Folate receptor alpha in ovarian cancer tissue and patient serum is associated with disease burden and treatment outcomes.

BACKGROUND: Survival rates for ovarian cancer remain poor, and monitoring and prediction of therapeutic response may benefit from additional markers. Ovarian cancers frequently overexpress Folate Receptor alpha (FRα) and the soluble receptor (sFRα) is measurable in blood. Here we investigated sFRα as a potential biomarker. METHODS: We evaluated sFRα longitudinally, before and during neo-adjuvant, adjuvant and palliative therapies, and tumour FRα expression status by immunohistrochemistry. The impact of free FRα on the efficacy of anti-FRα treatments was evaluated by an antibody-dependent cellular cytotoxicity assay. RESULTS: Membrane and/or cytoplasmic FRα staining were observed in 52.7% tumours from 316 ovarian cancer patients with diverse histotypes. Circulating sFRα levels were significantly higher in patients, compared to healthy volunteers, specifically in patients sampled prior to neoadjuvant and palliative treatments. sFRα was associated with FRα cell membrane expression in the tumour. sFRα levels decreased alongside concurrent tumour burden in patients receiving standard therapies. High concentrations of sFRα partly reduced anti-FRα antibody tumour cell killing, an effect overcome by increased antibody doses. CONCLUSIONS: sFRα may present a non-invasive marker for tumour FRα expression, with the potential for monitoring patient response to treatment. Larger, prospective studies should evaluate FRα for assessing disease burden and response to systemic treatments.

COVID-19 Detection Using Photoplethysmography and Neural Networks.

The early identification of microvascular changes in patients with Coronavirus Disease 2019 (COVID-19) may offer an important clinical opportunity. This study aimed to define a method, based on deep learning approaches, for the identification of COVID-19 patients from the analysis of the raw PPG signal, acquired with a pulse oximeter. To develop the method, we acquired the PPG signal of 93 COVID-19 patients and 90 healthy control subjects using a finger pulse oximeter. To select the good quality portions of the signal, we developed a template-matching method that excludes samples corrupted by noise or motion artefacts. These samples were subsequently used to develop a custom convolutional neural network model. The model accepts PPG signal segments as input and performs a binary classification between COVID-19 and control samples. The proposed model showed good performance in identifying COVID-19 patients, achieving 83.86% accuracy and 84.30% sensitivity (hold-out validation) on test data. The obtained results indicate that photoplethysmography may be a useful tool for microcirculation assessment and early recognition of SARS-CoV-2-induced microvascular changes. In addition, such a noninvasive and low-cost method is well suited for the development of a user-friendly system, potentially applicable even in resource-limited healthcare settings.

Improvising resilience: The unfolding of resilient leadership in COVID-19 times.

The spread of the COVID-19 pandemic in Italy has had disastrous effects on the national economy. The hospitality sector has experienced a significant impact from the crisis: starting from March 2020 it has literally collapsed. Experts believe it will take three years for the sector to recover. Confronted with a dramatic uncertainty, which imposed rapid action, hospitality leaders need to nurture resilience. To enrich current understanding of the way resilient leadership unfolds to respond to jolts, we draw on an exploratory qualitative research involving Italian hotel managers. Following in-depth interviews, we show that resilient leadership and improvisation are deeply interconnected. Their interdependence entails two practices, namely gardening and learning. This suggests a paradoxical tension: to exercise resilience, leaders need to be at the same time in the system, by actively learning from events, and outside the system, by zooming out as they focus on ongoing planning for the next move.

A Blocking Group Scan Using a Spherical Organometallic Complex Identifies an Unprecedented Binding Mode with Potent Activity In Vitro and In Vivo for the Opioid Peptide Dermorphin.

Herein, the selective enforcement of one particular receptor-ligand interaction between specific domains of the µ-selective opioid peptide dermorphin and the µ opioid receptor is presented. For this, a blocking group scan is described which exploits the steric demand of a bis(quinolinylmethyl)amine rhenium(I) tricarbonyl complex conjugated to a number of different, strategically chosen positions of dermorphin. The prepared peptide conjugates lead to the discovery of two different binding modes: An expected N-terminal binding mode corresponds to the established view of opioid peptide binding, whereas an unexpected C-terminal binding mode is newly discovered. Surprisingly, both binding modes provide high affinity and agonistic activity at the µ opioid receptor in vitro. Furthermore, the unprecedented C-terminal binding mode shows potent dose-dependent antinociception in vivo. Finally, in silico docking studies support receptor activation by both dermorphin binding modes and suggest a biological relevance for dermorphin itself. Relevant ligand-protein interactions are similar for both binding modes, which is in line with previous protein mutation studies.

Aldehyde dehydrogenase and estrogen receptor define a hierarchy of cellular differentiation in the normal human mammary epithelium.

INTRODUCTION: Although estrogen and progesterone play a key role in normal mammary development and in breast cancer, the potential for proliferation and lineage differentiation as well as origin of cells that express the estrogen receptor (ER) in normal breast epithelium are not known. Some evidence suggests that normal human mammary stem/progenitor cells are ER-, but the identity of these cells and the cellular hierarchy of breast epithelium are still subjects of controversy. It is likely that elucidation of these aspects will bring insight into the cellular origin of breast cancer subtypes. METHODS: We used fluorescence-activated cell sorting of primary human mammary epithelial cells along with in vitro and in vivo functional assays to examine the hierarchic relation between cells with aldehyde dehydrogenase enzymatic activity (ALDH+ cells) and ER+ cells in the normal human breast epithelium. We assessed the proliferation and lineage differentiation potential of these cells in vitro and in vivo. A gene reporter assay was used to separate live ER+ and ER- mammary epithelial cells. With shRNA-mediated knockdown, we investigated the role of ALDH isoforms in the functionality of mammary epithelial progenitor cells. RESULTS: We describe a cellular hierarchy in the normal human mammary gland in which ER-/ALDH+ cells with functional properties of stem/progenitor cells generate ER+ progenitor cells, which in turn give rise to cells of luminal lineage. We show that the ALDH1A1 isoform, through its function in the retinoic acid metabolism, affects the proliferation and/or early differentiation of stem/progenitor cells and is important for branching morphogenesis. CONCLUSIONS: This study presents direct evidence that ER+ cells are generated by ER-/ALDH+ stem/progenitor cells. We also show that ER+ cells are able to generate cell progeny of luminal lineage in vitro and in vivo. Loss of ALDH1A1 function impairs this process, as well as branching morphogenesis and clonogenicity in suspension culture. This latter effect is reversed by treatment with retinoic acid.

Are all hands-on activities equally effective? Effect of using plastic models, organ dissections, and virtual dissections on student learning and perceptions.

This study investigated the impact of three commonly used cardiovascular model-assisted activities on student learning and student attitudes and perspectives about science. College students enrolled in a Human Anatomy and Physiology course were randomly assigned to one of three experimental groups (organ dissections, virtual dissections, or plastic models). Each group received a 15-min lecture followed by a 45-min activity with one of the treatments. Immediately after the lesson and then 2 mo later, students were tested on anatomy and physiology knowledge and completed an attitude survey. Students who used plastic models achieved significantly higher overall scores on both the initial and followup exams than students who performed organ or virtual dissections. On the initial exam, students in the plastic model and organ dissection treatments scored higher on anatomy questions than students who performed virtual dissections. Students in the plastic model group scored higher than students who performed organ dissections on physiology questions. On the followup exam, when asked anatomy questions, students in the plastic model group scored higher than dissection students and virtual dissection students. On attitude surveys, organ dissections had higher perceived value and were requested for inclusion in curricula twice as often as any other activity. Students who performed organ dissections were more likely than the other treatment groups to agree with the statement that "science is fun," suggesting that organ dissections may promote positive attitudes toward science. The findings of this study provide evidence for the importance of multiple types of hands-on activities in anatomy laboratory courses.

Kinetics of nuclear phosphorylation (γ-H2AX) in human lymphocytes treated in vitro with UVB, bleomycin and mitomycin C.

After double-strand break induction, formation of γ-H2AX foci due to phosphorylation at Ser-139 of histone H2AX represents an early event of the DNA damage response (DDR). γ-H2AX foci are then rapidly dephosphorylated as signal for the subsequent recruitment of effector proteins. The induction and disappearance of the foci can be, therefore, used to monitor the functioning of the DDR machinery in a cell population exposed to genotoxic stress. Here, we investigated the time-course of γ-H2AX in unstimulated or cultured peripheral lymphocytes in vitro treated with UVB, bleomycin and mitomycin C (MMC). Once the mutagen exposure was performed, cells were harvested at different interval times from 0.5 to 5h. The results show that (i) in 20-h stimulated peripheral lymphocytes, UVB irradiation caused extensive and dose-dependent increases in nuclear phosphorylation, and disappearance of γ-H2AX foci progressed, proportionally to the UV fluence, with increasing the harvesting time; (ii) UVB-exposed G0 cells cultured for 20-h post-irradiation displayed low amounts of DNA phosphorylation, depicting a time-course in which the maximum effect was reached at 0.5h and dephosphorylation started after 1h; (iii) treatment of unstimulated lymphocytes with bleomycin sulphate induced an increase in nuclear phosphorylation of several folds higher than that of untreated cells, depicting kinetics comparable to those observed for UVB-exposed G1 cells; (iv) in stimulated cells, MMC caused a severe and dose-dependent high degree of H2AX phosphorylation together with a very slower kinetic of dephosphorylation with respect to the other experimental treatments. This study confirms the feasibility of the γ-H2AX focus assay as a genotoxic end-point and supports the view that the proposed type of analysis should be introduced in biomonitoring studies of human populations. This could also represent a feasible and useful tool in the screening and diagnosis of precancerous states or very early stages of other diseases.

Anti-cancer pro-inflammatory effects of an IgE antibody targeting the melanoma-associated antigen chondroitin sulfate proteoglycan 4.

Outcomes for half of patients with melanoma remain poor despite standard-of-care checkpoint inhibitor therapies. The prevalence of the melanoma-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) expression is ~70%, therefore effective immunotherapies directed at CSPG4 could benefit many patients. Since IgE exerts potent immune-activating functions in tissues, we engineer a monoclonal IgE antibody with human constant domains recognizing CSPG4 to target melanoma. CSPG4 IgE binds to human melanomas including metastases, mediates tumoricidal antibody-dependent cellular cytotoxicity and stimulates human IgE Fc-receptor-expressing monocytes towards pro-inflammatory phenotypes. IgE demonstrates anti-tumor activity in human melanoma xenograft models engrafted with human effector cells and is associated with enhanced macrophage infiltration, enriched monocyte and macrophage gene signatures and pro-inflammatory signaling pathways in the tumor microenvironment. IgE prolongs the survival of patient-derived xenograft-bearing mice reconstituted with autologous immune cells. No ex vivo activation of basophils in patient blood is measured in the presence of CSPG4 IgE. Our findings support a promising IgE-based immunotherapy for melanoma.

Effects of spindle poisons in peripheral human lymphocytes by the in vitro cytokinesis-block micronucleus assay.

The search for micronuclei (MN) in binucleated cells is not always the best choice to recognize microtubule-perturbing agents, as they give rise to (micronucleated) mononucleated cells, mainly via mitotic slippage. We therefore treated peripheral lymphocytes with vincristine (VCR), nocodazole (NOC) and colcemid (COL): (i) to quantify the formation of MN in mononucleated cells and the occurrence of abnormal mitoses (c-anaphases, endoreduplicated or tetraploid metaphases); (ii) to investigate the role of cytokinesis inhibition in determining or modulating the cytogenetic effects induced by the spindle poisons (we used either cytochalasin B (cyt B) or latrunculin A, a cytokinesis inhibitor that acts differently as compared with cyt B); (iii) to assess the ploidy of cells bearing MN by fluorescence in situ hybridisation (FISH) analysis; and (iv) to evaluate the levels of the mitotic arrest deficient (MAD2) protein, that blocks the cell at the metaphase-anaphase transition, by immunoblotting. We observed the induction of numerous abnormal mitoses and tetraploid interphase nuclei, as well as of MN in mononucleated cells, a high percentage of which had a diploid complement. We also found that the effects were generally not dose but chemical dependent, where NOC was proven to be more effective than COL and VCR in inducing overall MN formation and, specifically, diploid micronucleated lymphocytes. Aneugens damaged cells to a greater extent in the presence of cytokinesis inhibitors rather than in their absence. MAD2 protein was expressed in controls to an extent reflecting the amount of lymphocytes which were initially in the G2/M transition phase. The same trend was seen in aneugen-treated cells where MAD2 levels decreased with increasing spindle poison concentration. Here, we demonstrate that micronucleated mononucleated cells and aberrant mitoses can be considered useful markers of exposure to aneugens-like spindle poisons causing preferentially, but not exclusively, mitotic slippage. Assessment of MAD2 levels can be used to confirm the cell-damaging activity of the compounds.

TRIM proteins in cancer.

Some members of the tripartite motif (TRIM/RBCC) protein family are thought to be important regulators of carcinogenesis. This is not surprising as the TRIM proteins are involved in several biological processes, such as cell growth, development and cellular differentiation and alteration of these proteins can affect transcriptional regulation, cell proliferation and apoptosis. In particular, four TRIM family genes are frequently translocated to other genes, generating fusion proteins implicated in cancer initiation and progression. Among these the most famous is the promyelocytic leukaemia gene PML, which encodes the protein TRIM19. PML is involved in the t(15;17) translocation that specifically occurs in Acute Promyelocytic Leukaemia (APL), resulting in a PML-retinoic acid receptor-alpha (PML-RARalpha) fusion protein. Other members of the TRIM family are linked to cancer development without being involved in chromosomal re-arrangements, possibly through ubiquitination or loss of tumour suppression functions. This chapter discusses the biological functions of TRIM proteins in cancer.

Detecting sepsis from photoplethysmography: strategies for dataset preparation.

Sepsis is one of the most frequent causes of death in Intensive Care Units, and its prognosis greatly depend on timeliness of diagnosis. MIMIC-III database is a frequent source of data for developing method for automatic sepsis detection. However, the heterogeneity of data jeopardize the feasibility of the task. In this work we propose a selection strategy for generating high quality data suitable for training a sepsis detection system based on the utilization of only plethysmographic data. Clinical relevance A system for detecting sepsis based only on PPG may be potentially at virtually no cost in any case clinicians suspect the possibility of developing sepsis.

Classifying sepsis from photoplethysmography.

Sepsis is a life-threatening organ dysfunction. It is caused by a dysregulated immune response to an infection and is one of the leading causes of death in the intensive care unit (ICU). Early detection and treatment of sepsis can increase the survival rate of patients. The use of devices such as the photoplethysmograph could allow the early evaluation in addition to continuous monitoring of septic patients. The aim of this study was to verify the possibility of detecting sepsis in patients from whom the photoplethysmographic signal was acquired via a pulse oximeter. In this work, we developed a deep learning-based model for sepsis identification. The model takes a single input, the photoplethysmographic signal acquired by pulse oximeter, and performs a binary classification between septic and nonseptic samples. To develop the method, we used MIMIC-III database, which contains data from ICU patients. Specifically, the selected dataset includes 85 septic subjects and 101 control subjects. The PPG signals acquired from these patients were segmented, processed and used as input for the developed model with the aim of identifying sepsis. The proposed method achieved an accuracy of 76.37% with a sensitivity of 70.95% and a specificity of 81.04% on the test set. As regards the ROC curve, the Area Under Curve reached a value of 0.842. The results of this study indicate how the plethysmographic signal can be used as a warning sign for the early detection of sepsis with the aim of reducing the time for diagnosis and therapeutic intervention. Furthermore, the proposed method is suitable for integration in continuous patient monitoring.

Immunotherapy using IgE or CAR T cells for cancers expressing the tumor antigen SLC3A2.

BACKGROUND: Cancer immunotherapy with monoclonal antibodies and chimeric antigen receptor (CAR) T cell therapies can benefit from selection of new targets with high levels of tumor specificity and from early assessments of efficacy and safety to derisk potential therapies. METHODS: Employing mass spectrometry, bioinformatics, immuno-mass spectrometry and CRISPR/Cas9 we identified the target of the tumor-specific SF-25 antibody. We engineered IgE and CAR T cell immunotherapies derived from the SF-25 clone and evaluated potential for cancer therapy. RESULTS: We identified the target of the SF-25 clone as the tumor-associated antigen SLC3A2, a cell surface protein with key roles in cancer metabolism. We generated IgE monoclonal antibody, and CAR T cell immunotherapies each recognizing SLC3A2. In concordance with preclinical and, more recently, clinical findings with the first-in-class IgE antibody MOv18 (recognizing the tumor-associated antigen Folate Receptor alpha), SF-25 IgE potentiated Fc-mediated effector functions against cancer cells in vitro and restricted human tumor xenograft growth in mice engrafted with human effector cells. The antibody did not trigger basophil activation in cancer patient blood ex vivo, suggesting failure to induce type I hypersensitivity, and supporting safe therapeutic administration. SLC3A2-specific CAR T cells demonstrated cytotoxicity against tumor cells, stimulated interferon-γ and interleukin-2 production in vitro. In vivo SLC3A2-specific CAR T cells significantly increased overall survival and reduced growth of subcutaneous PC3-LN3-luciferase xenografts. No weight loss, manifestations of cytokine release syndrome or graft-versus-host disease, were detected. CONCLUSIONS: These findings identify efficacious and potentially safe tumor-targeting of SLC3A2 with novel immune-activating antibody and genetically modified cell therapies.

A Novel Kindred with Familial Gastrointestinal Stromal Tumors Caused by a Rare KIT Germline Mutation (N655K): Clinico-Pathological Presentation and TKI Sensitivity.

Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the gastrointestinal tract, are characterized by activating mutations in KIT or PDGFRA genes. The vast majority of GISTs are sporadic, but rare hereditary forms have been reported, often featuring multifocality and younger age of onset. We here report the identification of a novel kindred affected by familial GIST caused by a KIT germline mutation in exon 13 (N655K). No family affected by hereditary GIST due to this KIT variant has been reported in literature so far. We were able to track the mutation in three members of the family (proband, mother, and second-degree cousin), all affected by multiple GISTs. Due to its rarity, the N655K variant is poorly characterized. We conducted in vitro drug sensitivity assays that indicated that most tyrosine kinase inhibitors (TKIs) currently included in the therapeutic armamentarium for GISTs have a limited inhibitory activity toward this mutation. However, when compared to a classical imatinib-resistant KIT mutation (T670I), N655K was slightly more sensitive to imatinib, and encouraging responses were observed with last-generation TKIs.

Basophils from Cancer Patients Respond to Immune Stimuli and Predict Clinical Outcome.

Basophils are involved in manifestations of hypersensitivity, however, the current understanding of their propensity for activation and their prognostic value in cancer patients remains unclear. As in healthy and atopic individuals, basophil populations were identified in blood from ovarian cancer patients (n = 53) with diverse tumor histologies and treatment histories. Ex vivo basophil activation was measured by CD63 expression using the basophil activation test (BAT). Irrespective of prior treatment, basophils could be activated by stimulation with IgE- (anti-FcεRI and anti-IgE) and non-IgE (fMLP) mediated triggers. Basophil activation was detected by ex vivo exposure to paclitaxel, but not to other anti-cancer therapies, in agreement with a clinical history of systemic hypersensitivity reactions to paclitaxel. Protein and gene expression analyses support the presence of basophils (CCR3, CD123, FcεRI) and activated basophils (CD63, CD203c, tryptase) in ovarian tumors. Greater numbers of circulating basophils, cells with greater capacity for ex vivo stimulation (n = 35), and gene signatures indicating the presence of activated basophils in tumors (n = 439) were each associated with improved survival in ovarian cancer. Circulating basophils in cancer patients respond to IgE- and non-IgE-mediated signals and could help identify hypersensitivity to therapeutic agents. Activated circulating and tumor-infiltrating basophils may be potential biomarkers in oncology.

IgE Activates Monocytes from Cancer Patients to Acquire a Pro-Inflammatory Phenotype.

IgE contributes to host-protective functions in parasitic and bacterial infections, often by monocyte and macrophage recruitment. We previously reported that monocytes contribute to tumour antigen-specific IgE-mediated tumour growth restriction in rodent models. Here, we investigate the impact of IgE stimulation on monocyte response, cellular signalling, secretory and tumour killing functions. IgE cross-linking on human monocytes with polyclonal antibodies to mimic formation of immune complexes induced upregulation of co-stimulatory (CD40, CD80, CD86), and reduced expression of regulatory (CD163, CD206, MerTK) monocyte markers. Cross-linking and tumour antigen-specific IgE antibody-dependent cellular cytotoxicity (ADCC) of cancer cells by cancer patient-derived monocytes triggered release of pro-inflammatory mediators (TNFα, MCP-1, IL-10, CXCL-10, IL-1ß, IL-6, IL-23). High intratumoural gene expression of these mediators was associated with favourable five-year overall survival in ovarian cancer. IgE cross-linking of trimeric FcεRI on monocytes stimulated the phosphorylation of intracellular protein kinases widely reported to be downstream of mast cell and basophil tetrameric FcεRI signalling. These included recently-identified FcεRI pathway kinases Fgr, STAT5, Yes and Lck, which we now associate with monocytes. Overall, anti-tumour IgE can potentiate pro-inflammatory signals, and prime tumour cell killing by human monocytes. These findings will inform the development of IgE monoclonal antibody therapies for cancer.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Nociceptin/orphanin FQ antagonizes lipopolysaccharide-stimulated proliferation, migration and inflammatory signaling in human glioblastoma U87 cells.

Glioblastoma is among the most aggressive brain tumors and has an exceedingly poor prognosis. Recently, the importance of the tumor microenvironment in glioblastoma cell growth and progression has been emphasized. Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and endogenous ligands originating from dying cells or the extracellular matrix involved in host defense and in inflammation. G-protein coupled receptors (GPCRs) have gained interest in anti-tumor drug discovery due to the role that they directly or indirectly play by transactivating other receptors, causing cell migration and proliferation. A proteomic analysis showed that the nociceptin receptor (NOPr) is among the GPCRs significantly expressed in glioblastoma cells, including U87 cells. We describe a novel role of the peptide nociceptin (N/OFQ), the endogenous ligand of the NOPr that counteracts cell migration, proliferation and increase in IL-1ß mRNA elicited by LPS via TLR4 in U87 glioblastoma cells. Signaling pathways through which N/OFQ inhibits LPS-mediated cell migration and elevation of [Ca2+]i require ß-arrestin 2 and are sensitive to TNFR-associated factor 6, c-Src and protein kinase C (PKC). LPS-induced cell proliferation and increase in IL-1ß mRNA are counteracted by N/OFQ via ß-arrestin 2, PKC and extracellular signal-regulated kinase 1/2; furthermore, the contributions of the transcription factors NF-kB and AP-1 were investigated. Independent of LPS, N/OFQ induces a significant increase in cell apoptosis. Contrary to what was observed in other cell models, a prolonged exposure to this endotoxin did not promote any tolerance of the cellular effects above described, including NOPr down-regulation while N/OFQ loses its inhibitory role.