguez-Antolin More articles by this author Jesus M Paramio More articles by this author Marta Dueñas More articles by this author Expand All Advertisement PDF downloadLoading ...

Diagnostic yield of magnetic resonance imaging and intraoperative frozen section in the determination of deep myometrial invasion in endometrial cancer

Background and objectivesThe standard treatment for endometrial cancer is simple hysterectomy with bilateral salpingo-oophorectomy. Patients with high risk also benefit from lumbo-aortic lymphadenectomy. High risk patients include those with grades and histologic subtypes associated with poor prognosis and depth of myometrial invasion greater than 50% (M2). To determine which patients would benefit from lumbo-aortic lymphadenectomy, the depth of myometrial invasion can be assessed by intraoperative frozen section or by magnetic resonance imaging (MRI). We aimed to determine the diagnostic yield of intraoperative frozen section and MRI for detecting the presence of M2 in patients with endometrial cancer. Materials and methodsThis cross-sectional study included women with a histologically confirmed diagnosis of endometrial cancer who underwent baseline MRI and surgical intervention in our institution between 1 January 2010 and 31 December 2017. We reviewed the MRI studies and the intraoperative frozen section reports and compared them with the information in the histopathology report. We compared the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the two tests. We also calculated the diagnostic accuracy of each method and the percentages of underestimation and overestimation. Finally, we calculated the predictive value of MRI for the presence of M2, adjusting it for the histologic variables known to be associated with poor prognosis. ResultsTo detect M2, MRI had 63% sensitivity, 87% specificity, 73% PPV, and 81% NPV; the diagnostic accuracy was 78.8%, with 13.12% underestimation and 8.13% overestimation of M2. Intraoperative frozen section had 69% sensitivity, 86.7% specificity, 69% PPV, and 86% NPV; the diagnostic accuracy was 81.5%, with 9.24% underestimation and 9.24% overestimation of M2. The degree of concordance between the two methods was moderate (k=0.54, p<0.00001). ConclusionsIn our experience, MRI and intraoperative frozen section have adequate diagnostic yields for determining M2, though intraoperative frozen section is slightly better. The contribution of MRI in determining the presence and the site of deep myometrial invasion, as well as the factors that can confound the diagnosis, when added to the contribution of intraoperative frozen section, means that both methods help reduce the number of unnecessary lymph node dissections and the morbidity, mortality, and health costs associated with this practice.

Rédito diagnóstico de la resonancia magnética y el estudio por congelación intraoperatorio en la determinación de la invasión miometrial profunda en cáncer de endometrio

Background and objectivesThe standard treatment for endometrial cancer is simple hysterectomy with bilateral salpingo-oophorectomy. Patients with high risk also benefit from lumbo-aortic lymphadenectomy. High risk patients include those with grades and histologic subtypes associated with poor prognosis and depth of myometrial invasion greater than 50% (M2). To determine which patients would benefit from lumbo-aortic lymphadenectomy, the depth of myometrial invasion can be assessed by intraoperative frozen section or by magnetic resonance imaging (MRI). We aimed to determine the diagnostic yield of intraoperative frozen section and MRI for detecting the presence of M2 in patients with endometrial cancer. Materials and methodsThis cross-sectional study included women with a histologically confirmed diagnosis of endometrial cancer who underwent baseline MRI and surgical intervention in our institution between 1 January 2010 and 31 December 2017. We reviewed the MRI studies and the intraoperative frozen section reports and compared them with the information in the histopathology report. We compared the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the two tests. We also calculated the diagnostic accuracy of each method and the percentages of underestimation and overestimation. Finally, we calculated the predictive value of MRI for the presence of M2, adjusting it for the histologic variables known to be associated with poor prognosis. ResultsTo detect M2, MRI had 63% sensitivity, 87% specificity, 73% PPV, and 81% NPV; the diagnostic accuracy was 78.8%, with 13.12% underestimation and 8.13% overestimation of M2. Intraoperative frozen section had 69% sensitivity, 86.7% specificity, 69% PPV, and 86% NPV; the diagnostic accuracy was 81.5%, with 9.24% underestimation and 9.24% overestimation of M2. The degree of concordance between the two methods was moderate (k=0.54, p < 0.00001). ConclusionsIn our experience, MRI and intraoperative frozen section have adequate diagnostic yields for determining M2, though intraoperative frozen section is slightly better. The contribution of MRI in determining the presence and the site of deep myometrial invasion, as well as the factors that can confound the diagnosis, when added to the contribution of intraoperative frozen section, means that both methods help reduce the number of unnecessary lymph node dissections and the morbidity, mortality, and health costs associated with this practice.

Glioblastoma Recurrence Correlates With Increased APE1 and Polarization Toward an Immuno-Suppressive Microenvironment.

While treatment with surgery, radiotherapy and/or chemotherapy may prolong life for patients with glioblastoma, recurrence is inevitable. What is still being discovered is how much these treatments and recurrence of disease affect the molecular profiles of these tumors and how these tumors adapt to withstand these treatment pressures. Understanding such changes will uncover pathways used by the tumor to evade destruction and will elucidate new targets for treatment development. Nineteen matched pre-treatment and post-treatment glioblastoma tumors were subjected to gene expression profiling (Fluidigm, TaqMan assays), MGMT promoter methylation analysis (pyrosequencing) and protein expression analysis of the DNA repair pathways, known to be involved in temozolomide resistance (immunohistochemistry). Gene expression profiling to molecularly subtype tumors revealed that 26% of recurrent post-treatment specimens did not match their primary diagnostic specimen subtype. Post-treatment specimens had molecular changes which correlated with known resistance mechanisms including increased expression of APEX1 (p < 0.05) and altered MGMT methylation status. In addition, genes associated with immune suppression, invasion and aggression (GPNMB, CCL5, and KLRC1) and polarization toward an M2 phenotype (CD163 and MSR1) were up-regulated in post-treatment tumors, demonstrating an overall change in the tumor microenvironment favoring aggressive tumor growth and disease recurrence. This was confirmed by in vitro studies that determined that glioma cell migration was enhanced in the presence of M2 polarized macrophage conditioned media. Further, M2 macrophage-modulated migration was markedly enhanced in post-treatment (temozolomide resistant) glioma cells. These findings highlight the ability of glioblastomas to evade not only the toxic onslaught of therapy but also to evade the immune system suggesting that immune-altering therapies may be of value in treating this terrible disease.

Expression of M2 macrophage markers YKL-39 and CCL18 in breast cancer is associated with the effect of neoadjuvant chemotherapy.

High activity of enzyme TOP2a in tumor cells is known to be associated with sensitivity to anthracycline chemotherapy, but 20% of such patients do not show clinical response. Tumor microenvironment, including tumor-associated macrophages (TAM), is an essential factor defining the efficiency of chemotherapy. In the present study, we analyzed the expression of M2 macrophage markers, YKL-39 and CCL18, in tumors of breast cancer patients received anthracycline-based NAC. Patients were divided into two groups according to the level of doxorubicin sensitivity marker TOP2a: DOX-Sense and DOX-Res groups. Expression levels of TOR2a, CD68, YKL-39 and CCL18 genes were analyzed by qPCR, the amplification of TOR2a gene locus was assessed by the microarray assay. Clinical and pathological responses to neoadjuvant chemotherapy were assessed. We found that the average level of TOP2a expression in patients of DOX-Sense group was almost 10 times higher than in patients of DOX-Res group, and the expression of CD68 was 3 times higher in the DOX-Sense group compared to DOX-Res group. We demonstrated that expression levels of M2-derived cytokines but not the amount of TAM is indicative for clinical and pathological chemotherapy efficacy in breast cancer patients. Out of 8 patients from DOX-Sense group who did not respond to neoadjuvant chemotherapy (NAC), 7 patients had M2+ macrophage phenotype (YKL-39+CCL18- or YKL-39-CCL18+) and only one patient had M2- macrophage phenotype (YKL-39-CCL18-). In DOX-Res group, out of 14 patients who clinically responded to NAC 9 patients had M2- phenotype and only 5 patients had M2+ macrophage phenotype. Among pathological non-responders in DOX-Sense group, 19 (82%) patients had M2+ tumor phenotype and only 4 (18%) patients had M2- phenotype. In DOX-Res group, all 5 patients who pathologically responded to NAC had M2 phenotype (YKL-39-CCL18-). Unlike the clinical response to NAC, the differences in the frequency of M2+ and M2- phenotypes between pathologically responding and non-responding patients within DOX-Sense and DOX-Res groups were statistically significant. Thus, we showed that in patients with breast cancer who received anthracycline-containing NAC the absence of clinical response is associated with the presence of M2+ macrophage phenotype (YKL-39-CCL18 + or YKL-39 + CCL18-) based on TOP2a overexpression data.

Dissolved molecular hydrogen (H2) in Peritoneal Dialysis (PD) solutions preserves mesothelial cells and peritoneal membrane integrity

BackgroundPeritoneal dialysis (PD) is used as renal replacement therapy in patients with end-stage kidney disease. However, peritoneal membrane failure remains problematic and constitutes a critical cause of PD discontinuation. Recent studies have revealed the unique biological action of molecular hydrogen (H2) as an anti-oxidant, which ameliorates tissue injury. In the present study, we aimed to examine the effects of H2 on the peritoneal membrane of experimental PD rats.MethodEight-week-old male Sprague-Dawley rats were divided into the following groups (n = 8–11 each) receiving different test solutions: control group (no treatment), PD group (commercially available lactate-based neutral 2.5% glucose PD solution), and H2PD group (PD solution with dissolved H2 at 400 ppb). Furthermore, the influence of iron (FeCl3: 5 μM: inducer of oxidative cellular injury) in the respective PD solutions was also examined (Fe-PD and Fe-H2PD groups). The H2PD solution was manufactured by bathing a PD bag in H2-oversaturated water created by electrolysis of the water. Twenty mL of the test solutions were intraperitoneally injected once a day for 10 days. Parietal peritoneum samples and cells collected from the peritoneal surface following treatment with trypsin were subjected to analysis.ResultsIn the PD group as compared to controls, a mild but significant sub-mesothelial thickening was observed, with increase in the number of cells in the peritoneal surface tissue that were positive for apoptosis, proliferation and vimentin, as seen by immunostaining. There were significantly fewer of such changes in the H2PD group, in which there was a dominant presence of M2 (CD163+) macrophages in the peritoneum. The Fe-PD group showed a significant loss of mesothelial cells with sub-mesothelial thickening, these changes being ameliorated in the Fe-H2PD group.ConclusionH2-dissolved PD solutions could preserve mesothelial cells and peritoneal membrane integrity in PD rats. Clinical application of H2 in PD could be a novel strategy for protection of peritoneal tissue during PD treatment.

MP94-15 MUSCARINIC M3- AND M2- RECEPTOR-ACTIVATED SIGNALING IN THE BLADDER IS INVERSELY REGULATED BY CAVEOLAE.

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II1 Apr 2017MP94-15 MUSCARINIC M3- AND M2- RECEPTOR-ACTIVATED SIGNALING IN THE BLADDER IS INVERSELY REGULATED BY CAVEOLAE. Vivian Cristofaro and Maryrose P. Sullivan Vivian CristofaroVivian Cristofaro More articles by this author and Maryrose P. SullivanMaryrose P. Sullivan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2918AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder smooth muscle (BSM) caveolae are cholesterol-enriched membrane microdomains that augment or attenuate detrusor functional responses by differentially regulating specific receptor-activated signaling pathways. However, BSM contractions induced by cholinergic activation in the rat are not altered by depletion of caveolae, unlike other smooth muscle systems in which muscarinic receptor signaling is evidently mediated by caveolae. Although this discrepancy may reflect the highly specific regulation imparted by caveolae among different tissues and species, a differential regulation of muscarinic M3 and M2 receptor subtypes in the bladder cannot be excluded. This study examined the functional and molecular relationship between caveolae and muscarinic acetylcholine receptor (mAChR) subtypes M3 and M2. METHODS BSM tissue strips were prepared from Sprague Dawley rat bladders after removing the mucosa. Tissue was suspended in organ baths for isometric tension studies. Dose response curves to carbachol (CCh, 1nM-10μM) were generated at baseline, as well as in the presence of 4-DAMP (10nM) or AFDX (0.1μM) to inactive M3 or M2 receptors respectively. Responses to CCh were repeated after incubation with methyl-β-cyclodextrin (mβCD, 15mM), an agent that disrupts caveolae by depleting membrane cholesterol. Interaction between caveolin-1 (Cav-1, a protein required for caveolae biogenesis) and M3 or M2 mAChR subtype were investigated by co-immunoprecipitation. RESULTS Compared to baseline responses, 4-DAMP decreased CCh-induced contractions at each dose. After mβCD treatment and in the presence of 4-DAMP, contractile responses to CCh were significantly enhanced. AFDX had little effect on CCh dose-response curves. However, the subsequent disruption of caveolae in the presence of AFDX attenuated significantly contractions induced by CCh. Immunoreactive bands corresponding to M3 and M2 mAChR subtypes were detected in Cav-1 immunoprecipitates. CONCLUSIONS The opposite effect of mβCD on CCh responses in the presence of M2 or M3 antagonists suggests that caveolae negatively regulate M2- and positively regulate M3-mediated signaling respectively, but this interaction is masked when only the aggregate effect of CCh is examined. Molecular interaction of Cav-1 and mAChRs is consistent with their localization within caveolae. Changes in the balance among caveolin-mAChR interactions, due to loss of caveolae or changes in mAChR subtype expression, may alter responses to cholinergic activation or the efficacy of anti-muscarinic agents in the bladder. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1252 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Vivian Cristofaro More articles by this author Maryrose P. Sullivan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Monocytes, Macrophages, and Osteoclasts in Osteosarcoma.

Macrophages appear to have a fundamental role in the pathogenesis of osteosarcoma. These highly diverse plastic cells are subdivided into classical or inflammatory macrophages known as M1 and alternative macrophages, which decrease inflammation and are reparative, called M2. Although primary and metastatic osteosarcomas are infiltrated with M2 macrophages, targeting the M1 macrophages with the immune adjuvant muramyl tripeptide phosphatidyl ethanolamine (MTP-PE) has been the greatest recent therapeutic advance in osteosarcoma. This discrepancy between the presence of M2 and activation of M1 macrophages is intriguing and is likely explained either by the plasticity of M1 and M2 macrophages or nonclassical patrolling monocytes (PMos). To date, MTP-PE has been approved in combination with chemotherapy for nonmetastatic osteosarcoma, but its use in metastatic tumors has not been investigated. In this review, we focus on macrophages, monocytes, and osteoclasts, their role in osteosarcoma, and the potential for targeting these cells in this disease.

A novel multimode sensor showing cation-dependent fluorescence colour.

A novel sensor, 4-[2-(9-anthryl)ethynyl]-1,10-phenanthroline (1), exhibits highly intense fluorescent in the wavelength region of 440-600 nm (maximum wavelength (λf) = 470 nm) with the quantum yield (Φf) and lifetime (τf) being 0.90 and 4.2 ns, respectively, in CH3CN at 298 K. In the presence of a divalent cation (M2+ = Ba2+, Ca2+, Mg2+, or Zn2+) in CH3CN, sensor 1 can tightly bind M2+ and shows intense fluorescent (Φf = 0.90-0.19, τf = 2.1-6.9 ns) with the color being dependent on the nature of M2+ (λf = 514-584 nm). The results demonstrate that a single fluorescent sensor 1 is capable of simultaneous identification and quantitation of M2+ based on λf and the fluorescent intensity (Φf), respectively. The fluorescence maximum energy of the [1-M2+] complex is shown to correlate linearly with the pKa value of M2+. The spectroscopic and photophysical properties of sensor 1 in the absence and presence of M2+ are also discussed.

Selective Phosphonylation of 5'-Adenosine Monophosphate (5'-AMP) via Pyrophosphite [PPi(III)

We describe here experiments which demonstrate the selective phospho-transfer from a plausibly prebiotic condensed phosphorus (P) salt, pyrophosphite [H2P2O5 2−; PPi(III)], to the phosphate group of 5′-adenosine mono phosphate (5′-AMP). We show further that this P-transfer process is accelerated both by divalent metal ions (M2+) and by organic co-factors such as acetate (AcO−). In this specific case of P-transfer from PPi(III) to 5′-AMP, we show a synergistic enhancement of transfer in the combined presence of M2+ & AcO−. Isotopic labelling studies demonstrate that hydrolysis of the phosphonylated 5′-AMP, [P(III)P(V)-5′-AMP], proceeds via nuceophilic attack of water at the Pi(III) terminus.

A New Skull of Hyaenictis Gaudry, 1861 (Carnivora, Hyaenidae) Shows Incipient Adaptations to Durophagy

The European Miocene records a wide diversity of hyaenid ecomorphotypes represented by multiple genera. Among these, Hyaenictis Gaudry, 1861, is one of the least known. This genus includes four species from the late Miocene and Pliocene of the Old World, but in Europe Hyaenictis is only represented by two species, recorded by scarce and fragmentary remains: Hyaenictis graeca Gaudry, 1861, from Pikermi (MN12; Greece) and Hyaenictis almerai Villalta Comella and Crusafont Pairo, 1948, from Sant Miquel de Toudell (MN10; Valles-Penedes Basin, NE Iberia). Here, we describe a new skull of Hyaenictis aff. almerai from the Valles-Penedes site of Ronda Oest Sabadell Sector D (MN10), representing the most complete European specimen of the genus. In the presence of m2 and virtual lack of m1 metaconid, the described cranium more closely resembles Hyaenictis rather than any other medium- to large-sized European hyaenid. However, the new skull does not fit well with previously known Hyaenictis species, more closely resembling the bone-cracking Adcrocuta Kretzoi, 1938, in the development of premolar accessory cuspids and the possession of relatively broad cheek teeth. These and other features (strong mandibular muscular insertions and enamel microstructure) denote more durophagous adaptations than previously documented in Hyaenictis (considered a cursorial/dog-like hyaena), and favor the inclusion of H. aff. almerai in the transitional bone-cracking hyaenid ecomorphotype.

Investigation of the curvature induction and membrane localization of the influenza virus M2 protein using static and off-magic-angle spinning solid-state nuclear magnetic resonance of oriented bicelles.

A wide variety of membrane proteins induce membrane curvature for function; thus, it is important to develop new methods to simultaneously determine membrane curvature and protein binding sites in membranes with multiple curvatures. We introduce solid-state nuclear magnetic resonance (NMR) methods based on magnetically oriented bicelles and off-magic-angle spinning (OMAS) to measure membrane curvature and the binding site of proteins in mixed-curvature membranes. We demonstrate these methods on the influenza virus M2 protein, which not only acts as a proton channel but also mediates virus assembly and membrane scission. An M2 peptide encompassing the transmembrane (TM) domain and an amphipathic helix, M2(21-61), was studied and compared with the TM peptide (M2TM). Static (31)P NMR spectra of magnetically oriented 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles exhibit a temperature-independent isotropic chemical shift in the presence of M2(21-61) but not M2TM, indicating that the amphipathic helix confers the ability to generate a high-curvature phase. Two-dimensional (2D) (31)P spectra indicate that this high-curvature phase is associated with the DHPC bicelle edges, suggestive of the structure of budding viruses from the host cell. (31)P- and (13)C-detected (1)H relaxation times of the lipids indicate that the majority of M2(21-61) is bound to the high-curvature phase. Using OMAS experiments, we resolved the (31)P signals of lipids with identical headgroups based on their distinct chemical shift anisotropies. On the basis of this resolution, 2D (1)H-(31)P correlation spectra show that the amide protons in M2(21-61) correlate with the DMPC but not DHPC (31)P signal of the bicelle, indicating that a small percentage of M2(21-61) partitions into the planar region of the bicelles. These results show that the amphipathic helix induces high membrane curvature and localizes the protein to this phase, in good agreement with the membrane scission function of the protein. These bicelle-based relaxation and OMAS solid-state NMR techniques are generally applicable to curvature-inducing membrane proteins such as those involved in membrane trafficking, membrane fusion, and cell division.

A1.13 Increased presence of CD206+macrophage subset in peripheral blood of systemic sclerosis patients

Background and objectives In systemic sclerosis (SSc), the immune-inflammatory infiltrate is primarily made of macrophages and T cells. 1 Recently, alternatively activated macrophages (M2) have been shown to enhance fibrosis through the production of pro-fibrotic molecules (i.e. fibronectin, metalloproteinases and TGFβ. 2 M2 are characterised by an increased expression of specific markers, primarily CD206 (macrophage mannose receptor-1) and macrophage scavenger receptors (CD204 and CD163). The study investigated the presence of M2 in the peripheral blood (PB) of SSc patients. In addition, the capability of SSc serum to induce the expression of M2 markers and the production of pro-fibrotic metalloproteinase (MMP)-9 in cultured PB mononuclear cells (PBMCs) of healthy subjects (HS) were investigated. Materials and methods Eight SSc patients (mean age 65 ± 7 years) who fulfilled the new criteria of EULAR/ACR 3 and five age matched HS were enrolled into the study, after signing an Informed Consent. SSc patients were treated with vasodilators and endothelin-1 blockers. PB from SSc patients and HS was analysed by flow cytometry (FC) to detect the presence of cells positive for CD206, CD204 and CD14 (a marker of monocyte/macrophage lineage). PBMC-derived monocytes isolated from the enrolled HS (2 × 10 6 cells/well) were maintained for 12 h in growth medium at 10% of fetal bovine serum and then stimulated for 48 h with the serum from SSc patients and HS themselves. The expression of CD206, CD204 and CD68 (a macrophage marker) was evaluated by immunocytochemistry (ICC). MMP-9 production was evaluated by zymography. Results In the PB of SSc patients, the CD206 + cell percentage was found significantly higher than that of HS (p + cells co-expressed CD204, belonging to the monocyte/macrophage lineage due to their expression of CD14. SSc serum stimulated PBMC-derived monocytes to express CD206, CD204 and CD68 thus indicating their activation into macrophages. Conversely, serum from HS was unable to stimulate the expression of CD206 and it induced a limited expression of CD204 in these cells. Zymography showed that treatment with SSc serum induced an increased production and secretion of MMP-9 compared to the treatment with HS serum in PBMC-derived macrophages. Conclusion These preliminary results show an increased presence of the CD206 + cell subset in the PB of SSc patients. The ability of SSc serum to induce the expression of this M2 macrophage marker and to increase the production of pro-fibrotic MMP-9 might support a possible involvement of this cell subset in the pathogenesis of SSc. References Wynn TA, Ramalingam TR et al . Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med 2012; 18 :1028–40. Fairweather D, Cihakova D et al . Alternatively activated macrophages in infection and autoimmunity. J Autoimmun 2009; 33 :222–30. van den Hoogen F, Khanna D, Fransen J et al . 2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League against Rheumatism collaborative initiative. Arthrit Rheum 2013; 65 :2737–47.

Electrochemically Directed Synthesis of Co2+ and Ni2+ Complexes with TCNQF42– (TCNQF4 = 2,3,5,6‐Tetrafluoro‐7,7,8,8‐tetracyanoquinodimethane)

AbstractThe electrocrystallization of transition metal coordination polymers MTCNQF4 containing TCNQF42– dianions and M2+ cations (TCNQF4 = 2,3,5,6‐tetrafluoro‐7,7,8,8‐tetracyanoquinodimethane, M = Co or Ni) has been achieved in acetonitrile (0.1 M Bu4NPF6) at glassy carbon and indium tin oxide electrodes and the reaction pathway monitored by cyclic voltammetry and surface plasmon resonance. TCNQF4 is sequentially reduced to TCNQF4·– and then TCNQF42–. In the presence of M2+, the TCNQF42– formation is accompanied by the electrocrystallization of the sparingly soluble MTCNQF4 on the electrode surface. Cyclic voltammetry studies reveal that two phases of MTCNQF4 are formed. One is kinetically favoured and rapidly converts to a second thermodynamically stable phase. The formation of MTCNQF4 is possible because of the relatively high solubility of M(TCNQF4)2 derivatives. The solubilities of CoTCNQF4 and NiTCNQF4 are 8.6 × 10–6 and 9.2 × 10–6 M, respectively, in neat acetonitrile and 2.2 × 10–5 and 2.4 × 10–5 M, respectively, in an acetonitrile solution containing Bu4NPF6 (0.1 M). The presence of TCNQF42– dianions rather than TCNQF4·– monoanions in the coordination polymers was confirmed by UV/Vis, IR and Raman spectroscopy. Scanning electron microscopy images show that the morphology of the electrocrystallized MTCNQF4 depends on the applied potential for CoTCNQF4, but not for the NiTCNQF4 complex.