NLRs and inflammasome signaling in opioid-induced hyperalgesia and tolerance.

We investigated the role that innate immunological signaling pathways, principally nod-like receptors (NLRs) and inflammasomes, in the manifestation of the contradictory outcomes associated with opioids, namely hyperalgesia, and tolerance. The utilization of opioids for pain management is prevalent; nonetheless, it frequently leads to an increased sensitivity to pain (hyperalgesia) and reduced efficacy of the medication (tolerance) over an extended period. This, therefore, represents a major challenge in the area of chronic pain treatment. Recent studies indicate that the aforementioned negative consequences are partially influenced by the stimulation of NLRs, specifically the NLRP3 inflammasome, and the subsequent assembly of the inflammasome. This process ultimately results in the generation of inflammatory cytokines and the occurrence of neuroinflammation and the pathogenesis of hyperalgesia. We also explored the putative downstream signaling cascades activated by NOD-like receptors (NLRs) and inflammasomes in response to opioid stimuli. Furthermore, we probed potential therapeutic targets for modifying opioid-induced hyperalgesia, with explicit emphasis on the activation of the NLRP3 inflammasome. Ultimately, our findings underscore the significance of conducting additional research in this area that includes an examination of the involvement of various NLRs, immune cells, and genetic variables in the development of opioid-induced hyperalgesia and tolerance. The present review provides substantial insight into the possible pathways contributing to the occurrence of hyperalgesia and tolerance in individuals taking opioids.

The potential interplay between opioid and the toll-like receptor 4 (TLR-4).

CONTEXT: Opioids are available for the management of severe and chronic pain. However, long-term use of high-dose opioids could lead to physiologic tolerance, hyperalgesia, gastrointestinal immobility, addiction, respiratory depression, tumor progression, and inhibition of the immune system. It seems some of these adverse effects of opioids might be induced by TLR-4 signaling. OBJECTIVE: The review aims to investigate the potential interplay between opioids and TLR-4 in CNS, gastrointestinal, cancer, and immune system. METHODS: The search of PubMed, Embase, Scopus, web of sciences, and Google scholar was performed for all relevant studies published. From a total of 513 papers obtained at the initial database search, publications including in silico, in vitro, and in vivo studies were selected for the review. RESULTS: A comprehensive review of studies indicated that using opioids for the reduction of pain might induce adverse effects such as analgesic tolerance, hyperalgesia, cancer progression, and suppression of the immune system. Some studies have indicated these effects may be due to a change in the level of expression and signaling pathway of TLR-4. The generalizability of the results was limited due to the inconsistency of findings. CONCLUSIONS: More studies are needed to clarify TLR-4-mediated opioid effects on the biology or stages of the disease as well as the role of different types of opioids, appropriate dosage, and exposure in various contexts. Designing the drug candidate and doing many formulation studies for different diseases and various stages of disease could be associated with effective treatment and pain management.

Evaluation of Expression Level of miR-3135b-5p in Blood Samples of Breast Cancer Patients Experiencing Chemotherapy-Induced Cardiotoxicity.

The efficacy of chemotherapeutics in the treatment of breast cancer is limited by cardiotoxicity, which could lead to irreversible heart failure. The evaluation of miRNA levels as a vital biomarker could predict cardiotoxicity induced by chemotherapy. According to our previous meta-analysis study on patients with heart failure, we found that miR-3135b had a significant increase in patients with heart failure. Therefore, the present study aimed to evaluate the expression level of miR-3135b in the blood sample of patients experiencing chemotherapy-induced cardiotoxicity. Blood samples were collected from breast cancer patients or breast cancer patients who had received chemotherapy and had not experienced any chemotherapy-induced cardiotoxicity (N = 37, control group) and breast cancer patients experiencing chemotherapy-induced cardiotoxicity after chemotherapy (N = 33). The expression level of miR-3135b was evaluated using real-time polymerase chain reaction (RT-PCR). The 2-ΔCt values of miR-3135b were compared between two groups. We observed a significant increase in the expression level of miR-3135b between patients experiencing chemotherapy-induced cardiotoxicity and the control group (P = 0.0001). Besides, the ejection fraction parameter was correlated with the expression level of miR-3135b (r = 0.5 and P = 0.0001). To sum up, miR-3135b might be useful as a promising circulating biomarker in predicting cardiotoxicity induced by chemotherapy. However, more studies are needed to validate miR-3135b as a biomarker for the diagnosis of chemotherapy-induced cardiotoxicity. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01075-3.

A meta-analysis of microRNA expression profiling studies in heart failure.

Heart failure (HF) is a major consequence of many cardiovascular diseases with high rate of morbidity and mortality. Early diagnosis and prevention are hampered by the lack of informative biomarkers. The aim of this study was to perform a meta-analysis of the miRNA expression profiling studies in HF to identify novel candidate biomarkers or/and therapeutic targets. A comprehensive literature search of the PubMed for miRNA expression studies related to HF was carried out. The vote counting and robust rank aggregation meta-analysis methods were used to identify significant meta-signatures of HF-miRs. The targets of HF-miRs were identified, and network construction and gene set enrichment analysis (GSEA) were performed to identify the genes and cognitive pathways most affected by the dysregulation of the miRNAs. The literature search identified forty-five miRNA expression studies related to CHF. Shared meta-signature was identified for 3 up-regulated (miR-21, miR-214, and miR-27b) and 13 down-regulated (miR-133a, miR-29a, miR-29b, miR-451, miR-185, miR-133b, miR-30e, miR-30b, miR-1, miR-150, miR-486, miR-149, and miR-16-5p) miRNAs. Network properties showed miR-29a, miR-21, miR-29b, miR-1, miR-16, miR-133a, and miR-133b have the most degree centrality. GESA identified functionally related sets of genes in signaling and community pathways in HF that are the targets of HF-miRs. The miRNA expression meta-analysis identified sixteen highly significant HF-miRs that are differentially expressed in HF. Further validation in large patient cohorts is required to confirm the significance of these miRs as HF biomarkers and therapeutic targets.

Human adipose derived stem cell exosomes enhance the neural differentiation of PC12 cells.

Human adipose stem cells (hADSCs) are proper cell sources for tissue regeneration. They mainly mediate their therapeutic effects through paracrine factors as exosomes. The exosomes contents are protein, lipid and RNA. Exosomes are effective in restoring the function of neurons and astrocytes in neurodegenerative diseases, and improve the therapeutic outcomes. We investigated the effect of hADSCs derived exosomes on survival and neural differentiation of PC12 cells in vitro. The isolated hADSCs, were characterized by flow cytometry. Exosomes were separated from hADSC-condition medium using Exo-spinTM kit and characterized by DLS and TEM. Then acridine orange staining was performed to confirm entrance of exosomes into PC12 cells. PC12 cells were treated with culture medium containing NGF and exosome. Cell viability was assessed by MTT assay, and neural differentiation by ICC technique and qRT-PCR. TEM and DLS data confirmed the isolation of exosomes according to their size (30-100 nm) and acridine orange staining indicated entrance of exosomes to target cells. MTT assay showed that cell viability was significantly increased in exosome treated group. ICC technique revealed that the expression of Map2 was superior in the exosome treated group. Based on qRT-PCR data, Map2 and ß-tub III gene expression was increased in the exosome treated group. Significant expression of Gfap was seen in the NGF and NGF/EXO treated groups. Present study indicated that hADSCs derived exosomes might enhance cell viability and promote neuronal differentiation and expression of mature neural marker in PC12 cells.

The expression level of hsa-miR-146a-5p in plasma-derived exosomes of patients with diffuse large B-cell lymphoma.

BACKGROUND: The standard treatment for patients with diffuse large B-cell lymphoma (DLBCL) had been rituximab-based immunochemotherapy. However, the biological and clinical heterogeneity within DLBCL seems to affect treatment outcome. Therefore, the evaluation of miRNA levels might be useful in predicting treatment response and relapse risk. miR-146a is a modulator of innate and acquired immunity and may play an important role in predicting treatment response. The aim of the present study was to compare the expression level of miR-146a in plasma-derived exosomes of responsive DLBCL patients (response to R-CHOP (Rituximab, and Cyclophosphamide, Hydroxydaunorubicin, Oncovine and Prednisone)), refractory DLBCL patients (resistant to R-CHOP), patients receiving R-CHOP, and healthy donors. MATERIALS AND METHODS: After the preparation of plasma and isolation of exosomes, the presence of plasma-derived exosome was confirmed by Zetaseizer, electron microscope, and Western blot. The patients' medical records were collected and analyzed. The expression level of exosomal miR-146a was evaluated in DLBCL patients and healthy donors using real-time polymerase chain reaction (PCR). The -ΔCt values of miR-146a were compared among responsive patients (n = 17), refractory patients (n = 16), patients receiving R-CHOP therapy (n = 15), and healthy donors (n = 6). RESULTS: The presence and size of plasma-derived exosomes were confirmed. Our findings did not show any significant difference in the expression level of exosomal miR-146a between DLBCL patients and healthy donors (P = 0.48). As well, the clinical and histopathological parameters were not correlated with the expression level of exosomal miR-146a or plasma miR-146a. The expression level of plasma miR-146 was lower than the expression level of exosomal miR-146 (P = 0.01). CONCLUSION: Exosomal miR-146a might be useful as a promising "liquid biopsy" biomarker in predicting treatment response and relapse risk; however, we could not find significant differences due to small sample size.

Antibodies to interferon beta in patients with multiple sclerosis receiving CinnoVex, rebif, and betaferon.

Treatment with interferon beta (IFN-ß) induces the production of binding antibodies (BAbs) and neutralizing antibodies (NAbs) in patients with multiple sclerosis (MS). NAbs against IFN-ß are associated with a loss of IFN-ß bioactivity and decreased clinical efficacy of the drug. The objective of this study was to evaluate the incidence and the prevalence of binding antibodies (BAbs) and neutralizing antibodies (NAbs) to IFN-ß in MS patients receiving CinnoVex, Rebif, or Betaferon. The presence of BAbs was studied in serum samples from 124 MS patients using one of these IFN-ß medications by ELISA. The NAbs against IFN-ß were measured in BAb-positive MS patients receiving IFN-ß using an MxA gene expression assay (real-time RT-PCR). Of the 124 patients, 36 (29.03%) had BAbs after at least 12 months of IFN-ß treatment. The proportion of BAb+ was 38.1% for Betaferon, 21.9% for Rebif, and 26.8% for CinnoVex. Five BAb-positive MS patients were lost to follow-up; thus 31 BAb-positive MS patients were studied for NAbs. NAbs were present in 25 (80.6%) of BAb-positive MS patients receiving IFN-ß. In conclusion, the three IFN-ß preparations have different degrees of immunogenicity.

Multiscale modeling of collective cell migration elucidates the mechanism underlying tumor-stromal interactions in different spatiotemporal scales.

Metastasis is the pathogenic spread of cancer cells from a primary tumor to a secondary site which happens at the late stages of cancer. It is caused by a variety of biological, chemical, and physical processes, such as molecular interactions, intercellular communications, and tissue-level activities. Complex interactions of cancer cells with their microenvironment components such as cancer associated fibroblasts (CAFs) and extracellular matrix (ECM) cause them to adopt an invasive phenotype that promotes tumor growth and migration. This paper presents a multiscale model for integrating a wide range of time and space interactions at the molecular, cellular, and tissue levels in a three-dimensional domain. The modeling procedure starts with presenting nonlinear dynamics of cancer cells and CAFs using ordinary differential equations based on TGFß, CXCL12, and LIF signaling pathways. Unknown kinetic parameters in these models are estimated using hybrid unscented Kalman filter and the models are validated using experimental data. Then, the principal role of CAFs on metastasis is revealed by spatial-temporal modeling of circulating signals throughout the TME. At this stage, the model has evolved into a coupled ODE-PDE system that is capable of determining cancer cells' status in one of the quiescent, proliferating or migratory conditions due to certain metastasis factors and ECM characteristics. At the tissue level, we consider a force-based framework to model the cancer cell proliferation and migration as the final step towards cancer cell metastasis. The ability of the multiscale model to depict cancer cells' behavior in different levels of modeling is confirmed by comparing its outputs with the results of RT PCR and wound scratch assay techniques. Performance evaluation of the model indicates that the proposed multiscale model can pave the way for improving the efficiency of therapeutic methods in metastasis prevention.

Exosomes and COVID-19: challenges and opportunities.

Coronavirus disease 2019 or COVID-19, starting from Wuhan, China, in December 2019, is a pandemic situation affecting millions worldwide and has exerted a huge burden on healthcare infrastructure. Therefore, there is an urgent need to understand the molecular mechanisms underlying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and design novel effective therapeutic strategies for combating this pandemic. In this regard, special attention has been paid to the exosomes. These nanoparticles are extracellular vesicles with critical function in the pathogenesis of several diseases including viral sepsis. Therefore, they may be involved in the pathogenesis of COVID-19 infection and also may be a way for transferring viral components and infecting other neighbor cells. Exosomes also can be considered as a therapeutic strategy for treating COVID-19 patients or used as a carrier for delivering effective therapeutic agents. Therefore, in this review, we discussed the biogenesis and contents of exosomes, their function in viral infection, and their potential as a therapeutic candidate in treating COVID-19.

Effects of Methadone on the Toll-like Receptor 4 Expression in Human Non-Small Cell Lung Carcinoma A549 Cell Line Using In-silico and In vitro Techniques.

Background: In this study, the effects of methadone and naloxone on the expression of toll-like receptor 4 (TLR4) gene have been evaluated in human non-small cell lung carcinoma A549 cell line migration using in-silico and in vitro techniques. Materials and Methods: Lung cancer A549 cell cultures were stimulated for 24 h with methadone (5, 10, and 20 µM) and naloxone (20 and 40 µM) concentrations. The level of TLR4 expression was determined by the quantitative real-time polymerase chain reaction. Migration of the A549 cells was investigated after a 4-h incubation period with methadone using the Boyden Chamber assay. Results: Migration rate of the A549 cells treated with 5 (P < 0.05) and 20 (P < 0.01) µM methadone was, respectively, increased and decreased with 20 µM naloxone (P < 0.05). Furthermore, the TLR4 expression was enhanced with 5 (P < 0.05) and 20 (P < 0.01) µM methadone and decreased with 20 (P < 0.05) and 40 µM naloxone (P < 0.01). In addition, in silico docking analysis revealed docking of methadone to MD-2 and TLR4. Conclusion: According to the present DATA, methadone affects the TLR4 expression. It may however cause adverse consequences by increasing the TLR4 expression. Therefore, the useful analgesic properties of methadone should be separated from the unwanted TLR4-mediated side effects.

Evaluation of Radiation and Ammonium Lactate Effects on Hyaluronic Acid Expression as a Pro-cancerous Factor in Supernatant and Exosome Isolated from Supernatant of Primary Mouse Fibroblast Cell Culture.

BACKGROUND: Previous studies show that aberrant synthesis of Hyaluronan accelerates tumor growth, angiogenesis, and metastasis. The fibroblasts are probably responsible for most of the hyaluronic acid (HA) accumulation in tumor microenvironment after radiotherapy. Our goal is to investigate and compare radiation and lactate effects on HA levels in supernatant and exosome isolated from supernatant of primary mouse fibroblast cell culture. METHODS: Fibroblast cells were prepared from skin of C57BL6 mouse. These cells were divided into three groups (no treatment, cells treated with 10 mM ammonium lactate, and irradiated cells). Then supernatant was harvested from FBS-free culture media after 48 h. Exosomes were purified by differential centrifugation (300 × g for 10 min, 2000 × g for 30 min, 16500 g for 30 min) and were pelleted by ultracentrifugation (150,000 × g for 180 min). Size of exosomes was determined using a Zetasizer. HA concentration measured using a HA ELISA Kit. Data were analyzed using one-way ANOVA. RESULTS: There was a significant increase in HA-coated exosomes isolated from supernatants of irradiated cells compared to untreated cell and cells treated with 10 mM ammonium lactate (P < 0.001). As well, there was a significant increase in the HA concentration in the supernatants of cells treated with 10 mM ammonium lactate relative to untreated cells and irradiated cells (P < 0.05). CONCLUSIONS: It seems that routine radiation therapy leads to massive shedding of HA-coated exosomes by normal fibroblast cells and thus exosomes-HA may contribute to tumor promotion and induce of the premetastatic niche.

Effect of Plasma-Derived Exosomes of Refractory/Relapsed or Responsive Patients with Diffuse Large B-Cell Lymphoma on Natural Killer Cells Functions.

OBJECTIVE: The purpose of this study was to investigate effect of plasma-derived exosomes of refractory/relapsed or responsive diffuse large B-cell lymphoma (DLBCL) patients on natural killer (NK) cell functions. MATERIALS AND METHODS: In this cross-sectional and experimental study, NK cells were purified from responsive patients (n=10) or refractory/relapsed patients (n=12) and healthy donors (n=12). NK cells were treated with plasma-derived exosomes of responsive or refractory/relapsed patients. We examined the expression levels of hsa-miR-155-5p, hsalet- 7g-5p, INPP5D(SHIP-1) and SOCS-1 in NK cells quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Percentages of NK cells expressing CD69, NKG2D and CD16, NK cell cytotoxicity and NK cell proliferation (using flow-cytometry) as well as interferon-gamma (IFN-γ) level in the supernatant of NK cells using ELISA were also investigated. RESULTS: We observed an increased level of hsa-miR-155-5p and a decreased level of SOCS-1 in NK cells treated with exosomes compared to untreated NK cell in healthy donors and DLBCL patients. An increase in hsa-miR-155-5p level was associated with an increased level of IFN-γ in healthy donors. The decreased levels of hsa-let-7g-5p were observed in NK cells treated with exosomes in comparison with untreated NK cells in DLBCL patients (P<0.05). There was no significant difference in the percentage of CD69+ NK cells and NKG2D+ NK cells in the absence or presence of exosomes of DLBCL patients in each group. Furthermore, we observed significant reduction of NK cell proliferation in DLBCL patients and healthy donors in the presence of exosomes of refractory/relapsed patients (P<0.05). A significant decrease was observed in cytotoxicity of NK cell in patients with DLBCL treated with exosomes of responsive patients. CONCLUSION: Our findings demonstrated adverse effect of plasma-derived exosomes of DLBCL patients on some functions of NK cell. It was also determined that low NK cell count might be associated with impaired response to R-CHOP and an increased recurrence risk of cancer.

Comparison of Expression Levels of miR-29b-3p and miR-326 in T Helper-1 and T Helper-17 Cells Isolated from Responsive and Non-responsive Relapsing-remitting Multiple Sclerosis Patients Treated with Interferon-beta.

T helper type 1 (Th1) and Th17 Cells with distinct cytokine profiles including interferon-gamma (IFN-γ) and interleukin 17 (IL-17) have a pivotal role in neuroinflammation and myelin destruction in the central nervous system (CNS) in MS. MicroRNA-29b (MiR-29b) and miR-326 contribute to regulating Th1 and Th17 differentiation and altered expression of the miRNAs could be associated with response to treatment in multiple sclerosis (MS). Therefore, our study aimed to evaluate the percentage of Th1 and Th17 and determining the expression levels of miR-29b-3p and miR-326 in these lymphocyte subpopulations between responsive and non-responsive to interferon beta (IFN-ß) therapy in relapsing-remitting multiple sclerosis (RRMS) patients. The present study was performed on 40 RRMS patients following treatment with IFN-ß. The percentage of Th1 cells and Th17 cells were determined by flow cytometry in responsive and non-responsive patients. The expression levels of miR-29b-3p and miR-326 were assessed in Th1 and Th17 cells by quantitative polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was applied to evaluate the plasma levels of IFN-γ and IL-17A. No significant difference was observed in the percentage of Th1 and Th17 cells as well as the expression levels of miR-29b-3p and miR-326 (in Th1 and Th17, respectively) in treated patients. Also, we did not find any significant difference in IFN-γ and IL-17A plasma concentration between responsive or non-responsive to IFN-ß therapy in patients with RRMS. IFN-ß may regulate other miRNAs in Th1 and Th17 cells than miR29b-3p and miR-326 in MS patients.

Evaluation of exosomal miR-155, let-7g and let-7i levels as a potential noninvasive biomarker among refractory/relapsed patients, responsive patients and patients receiving R-CHOP.

This study aimed to investigate and compare exosomal miR-155, let-7g and let-7i levels as a noninvasive biomarker among patients with refractory/relapsed or responsive DLBCL after R-CHOP treatment and patients receiving R-CHOP. Plasma was collected and exosomes were isolated from plasma. Exosomes confirmed by zeta-seizer, electron microscope, and western blot. Exosomes concentration was investigated by BCA assay. MiR-155, let-7g and let-7i levels were evaluated in plasma-derived exosomes by real-time PCR. Plasma IFN-γ and IL-4 level were measured by ELISA assay. We observed the significant increase in the exosomal miR-155 levels (p = .002) and exosomes concentration (p = .001) in refractory/relapsed patients compared to responsive patients and patients receiving R-CHOP. No association was not observed between exosomal miR-155 levels and IPI and disease stage. The significant decrease in IFN-γ levels was observed in patients receiving R-CHOP compared to refractory/relapsed or responsive patients (p=.001). Therefore, exosomal miR-155 might be useful as potential prognostic biomarkers to predict response to treatment in DLBCL patients.