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Supplementary Figures S1-S4; Supplementary Table S1.

Purpose: This study characterizes, biologically and clinically, a novel type of dendritic cells (DC) produced in the short term and called tumor antigen–presenting cells (TAPCells). In particular, we identified factors present in a lysate derived from heat-shocked allogeneic melanoma cells (TRIMEL) that are associated with TAPCells' enhanced capability to induce CD8+ T-cell responses in vitro and in vaccinated melanoma patients.Experimental Design: First, extensive phenotypic and functional characterization of TAPCells was performed, followed by vaccination of 45 melanoma patients with four doses of TAPCells over a period of 2 months. Specific delayed-type hypersensitivity (DTH) reaction was analyzed posttreatment and correlated with overall survival rates. Furthermore, heat-shock (HS)-induced factors present in TRIMEL and their effects on DC activation were identified and studied.Results: TRIMEL induced a committed, mature, DC-like phenotype in TAPCells and effectively activated melanoma-specific CD4+ and CD8+ T cells. Clinically, 64% of vaccinated patients showed positive DTH reaction against TRIMEL, and this was associated with improved overall survival. HS treatment of tumor cells increased calreticulin (CRT) plasma membrane translocation and induced the release of high-mobility group box 1 proteins (HMGB1). Both CRT and HMGB1 mobilization were associated with enhanced TAPCells' maturation and antigen (Ag) cross-presentation, respectively. DTH infiltration analysis revealed the presence of CD8+/CD45RO+ T cells, thus confirming TAPCells' ability to cross-present Ags in vivo.Conclusions: Our results indicate that lysates derived from heat-shocked tumor cells are an optimal source of tumor-associated Ags, which are crucial for the generation of DCs with improved Ag cross-presentation capacity and clinically effective immunogenicity. Clin Cancer Res; 17(8); 2474–83. ©2011 AACR.

Introduction Osimertinib has shown promising activity in patients with leptomeningeal metastases (LMs) of EGFR-positive NSCLC at 160 mg once daily (qd) (BLOOM; NCT02228369). We report LM activity with osimertinib (80 mg qd) in a retrospective analysis of studies across the AURA program (AURA extension, AURA2, AURA17, and AURA3). Methods Patients with EGFR T790M-positive advanced NSCLC and progression after previous EGFR-tyrosine kinase inhibitor therapy received osimertinib (80 mg qd). Patients with central nervous system (CNS) metastases (including LMs) were eligible if the lesions were neurologically asymptomatic and stable. Patients with evidence of LMs at the study entry were retrospectively included for the analysis; brain scans were assessed for radiologic LM response by neuroradiologically blinded, independent central review per the modified Response Assessment in Neuro-Oncology LM criteria. LM objective response rate, duration of response, progression-free survival, and overall survival were assessed. A longitudinal analysis was performed to investigate the relationship between changes from the baseline in non-CNS tumor sizes and LM responses at each visit of patients in AURA LM and BLOOM studies. Results For the 22 patients included in the analysis, LM objective response rate was 55% (95% confidence interval [CI]: 32–76). Median LM duration of response was not reached (95% CI: 2.8–not calculable [NC]). Median LM progression-free survival and overall survival were 11.1 months (95% CI: 4.6–NC) and 18.8 months (95% CI: 6.3–NC), respectively. The longitudinal analysis revealed similar non-CNS and LM responses between the patients in AURA LM and BLOOM programs. Conclusions Patients with EGFR T790M-positive NSCLC and radiologically detected LM obtained clinical benefit from osimertinib (80 mg qd).

PURPOSE In this phase I study (BLOOM), osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), was evaluated in patients with leptomeningeal metastases (LMs) from EGFR-mutated (EGFRm) advanced non–small-cell lung cancer (NSCLC) whose disease had progressed on previous EGFR-TKI therapy. PATIENTS AND METHODS Patients with cytologically confirmed LM received osimertinib 160 mg once daily. Objectives were to assess confirmed objective response rate (ORR), duration of response (DoR), progression-free survival (PFS), overall survival (OS), pharmacokinetics (PK), and safety. Additional efficacy evaluations included changes from baseline in CSF cytology and neurologic examination. Measurable lesions were assessed by investigator according to RECIST version 1.1. LMs were assessed by neuroradiologic blinded central independent review (BICR) according to Response Assessment in Neuro-Oncology LM radiologic criteria and by investigator. RESULTS Forty-one patients were enrolled. LM ORR and DoR by neuroradiologic BICR were 62% (95% CI, 45% to 78%) and 15.2 months (95% CI, 7.5 to 17.5 months), respectively. Overall, ORR by investigator was 41% (95% CI, 26% to 58%), and median DoR was 8.3 months (95% CI, 5.6 to 16.5 months). Median investigator-assessed PFS was 8.6 months (95% CI, 5.4 to 13.7 months) with 78% maturity; median OS was 11.0 months (95% CI, 8.0 to 18.0 months) with 68% maturity. CSF tumor cell clearance was confirmed in 11 (28%; 95% CI, 15% to 44%) of 40 patients. Neurologic function was improved in 12 (57%) of 21 patients with an abnormal assessment at baseline. The adverse event and PK profiles were consistent with previous reports for osimertinib. CONCLUSION Osimertinib showed meaningful therapeutic efficacy in the CNS and a manageable safety profile at 160 mg once daily in patients with EGFRm NSCLC and LM.

Instituto de Investigaciones Filosóficas. Facultad de Filosofía. Programa Institucional de Maestría en Filosofía de la Cultura The thesis work presented here is the result of the investigation of the concept of «thought synthesizer» in the philosophy of Deleuze and Guattari, which, in the first place, is an epistemological concept, since it is the active form of subjectivity through the one force that adds knowledge to experience, and thus creates a unique way of existing in the world. Secondly, we find that synthesis is a concept that matches the musical ontology described by Deleuze and Guattari in Thousand Plateaus, as the music as a physical phenomenon of mechanical waves that it is intuitively described, as imperceptible forces for the perceptual spectrum of the human body and that, nevertheless, occur and are constitutive of its own being, of nature, the species that inhabit there and in the cosmos itself. The Being is music because all its phenomena are expressed by creating their rhythms, that is, the time and space that they open to inhabit. In that sense, History is also the expression of synthesis of time that is added to each other and creates new synthesis, like a pulsed, immeasurable rhythm that does not cease. The history of art, for example, is the expression of the «Synthesizer of thought», since all avant-garde is created from the gathering of forces, affects, perceptions and already existing concepts and adding their own, that is Ritornelos. The concept of «Synthesizer of thought» is seen from an epistemological question, tracing separately the implications it has. In Deleuze, the development from Empiricism and subjectivity, as an epistemological problem, constituting a type of subjectivation, passing through Capitalism and schizophrenia where the philosophy of subjectivity and the event converges with Guattari's social and musical perspective. El trabajo de tesis aquí presentado, es el resultado de la investigación del concepto de «sintetizador de pensamiento» en la filosofía de Deleuze y Guattari que, en primer lugar, es un concepto epistemológico, pues es la forma activa de la subjetividad por medio de la que agrega conocimiento a su experiencia, y así crea su modo único de existir en el mundo. En segundo lugar, encontramos que la síntesis es un concepto que se empata con la ontología musical descrita por Deleuze y Guattari en Mil Mesetas, donde lo musical como fenómeno físico de ondas mecánicas es descrito intuitivamente, como fuerzas imperceptibles para el espectro perceptivo del cuerpo humano y que, sin embargo, ocurren y son constitutivas de su propio ser, de la naturaleza, las especies que ahí habitan y en el cosmos mismo. El Ser es pensado como música ya que todos sus fenómenos se expresan creando ritmos, es decir, el tiempo y el espacio que abren para habitar. En ese sentido es que la historia es, también, la expresión de síntesis de tiempo que se agregan unas a otras y crean nuevas sintesis, como un ritmo pulsado, inconmensurable, y que no cesa. La historia del arte, por ejemplo, es la expresión del Sintetizador de pensamiento, pues toda vanguardia se crea a partir de la reunión de fuerzas, de afectos, perceptos y conceptos ya existentes y agregando los propios, es decir los Ritornelos. El concepto de Sintetizador de pensamiento es visto desde una cuestión epistemológica rastreando de manera separada las implicaciones que tiene. En Deleuze, el desarrollo desde Empirismo y subjetividad, como problema epistemológico, constituyente de un tipo de subjetivación, pasando por Capitalismo y esquizofrenia donde la filosofía de subjetividad y el acontecimiento confluye con la perspectiva social y musical de Guattari.

TPS3162 Background: There are substantial data suggesting that a subset of human epidermal growth factor receptor 2 (HER2)–activating mutations induce ligand-independent constitutive HER2 signaling and promote oncogenesis. Direct therapeutic targeting of HER2 has transformed the treatment of patients with HER2-overexpressing breast and gastric cancers. However, currently no targeted treatments are approved for patients with tumors harboring HER2-activating mutations. T-DXd is an antibody-drug conjugate consisting of an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a membrane-permeable topoisomerase I inhibitor payload that may be selectively internalized in tumors with HER2 mutations (Li BT, et al. Cancer Discov. 2020;10:674-687). In a phase 1 study (DS8201-A-J101), T-DXd demonstrated preliminary antitumor activity in patients with tumors harboring HER2 mutations, with confirmed responses observed in 9 of 19 patients (47.4%) (Tsurutani J, et al. Cancer Discov. 2020;10:688-701). Here we describe the DESTINY-PanTumor01 trial (NCT04639219). Methods: DESTINY-PanTumor01 is an open-label, multicenter, single-arm, phase 2 study evaluating T-DXd for the treatment of patients with unresectable and/or metastatic solid tumors (excluding non-small cell lung cancer) harboring prespecified HER2-activating mutations. Patients (N≈100) are required to have progressed following prior treatment for advanced or metastatic disease or have no satisfactory alternative treatment options. Prior HER2-targeting therapy is allowed. HER2 mutation status will be determined locally using next-generation sequencing or a validated nucleic acid–based methodology. The primary endpoint is confirmed objective response rate according to independent central review per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Secondary endpoints include duration of response, disease control rate, progression-free survival, investigator-assessed confirmed objective response rate, overall survival, safety, pharmacokinetics, and immunogenicity. Clinical trial information: NCT04639219.

Objective\ud We conducted a feasibility trial of acupuncture in cancer patients undergoing radiotherapy treatment. The trial included training radiographers to deliver acupuncture within patients’ routine NHS care.\ud \ud Methods\ud Mixed methods pragmatic randomized parallel-group exploratory feasibility trial comparing standard care to standard care plus acupuncture.\ud \ud Results\ud Most aspects of the research design and acupuncture intervention were acceptable to the 101 participants. Participants’ valued the opportunity to receive acupuncture within their NHS care, perceived the treatment as eliciting a number of beneficial effects, and had a positive impact on their NHS cancer treatment. However, quantitative analysis of outcome measure data revealed no consistent significant differences between those receiving standard care and those receiving standard care plus acupuncture.\ud \ud Conclusion\ud It is feasible to implement acupuncture in a busy radiotherapy unit provided by specially trained radiographers. The methodology employed appears acceptable for the evaluation of acupuncture for radiotherapy patients.

TPS261 Background: For patients (pts) with HER2-overexpressing metastatic gastric cancer, trastuzumab + chemotherapy is a standard first-line option but provides only a modest overall survival (OS) benefit vs chemotherapy. T-DXd is an antibody-drug conjugate consisting of an anti-HER2 antibody, cleavable tetrapeptide-based linker, and a membrane-permeable topoisomerase I inhibitor payload. Results from a phase 1 trial showed promising antitumor activity (confirmed objective response rate [ORR], 43.2%) in pts with heavily pretreated HER2+ metastatic gastric cancer who received T-DXd (5.4 or 6.4 mg/kg; Shitara K, et al. Lancet Oncol. 2019;20:827-836). Here we describe the phase 1b/2 DESTINY-Gastric03 trial (NCT04379596) evaluating T-DXd monotherapy and combinations in pts with HER2-overexpressing gastric cancer. Methods: This is an open-label, multicenter, 2-part, phase 1b/2 study in pts with HER2-overexpressing (immunohistochemistry [IHC] 3+ or IHC 2+/in situ hybridization positive) locally advanced, unresectable or metastatic gastric or gastroesophageal junction cancer. In part 1 (dose escalation), pts who had received prior trastuzumab-containing therapy will be assigned to 1 of 5 arms: (1) T-DXd + 5-fluorouracil (5-FU); (2) T-DXd + capecitabine (C); (3) T-DXd + durvalumab; (4) T-DXd + 5-FU or C + oxaliplatin (Ox); or (5) T-DXd + 5-FU or C + durvalumab. In part 2 (dose expansion), pts with no prior treatment for metastatic disease will be randomized across 4 arms: (1) T-DXd; (2) trastuzumab + 5-FU or C + Ox or cisplatin; (3) T-DXd + 5-FU or C ± Ox; or (4) T-DXd + 5-FU or C + durvalumab. In part 2, pts will be stratified by HER2 status. Primary endpoints are safety, determination of recommended phase 2 doses (part 1), and investigator-assessed confirmed ORR per RECIST v1.1 (part 2). Secondary endpoints include confirmed ORR (part 1), disease control rate, duration of response, progression-free survival (all per investigator), OS, safety (part 2), pharmacokinetics, and immunogenicity. Clinical trial information: NCT04379596.

EDXRD apparatus can be improved by multiple angle scanning implementing a hybrid system that take advantage of shorter acquisition time and higher energy penetration in EDXRD, and of higher inter-planar distance resolution in ADXRD. Methods for hybrid data processing at low energy range using reflection detection mode are presented. Additional to the ED XRD-XRF spectrum directly acquired, a specially developed software extracts ADXRD from angle scanning data and builds 3D/2D plots showing the Iso-d curves, builds a hybrid diffractogram and identifies crystalline phase by dealing with corrections of line shifts due to different positioning of the irradiated spot on the cultural heritage object.

The purpose of this study is to investigate several artificial Neural Network (NN) architectures in order to design a cognitive radar system capable of optimally distinguishing linear Frequency-Modulated (FM) signals from bandlimited Additive White Gaussian Noise (AWGN). The goal is to create a theoretical framework to determine an optimal NN architecture to achieve a Probability of Detection (PD) of 95% or higher and a Probability of False Alarm (PFA) of 1.5% or lower at 5 dB Signal to Noise Ratio (SNR). Literature research reveals that the frequency-domain power spectral densities characterize a signal more efficiently than its time-domain counterparts. Therefore, the input data is preprocessed by calculating the magnitude square of the Discrete Fourier Transform of the digitally sampled bandlimited AWGN and linear FM signals to populate a matrix containing N number of samples and M number of spectra. This matrix is used as input for the NN, and the spectra are divided as follows: 70% for training, 15% for validation, and 15% for testing. The study begins by experimentally deducing the optimal number of hidden neurons (1-40 neurons), then the optimal number of hidden layers (1-5 layers), and lastly, the most efficient learning algorithm. The training algorithms examined are: Resilient Backpropagation, Scaled Conjugate Gradient, Conjugate Gradient with Powell/Beale Restarts, Polak-Ribiere Conjugate Gradient, and Variable Learning Rate Backpropagation. We determine that an architecture with ten hidden neurons (or higher), one hidden layer, and a Scaled Conjugate Gradient for training algorithm encapsulates an optimal architecture for our application.

We propose an approach based on artificial Neural Networks (NN) to classify wideband radar jammer signals for more efficient use of countermeasures. A robust NN is be used to correctly differentiate Frequency-Modulated (FM) signals from bandlimited Additive White Gaussian Noise (AWGN). We compare the performance of the NN using the samples of the power spectrum versus the autocorrelation. Prior experiments showed that frequency-domain moments of the FM signal itself are better descriptors than time-domain moments. Using simulated wideband FM radar signals, we compute a set of N autocorrelation and spectra and feed them to the NN which has ten hidden layers. For training purposes, the autocorrelations or spectra sets are divided into three groups, 75% for training, 15% for validating and 15% for testing. For the power spectra set, we observe that a Signal to Noise Ratio (SNR) of 5dB allows the network to approach an average of 5% percent Probability of Error (PE). Training with the autocorrelation set yields comparable results. For an SNR of 5dB, the average PE reached an average of 0.3%. In both instances, the NN reaches zero percent PE at an SNR of 10dB.

A non-invasive archaeometrical study with a portable X-ray system is presented. A setup for XRF-XRD (X-ray fluorescence-X-ray diffraction) based on polychromatic excitation and energy dispersive detection in the reflection mode has been utilized for in situ characterization of artistic and archaeological artifacts. This portable X-ray system allows analysis of chemical elements and chemical crystalline phases and internal structural analysis by radiography. As representative examples is presented: the XRD-XRF identification and characterization of minerals in green stone axes, the provenance XRF study of archaeological ceramics and the possibilities in the radiography examination of paintings., Restaurierung und Archäologie, Bd. 8 (2015): Restaurierung und Archäologie

Gap junctions (GJs) mediate intercellular communication between adjacent cells. Previously, we showed that connexin 43 (Cx43), the main GJ protein in the immune system, mediates Ag transfer between human dendritic cells (DCs) and is recruited to the immunological synapse during T cell priming. This crosstalk contributed to T cell activation, intracellular Ca2+ responses, and cytokine release. However, the role of GJs in NK cell activation by DCs and NK cell–mediated cytotoxicity against tumor cells remains unknown. In this study, we found polarization of Cx43 at the NK/DC and NK/tumor cell-contact sites, accompanied by the formation of functional GJs between NK/DCs and NK/tumor cells, respectively. Cx43–GJ-mediated intercellular communication (GJIC) between human NK and DCs was bidirectional. Blockage of Cx43-GJIC inhibited NK cell activation, though it affected neither the phenotype nor the function of DCs. Cx43 knockdown or inhibition using mimetic peptides greatly reduced CD69 and CD25 expression and IFN-γ release by DC-stimulated NK cells. Moreover, blocking Cx43 strongly inhibited the NK cell–mediated tumor cell lysis associated with inhibition of granzyme B activity and Ca2+ influx. Our data identify a novel and active role for Cx43-GJIC in human NK cell activation and antitumor effector functions that may be important for the design of new immune therapeutic strategies.

Poor healing of DFUs (diabetic foot ulcers) is a major clinical problem that can be extremely debilitating and lead to lower limb amputation. In the normal acute wound, the Cx43 (connexin 43) gap junction protein is down-regulated at the wound edge as a precursor to cell migration and healing. In fibroblasts from the human chronic DFU wound edge there was a striking and significant 10-fold elevation of Cx43 protein, as well as a 6-fold increase in N-cadherin and a 2-fold increase in ZO-1 (zonular occludin-1), compared with unwounded skin. In streptozotocin diabetic rats, Cx43 was found to be up-regulated in intact dermal fibroblasts in direct proportion to blood glucose levels and increased 2-fold further in response to wounding of the skin. To mimic diabetes, NIH 3T3 fibroblasts were cultured under different concentrations of glucose or mannitol and Cx43 protein intercellular communication and migration rates were determined. Cultures of fibroblasts in very high (40 mM) glucose conditions showed significantly elevated Cx43 protein levels, as shown by immunostaining and Western blotting, and significantly increasing gap junctional communication, as shown by dye transfer. In scratch wound-healing assays, increased levels of Cx43 from high glucose resulted in repressed filopodial extensions and significantly slower migration rates than in either standard conditions (5.5 mM glucose) or the osmotic control of mannitol. Conversely, when glucose-induced Cx43 up-regulation was prevented with Cx43shRNA (Cx43 short-hairpin RNA) transduction, the fibroblasts extended long filopodia and migrated significantly faster. Cx43 protein was up-regulated in fibroblasts in DFUs as well as after high glucose exposure in culture which correlated with inhibition of fibroblast migration and is likely to contribute to impaired wound healing.

Starting on a laboratory developed portable X-ray fluorescence (PXRF) spectrometer; three different analytical results can be performed: analysis of chemical elements, analysis of major chemical crystalline phase and structural analysis, which represents a contribution to a new, low cost development of portable X-ray analyzer; since these results are respectively obtained with independent equipments for X-ray fluorescence, X-ray diffraction and radiography. Detection limits of PXRF were characterized using standard reference materials for ceramics, glass, bronze and bones, which are the main materials requiring quantitative analysis in art and archeological objects. A setup for simultaneous energy dispersive X-ray fluorescence and diffraction (ED (XRF–XRD)) in the reflection mode has been tested for in situ and non-destructive analysis according to the requirements of art objects inspection. The system uses a single low power X-ray tube and an X-ray energy dispersive detector to measure X-ray diffraction spectrum at a fixed angle. Application to the identification of jadeite-jade mineral in archeological objects by XRD is presented. A local high resolution radiography image obtained with the same low power X-ray tube allows for studies in painting and archeological bones.

Learning Objectives After completing this course, the reader will be able to: Explain the importance of resistance to anoikis in the development of metastases.Describe the mechanisms of anoikis resistance in EFTs and osteosarcoma and their potential use in development of new therapies. CME This article is available for continuing medical education credit at CME.TheOncologist.com Detection of micrometastatic tumor cells in the bone marrow or peripheral blood of patients with Ewing family of tumors (EFTs) and osteosarcoma has been shown to correlate with poor outcome. Although one of the aims of chemotherapy is eradication of micrometastatic disease, these cells vary phenotypically from primary tumor cells and appear to be more resistant to chemotherapy. As a barrier to metastasis, cells normally undergo a form of cell death termed anoikis after they lose contact with the extracellular matrix or neighboring cells. Tumor cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive after detachment from their primary site and while traveling through the circulation. Investigating mechanisms of resistance to anoikis, therefore, provides a valuable model to investigate regulation of micrometastatic disease. This review focuses on the current understanding of the mechanisms involved in mediating cell survival and resistance to anoikis in EFTs and osteosarcoma and discusses future studies that may help to identify novel therapeutics targeted at micrometastatic disease.

Previously, we found that human dendritic cells (hDCs) pulsed with a melanoma cell lysate (MCL) and stimulated with TNF-α (MCL/TNF) acquire a mature phenotype in vitro and are able to trigger tumor-specific immune responses when they are used in melanoma immunotherapy in patients. In this study, we describe that MCL/TNF induces gap junction (GJ)-mediated intercellular communications and promotes melanoma Ag transfer between ex vivo produced hDCs from melanoma patients. hDCs also exhibit increased expression of the GJ-related protein connexin 43, which contributes to GJ plaque formation after MCL/TNF stimulation. The addition of GJ inhibitors suppresses intercellular tumor Ag transfer between hDCs, thus reducing melanoma-specific T cell activation. In summary, we demonstrate that MCL/TNF-stimulated hDCs can establish functional GJ channels that participate in melanoma Ag transfer, facilitating Ag cross-presentation and an effective dendritic cell-mediated melanoma-specific T cell response. These results suggest that GJs formed between hDCs used in cancer vaccination protocols could be essentials for the establishment of a more efficient antitumor response.

In this study we have used the transgenic mouse model Tg2576 to analyze the involvement of anomalous loss of regulation of cdk5 and the stress kinases JNK and p38 in brain neuronal death as related to neurodegenerative disorders such as Alzheimer's disease. Previous studies on hippocampal cells led us to the discovery that the cdk5/p35 complex is activated in neurodegeneration, a finding that was confirmed later in the transgenic mouse model. Here we show a link between the cdk5 system and JNK and p38 phosphoproteins, as an alternative pathway to neuronal death. Brains of the Tg2576 transgenic mice overexpressing amyloid precursor protein exhibited immunoreactivity with the phosphoproteins p-JNK, p-p38 and the GTPase protein Rac1 surrounding neuritic plaques. A significant increase in the immunodetection of JNK and p38 phosphoproteins in the Tg2576 mouse compared with wild type controls confirmed these findings. The significant increase in co-immunoprecipitation of p-JNK, p-p38 and Rac1 proteins with cdk5 in the transgenic mouse provided evidence for these interactions. At the cellular level, p-JNK and cdk5 colocalized in the cytoplasm of the cell bodies and neurites of brain cortical areas of the transgenic mouse. The present evidence suggests a cellular link between the cdk5 system and the stress kinase JNK and p38 pathways in an in vivo model. This study sheds new light on the pathogenesis of neuronal degeneration processes such as those occurring in Alzheimer's disease.

Background: Alterations in connexin 43 (Cx43) expression and/or gap junction (GJ)-mediated intercellular communication are implicated in cancer pathogenesis. Herein, we have investigated the role of Cx43 in melanoma cell proliferation and apoptosis sensitivity in vitro, as well as metastatic capability and tumour growth in vivo. Methods: Connexin 43 expression levels, GJ coupling and proliferation rates were analysed in four different human melanoma cell lines. Furthermore, tumour growth and lung metastasis of high compared with low Cx43-expressing FMS cells were evaluated in vivo using a melanoma xenograft model. Results: Specific inhibition of Cx43 channel activity accelerated melanoma cell proliferation, whereas overexpression of Cx43 increased GJ coupling and reduced cell growth. Moreover, Cx43 overexpression in FMS cells increased basal and tumour necrosis factor-α-induced apoptosis and resulted in decreased melanoma tumour growth and lower number and size of metastatic foci in vivo. Conclusions: Our findings reveal an important role for Cx43 in intrinsically controlling melanoma growth, death and metastasis, and emphasise the potential use of compounds that selectively enhance Cx43 expression on melanoma in the future chemotherapy and/or immunotherapy protocols.

The AXIL computer program, aimed at x-ray spectral deconvolution, was extended in order to take into account the broadening of peaks of K-series radiation of high-Z elements (Z > 50) due to the increasing contribution of the natural linewidth to the peak width. This effect needs to be taken into account in energy-dispersive spectrometry of K-series radiation of heavy elements, such as W, Au, Pb and U. The so-called Voigt peak profile function was implemented into the AXIL computer code. Based on the published literature data, a library of natural atomic level widths was created, containing the data for K, L1, L2, L3 atomic level widths of elements up to Z = 94. The database was necessary to calculate the Voigt peak profiles. The AXIL program user interface and the fitting procedures were adapted, allowing us to perform spectral evaluation using either Gaussian or Voigt peak profiles. The relevance of fitting the x-ray spectra of K-series radiation of high-Z elements with the Voigt peak shape model versus Gaussian peak approximation was thoroughly examined with the aid of simulated and measured spectra. The experimental x-ray spectra were measured using a high-voltage x-ray tube spectrometer utilizing a 130 kV/65 W tungsten anode x-ray tube and an ultra-low energy germanium detector. The results obtained showed that when using the Gaussian peak model the calculated peak areas and fitted peak widths are systematically biased. It was found that the value of the systematic error is a linear function of the ratio of the natural linewidth to the spectrometer energy resolution at the peak position. Copyright © 2001 John Wiley & Sons, Ltd.

The neuronal cytoskeleton regulates numerous processes that occur in normal homeostasis. Under pathological conditions such as those of Alzheimer's disease (AD), major alterations in cytoskeleton organization have been observed and changes in both microtubules and actin filaments have been reported. Many neurodegenerative consequences of AD are linked to the production and accumulation of amyloid peptides (Aβ) and their oligomers, produced from the internal cleavage of the amyloid-β protein precursor. We previously reported that fibrillar Aβ1-42 (fAβ) treatment of hippocampal neurons induced an increase in Rac1 and Cdc42 activities linking fAβ effects with changes in actin dynamics. Here we show fAβ-induces increased activity of PAK1 and cyclin-dependent kinase 5, and that p21-activated kinase (PAK1) activation targets the LIMK1-cofilin signaling pathway. Increased cofilin dephosphorylation under conditions of enhanced LIM-Kinase 1 (LIMK1) activity suggests that fAβ co-stimulates bifurcating pathways impacting cofilin phosphorylation. Overexpression of slingshot (SSH) prevents the augment of F-actin induced by fAβ after 24 h, suggesting that fAβ-induced changes in actin assembly involve both LIMK1 and SSH. These results suggest that fAb may alter the PAK1/LIMK1/cofilin axis and therefore actin organization in AD.

Purpose: This study characterizes, biologically and clinically, a novel type of dendritic cells (DC) produced in the short term and called tumor antigen–presenting cells (TAPCells). In particular, we identified factors present in a lysate derived from heat-shocked allogeneic melanoma cells (TRIMEL) that are associated with TAPCells' enhanced capability to induce CD8+ T-cell responses in vitro and in vaccinated melanoma patients. Experimental Design: First, extensive phenotypic and functional characterization of TAPCells was performed, followed by vaccination of 45 melanoma patients with four doses of TAPCells over a period of 2 months. Specific delayed-type hypersensitivity (DTH) reaction was analyzed posttreatment and correlated with overall survival rates. Furthermore, heat-shock (HS)-induced factors present in TRIMEL and their effects on DC activation were identified and studied. Results: TRIMEL induced a committed, mature, DC-like phenotype in TAPCells and effectively activated melanoma-specific CD4+ and CD8+ T cells. Clinically, 64% of vaccinated patients showed positive DTH reaction against TRIMEL, and this was associated with improved overall survival. HS treatment of tumor cells increased calreticulin (CRT) plasma membrane translocation and induced the release of high-mobility group box 1 proteins (HMGB1). Both CRT and HMGB1 mobilization were associated with enhanced TAPCells' maturation and antigen (Ag) cross-presentation, respectively. DTH infiltration analysis revealed the presence of CD8+/CD45RO+ T cells, thus confirming TAPCells' ability to cross-present Ags in vivo. Conclusions: Our results indicate that lysates derived from heat-shocked tumor cells are an optimal source of tumor-associated Ags, which are crucial for the generation of DCs with improved Ag cross-presentation capacity and clinically effective immunogenicity. Clin Cancer Res; 17(8); 2474–83. ©2011 AACR.

Natural-killer group 2, member D (NKG2D) binds to a variety of ligands, including the major histocompatibility complex (MHC) class I chain-related proteins (MIC) and UL16-binding proteins (ULBP). It is regarded as a co-activating receptor on NK cells, having an important role in the cell-mediated immune response to tumours. We studied the influence of interleukin (IL)-10 on the regulation of MIC and ULBP expression on melanoma cells, and its effect on the cytotoxic function of NK cells in vitro. Here, we show that, in the presence of IL-10, FMS mel and BL mel cell lines decreased MICA and ULBP2 surface expression, whereas MHC class I did not change substantially on the cell surface. MICA mRNA levels decreased in IL-10-treated FMS and IL-10-transduced BL cell lines. Interestingly, we observed that MICB surface expression and its mRNA levels increased upon IL-10 treatment in a melanoma cell line. These changes in NKG2D ligands surface expression patterns owing to IL-10 treatment resulted in an effect on lysis susceptibility mediated by lymphocyte-activated killer cells, as tumour cell lines that displayed a higher decrease of MICA on their surface had lower levels of lysis. In addition, expression of CD107a was downregulated on the surface of NK cells following stimulation with IL-10-treated FMS cells. Our results suggest a novel function for IL-10 in the modulation of NKG2D ligand expression and in the control of cytotoxicity mediated by NKG2D/NKG2D ligand axis.

Rearrangement of the cytoskeleton in T cells plays a critical role in the organization of a complex signaling interface referred to as immunologic synapse (IS). Surprisingly, the contribution of antigen presenting cells, in particular dendritic cells (DCs), to the structure and function of the IS has not been investigated in as much detail. We have used a natural model of cytoskeletal dysfunction caused by deficiency of the Wiskott-Aldrich syndrome protein (WASp) to explore the contribution of the DC cytoskeleton to IS formation and to T-cell priming. In an antigen-specific system, T-DC contacts were found to be less stable when DCs alone lacked WASp, and associated with multiple defects of IS structure. As a consequence, DCs were unable to support normal IL-12 secretion, and events downstream of TCR signaling were abrogated, including increased calcium flux, microtubule organizing center (MTOC) polarization, phosphorylation of ZAP-70, and T-cell proliferation. Formation of an effective signaling interface is therefore dependent on active cytoskeletal rearrangements in DCs even when T cells are functionally competent. Deficiency of DC-mediated activities may contribute significantly to the varied immunodysregulation observed in patients with WAS, and also in those with limited myeloid reconstitution after allogeneic hematopoietic stem cell transplantation.

We have previously demonstrated that IT9302, a nonameric peptide homologous to the C-terminal domain of human IL-10, mimics several effects of the cytokine including down-regulation of the antigen presentation machinery and increased sensitivity of tumor cells to NK-mediated lysis. In the present report, we have explored a potential therapeutic utility for IT9302 related to the ex vivo production of tolerogenic dendritic cells (DCs). Our results indicate that IT9302 impedes human monocyte response to differentiation factors and reduces antigen presentation and co-stimulatory capacity by DCs. Additionally, peptide-treated DCs show impaired capacity to stimulate T-cell proliferation and IFN-gamma production. IT9302 exerts its effect through mechanisms, in part, distinct from IL-10, involving STAT3 inactivation and NF-kappa B intracellular pathway. IT9302-treated DCs display increased expression of membrane-associated TGF-beta, linked to a more effective induction of foxp3+ regulatory T cells. These results illustrate for the first time that a short synthetic peptide can promote monocytes differentiation to tolerogenic DCs with therapeutic potential for the treatment of autoimmune and transplantation-related immunopathologic disease. (c) 2011 Elsevier GmbH. All rights reserved.

Gap junction (GJ) mediates intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models, we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was Ag specific and time dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43-specific inhibitors were used to prove that intercellular communication between T cells and dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca2+ responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the dendritic cell–T cell signaling machinery that regulates T cell activation.

Purpose The aim of this work was to assess immunologic response, disease progression, and post-treatment survival of melanoma patients vaccinated with autologous dendritic cells (DCs) pulsed with a novel allogeneic cell lysate (TRIMEL) derived from three melanoma cell lines. Patients and Methods Forty-three stage IV and seven stage III patients were vaccinated four times with TRIMEL/DC vaccine. Specific delayed type IV hypersensitivity (DTH) reaction, ex vivo cytokine production, and regulatory T-cell populations were determined. Overall survival and disease progression rates were analyzed using Kaplan-Meier curves and compared with historical records. Results The overall survival for stage IV patients was 15 months. More than 60% of patients showed DTH-positive reaction against the TRIMEL. Stage IV/DTH-positive patients displayed a median survival of 33 months compared with 11 months observed for DTH-negative patients (P = .0014). All stage III treated patients were DTH positive and remained alive and tumor free for a median follow-up period of 48 months (range, 33 to 64 months). DTH-positive patients showed a marked reduction in the proportion of CD4+ transforming growth factor (TGF) β+ regulatory T cells compared to DTH-negative patients (1.54% v 5.78%; P < .0001). Conclusion Our findings strongly suggest that TRIMEL-pulsed DCs provide a standardized and widely applicable source of melanoma antigens, very effective in evoking antimelanoma immune response. To our knowledge, this is the first report describing a correlation between vaccine-induced reduction of CD4+TGFβ+ regulatory T cells and in vivo antimelanoma immune response associated to improved patient survival and disease stability.

Gap junctions (GJs) are highly specialized membrane structures which allow the passage of small molecules and ions between neighboring cells. Intercellular communication via GJs is a crucial mechanism that plays a central role in several pathologies. This review focuses on: i) the role of connexins (Cxs, transmembrane proteins that form GJ channels) in the pathophysiology of neuronal injury after brain hypoxia-ischemia, ii) the opposing theories regarding whether Cxs are protective agents or contribute to the spread of damage, and iii) recent patent applications and registrations showing Cxs as key targets in regulating GJ-mediated intercellular communication.

The role of cytokines in modulating the formation of new tumors is mediated by their ability to regulate antigen-specific anti-tumor responses and by the activation of non-specific mechanisms, including those involved in the processes of inflammation and innate resistance. Cytokines may influence the growth of tumors by acting directly on tumor cells as growth promoting or growth inhibiting factors or indirectly by attracting inflammatory cell types and affecting angiogenesis. Due to the potency and complexity of cytokine activity against tumor growth, the improvement of cloning techniques and the availability of recombinant forms of different cytokines, a great effort has been made in the recent years to exploit this anti-tumor potential for cancer therapy. This important goal has been difficult to achieve in most cases due to toxicity of most cytokines which could not be dissociated from their anti-tumoral functions. Nevertheless, if well designed, treatment protocols and/or modifications of the cytokine molecules may in some situations augment the anti-tumor effects while limiting the toxicity. One of these molecular approaches could be the design of peptides containing the functional domain of certain cytokines, exemplified by IT9302, a peptide homologous to the functional domain of IL-10, which has demonstrated to increase tumor NK cell sensitivity. (c) 2007 Elsevier Ltd. All rights reserved.

A number of psychiatric and neurodegenerative disorders, such as Alzheimer's disease, are characterized by abnormalities in the neuronal cytoskeleton. Here, we find that the enhancement in actin polymerization induced by fibrillar amyloid-beta peptide (Aβ) is associated with increased activity of Rac1/Cdc42 Rho GTPases. Rac1 upregulation involves the participation of Tiam1, a Rac guanine-nucleotide exchange factor, where Aβ exposure leads to Tiam1 activation by a Ca2+-dependent mechanism. These results point to Rho GTPases as one of the targets in Aβ-induced neurodegeneration in Alzheimer's disease pathology, with a role in mediating changes in the actin cytoskeletal dynamics.

PURPOSE. Uveal melanoma is the most common primary malignant ocular cancer in adults. This tumor has a distinct expression pattern of markers compared with cutaneous melanoma. MC1R is under study as a potential target for antitumor immunity. Because of the potential immunogenicity of MC1R, it is important to evaluate its expression on uveal melanomas.METHODS. Two novel monoclonal antibodies (MP1.1C11 and MP1.1B7) were used to examine the expression of MC1R in uveal melanomas. Tissue samples obtained from 17 patients were analyzed for expression of MC1R by immunohistochemistry. Additionally, uveal melanoma cell lines were treated with proinflammatory cytokines, after which MC1R cell surface expression was analyzed by flow cytometry.RESULTS. Results demonstrated that MC1R is expressed by uveal melanoma to a significantly greater extent than other melanoma markers. With the use of MP1.1C11 or MP1.1B7, MC1R was detected in 95% of the tested melanoma tissues, including one liver metastasis. In contrast, MART-1, S100-specific protein, and gp-100 were only expressed by 66%, 33%, and 67% of the analyzed samples, respectively. Results also demonstrated that even though MC1R is mainly located intracellularly, its cell surface expression can be promoted by cytokines such as IFN-gamma, TNF-alpha, IL-4, and IL-10.CONCLUSIONS. These observations support the inclusion of MC1R in the panel of markers for the diagnosis of uveal melanoma. Therapeutic use of MC1R-specific antibodies targeting cytokine-induced MC1R potentially requires expression of the target molecule on the surfaces of tumor cells. Data presented here support MC1R as a new marker and a putative therapeutic target for uveal melanoma.

A portable X-ray diffraction (XRD) and X-ray fluorescence (XRF) system, based on energy dispersive detection in reflection geometry, has been developed for the non-invasive study of cultural heritage materials. This analytical system is an open-work platform based on a low-power miniaturized X-ray tube (Ag anode), a single Si-drift detector, and a set of collimators that have been mounted on a compact and versatile manual or motorized stage, which allows independent and precise angular positioning of both source and detector and the study of complex geometry objects. The design of two instruments and experimental setups, and their advantages are discussed. The XRF and XRD analytical performance has been tested through the analysis of standard reference materials and some applications, including provenance study of obsidian and ‘green stone’ artifacts, identification of pigments in a model of fresco painting, and chemical element analysis of bones. The main advantages of portable energy dispersive X-ray diffraction systems compared with commonly used angle dispersive X-ray diffraction (ADXRD) instruments are shorter measurement times and compactness. Moreover, optimum XRF spectra can be collected because a polychromatic beam is used instead of a (quasi) monochromatic beam as in an angle dispersive X-ray diffraction-X-ray fluorescence portable system. Results demonstrate that it is possible to identify the main crystalline phases in a measurement time of 100 s–600 s and the system is sensible enough for answering common questions, as the ones presented, in the cultural heritage field. Copyright © 2015 John Wiley & Sons, Ltd.

Immunotherapy has become a novel therapeutic alternative for various kinds of tumours. Recently, we have finalized the first phase I clinical study in Chile for the treatment of advanced malignant melanoma, using dendritic cells (DCs) loaded with allogencic melanoma cell lysate. This study included 20 patients and the obtained results, pioneer in Latin America, showed that DC-based immunotherapy is innocuous, even provided in combination with IL-2. In addition, immunological responses were detected in 50% of the treated patients, establishing a positive correlation between the delayed type hypersensitivity (DTH) reaction, which indicates induction of in vivo immunological memory, and patients surviving. Nevertheless, objective clinical responses in vaccinated patients are still insufficient. Only sporadic objective metastasis regressions have been registered and an important proportion of the treated patients did not respond, or their responses were weak. Several strategies have been described to be used by tumours to escape from the immune response. Actually, we have demonstrated that IL-10 inhibits antigen presentation in melanoma, reducing tumour sensitivity to melanoma-specific cytotoxic T lymphocytes (CTLs). Regulation of the immunological response by inhibitory cells could be another possible cause of clinical unresponsiveness. Lately, the existence of subpopulations of regulatory T lymphocytes (RTL) able to limit the immune response in a specific form has been established, specially inhibiting the proliferation and activity of CD4+ and CD8+ effector T lymphocytes. These cellular subpopulations, mostly CD4(+)/CD25(+)/Foxp3(+) T lymphocytes (Treg) of thymic origin, or TR1 lymphocytes able to release IL-10, and tumour growth factor beta (TGF-beta) producing TH3 lymphocytes, would be accumulated in the body during tumour growth, inhibiting the immune response. In relation to RTL and cancer, evidence indicates that Treg cell numbers are increased in blood and other tissues in different types of cancer. Additionally, it has been demonstrated that in patients with refractory metastatic melanoma, the adoptive transference of anti-tumour CD8+ T lymphocytes after non-myeloablative chemotherapy was able to induce important tumour regressions that would be due to elimination of RTL populations. Additionally, chemotherapeutical drugs like decarbazine, besides their effect on tumour proliferation, also have an immunosuppressive effect on T lymphocyte populations, as well as on accumulated RTL. In this article, a novel strategy for the study of RTL is proposed, including potential therapeutic innovations, which is being pioneered in current clinical trials. (c) 2005 Elsevier GmbH. All rights reserved.

The diagnosis of Alzheimer's disease (AD) is mainly performed by excluding other disorders with similar clinical features. In addition, an analysis of symptoms and signs, blood studies and brain imaging are major ingredients of the clinical diagnostic work-up. However, the diagnosis based on these instruments is unsatisfactory, indicating the need of a highly sensitive and reliable approaches, selective for AD and based on biological markers. Ideally, such markers should reflect the pathophysiological mechanisms of AD, which according to the current hypotheses, derive from the actions of two major protein aggregates, the extracellular beta-amyloid (Abeta) plaques and the neurofibrillary tangles. Since AD is a multifactorial disease, other factors that cause neuronal insult and that contribute to neuronal degeneration in AD include free radical and oxidative stress promoting molecules, proinflammatory cytokines and neurotoxic agents. In this context, the search for anomalous levels or changes in the molecular patterns of Abeta(1-42) or Abeta(1-40), hyperphosphorylated tau isoforms, oxidation products in the cell or cytokines such as interleukin-1 or 6 facilitates the selection of biomarkers in AD. There is clear evidence that the cerebrospinal fluid (CSF) levels of beta(1-42) are significantly reduced in AD patients as compared with senile controls, while increased levels of tau have been revealed. The CSF levels of these proteins reflect their metabolism in the central nervous system. Approaches using ELISA and immunochemical methods for the quantification of these markers in CSF have been preferentially used. Diagnosis criteria and number of patients exhibits variations in the different reports, while clinico-pathological studies are scarce. An increasing number of studies suggest that supplementary use of these CSF markers preferably in combination, adds to the accuracy of an AD diagnosis.