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Objective. Natural killer (NK) cell-deficient mice are useful models in biomedical research. NOD/SCID mice have been used as a model of this type in research. However, the actual status of NK cells in NOD/SCID mice and CB17/SCID mice in comparison with that in BALB/c mice has not been sufficiently evaluated. Methods. Splenocytes from naïve or poly(I:C)-treated mice were isolated for phenotyping and analysis of cytotoxicity-related molecules and inhibitory receptors; for cytotoxicity assay, purified NK cells were also used. Results. The proportion of splenic NK cells did not differ significantly between NOD/SCID and CB17/SCID mice. The perforin levels in NK cells were similar between the poly(I:C)-treated CB17/SCID and NOD/SCID mice, while the granzyme B and NKG2A/C/E levels in NK cells from NOD/SCID mice were significantly lower than those from CB17/SCID mice. Moreover, the NKG2D and Ly49A levels in NK cells from NOD/SCID mice were higher than those from CB17/SCID. The splenocytes from CB17/SCID mice showed higher cytotoxicity than those from NOD/SCID mice, while the cytotoxicity of purified NK cells basically did not differ between the two strains. After in vitro stimulation with cytokines, the splenocytes from CB17/SCID mice showed higher IFN-γ production than those from NOD/SCID mice; however, NK cells did not. Conclusion. There was no significant difference in the proportion of splenic NK cells between CB17/SCID and NOD/SCID mice, and the function of NK cells was only partially compromised in NOD/SCID mice. Caution should be taken when considering the use of NOD/SCID mice as an NK-deficient model.

There is evidence that methionine enkephalin (MENK), an opioid peptide, promotes anti-tumor immune responses. In this study, the effect of MENK on colorectal cancer (CRC) and its mechanisms of action were examined in vivo. The intraperitoneal administration of 20 mg/kg MENK effectively inhibited MC38 subcutaneous colorectal tumor growth in mice. MENK inhibited tumor progression by increasing the immunogenicity and recognition of MC38 cells. MENK down-regulated the oncogene Kras and anti-apoptotic Bclxl and Bcl2, suppressed Il1b, Il6, iNOS, and Arg1 (encoding inflammatory cytokines), and increased Il17a and Il10 levels. MENK promoted a tumor suppressive state by decreasing the immune checkpoints Pd-1, Pd-l1, Lag3, Flgl1, and 2b4 in CRC. MENK also altered the immune status of the tumor immune microenvironment (TIME). It increased the infiltration of M1-type macrophages, CD8

Interleukin (IL)-10 family cytokines include IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B, and IL-29. These cytokines play crucial regulatory roles in various biological reactions and diseases. In recent years, several studies have shown that the IL-10 family plays a vital role in bone and joint diseases, including bone metabolic diseases, fractures, osteoarthritis, rheumatoid arthritis, and bone tumors. Herein, the recent progress on the regulatory role of IL-10 family of cytokines in the occurrence and development of bone and joint diseases has been summarized. This review will provide novel directions for immunotherapy of bone and joint diseases.

Immunotherapy for cervical carcinoma is becoming increasingly important recently. In these studies methionine enkephalin (menk) is shown to inhibit cervical tumor cell proliferation in vitro in association with an increase in the expression of apoptosis markers and mediators, including an increase in fas, caspase 8, and caspase 3 expression and intrinsic expression of the signaling pathway mediator bax. In vivo, tumor growth was restrained in mice xenotransplant model with typical pathological features of apoptosis. Furthermore, myeloid derived suppressor cells (MDSCs) had a significant decrease in circulation and in tumor site. In brief, these findings showed menk could inhibit tumor growth in vitro and in vivo, providing direction of further research and clinical application prospect.

The incidence and mortality of type 2 diabetes mellitus (T2DM) rank among the top ten worldwide. Emerging studies indicate pathological roles for the immune system in inflammation, insulin resistance and islet β-cell damage in subjects with T2DM. Methionine enkephalin (MENK) is present in endocrine cells of the pancreas and has been suggested to be an important mediator between the immune and neuroendocrine systems. Therefore, it may play a role in modulating insulin secretion from islet cells. Since little is known about the effect of MENK on T2DM, therefore it was the aim of this study to characterize the role and possible mechanism of action of MENK on plasma glucose and serum insulin levels in T2DM rats and INS-1 cells in vivo and in vitro. MENK significantly decreased the plasma glucose level and increased the serum insulin concentration in T2DM rats. It also increased the serum levels of the cytokines IL-5 and IL-10, while decreased TNF-α and IL-2 levels. We further confirmed that MENK regulated glucose metabolism by upregulating opioid receptor expression and modulating the IL-33/ST2 and MyD88-TRAF6-NF-κB p65 signaling pathways. Based on these results, an intraperitoneal injection of MENK represents a potentially new approach for T2DM.

The purpose of this study was to identify the modulatory effect of MENK on MDSCs and to investigate the relationship between this modulation and the expression of opioid receptors. Our results showed that MENK could inhibit the proliferation of MDSCs from both marine bone marrow and spleen. MENK also could promote the expression of opioid receptors MOR and DOR. MENK suppressed the PMN-MDSCs generated from splenocyte while up-regulated M-MDSCs. The stimulation of MENK increased the production of IL-4 secreted by MDSCs from slpenocytes. Our currently data indicated that MENK could suppress the accumulation of MDSCs in tumor-bearing mice via binding to and up-regulating expressions of subunits of opioid receptors. Therefore, it is concluded that MENK, through triggering opioid receptors could exert inhibiting modulation on MDSCs.

The antitumor effects of methionine enkephalin (MENK), also known as opioid growth factor (OGF), including its inhibitory effects on cutaneous squamous cell carcinoma (CSCC), have been established. In this study, we determined the precise mechanism by which MENK suppresses CSCC cell growth. In particular, MENK induced G0/G1 cell cycle arrest and promoted apoptosis in CSCC cells via the Bcl-2/Bax/Caspase-3 signaling pathway. Moreover, MENK reduced immunosuppression by downregulating the number of myeloid-derived suppressor cells (MDSCs) and regulating the polarization of tumor-associated macrophages from M2 to M1 in vivo. Furthermore, JAK2/STAT3, an important tumor-promotion and immunosuppression signaling pathway that is involved in MDSC expansion in tumors and macrophage polarization, was inhibited. These findings highlight the potential of the JAK2/STAT3 signaling pathway as a therapeutic target and suggest the clinical application of MENK for CSCC.

Highlights • LDN inhibited proliferation in cervical cancer. • LDN inhibited migration and invasion in cervical cancer cells. • LDN mediated the propagation property in cervical cancer through PI3K/AKT/mTOR signaling pathway., The incidence of cervical cancer is increasing annually worldwide. Low-dose naltrexone (LDN) has been reported to delay tumor progression, but the mechanism remains unclear. Here, we found that low-dose naltrexone could upregulate the expression of OGFr. Additionally, LDN could suppress the abilities of colony formation, migration and invasion in cervical cancer cells. LDN could also inhibit cervical cancer progression in mice model. Moreover, LDN indirectly reduced the expressions of PI3K, pAKT and mTOR in vitro and in vivo. Therefore, LDN may be considered a potential treatment option for cervical cancer.

Methionine enkephalin (MENK) has an important role in both neuroendocrine and immune systems. MENK was known as an opioid growth factor (OGF) for its growth regulatory characteristics. OGF interacts with the OGF receptor (OGFr) to inhibit DNA synthesis by upregulating p16 and/or p21, which delays the cell cycle transition from G0/G1 to S phase, and inhibits cell proliferation. In addition, OGF combines with OGFr in immune cells to exert its immunomodulatory activity and regulate immune function. OGF has been studied as an immunomodulator in a variety of autoimmune diseases, including multiple sclerosis, inflammatory bowel disease, diabetes and viral infections, and has been proven to relieve symptoms of certain diseases in animal and in vitro experiments. Also, OGF and OGFr have various anti-tumor molecular mechanisms. OGF can be used as the primary therapy alone or combined with other drugs to treat tumors. This article summarizes the research progress of OGF in immune-related diseases and cancer diseases.

Additional file 1: Figure S1. Quality confirmation of the peptides. Figure S2. Elevated expression of ECM1 in multiple tumours. Figure S3. Cytotoxicity of YL/DC-CTLs, LA/DC-CTLs, or LA/DC-NK cells against tumour cell lines. Figure S4. Cytotoxicity of YL/DC-CTLs, LA/DC-CTLs, or LA/DC-NK cells against primary tumour cells from HLA-A2.1+/ECM1+ patients. Figure S5. No cytotoxicity of LA- or YL-induced DC-CTLs or DC-NK cells on immune cells. Figure S6. ECM1-LA/DCs induce the activation of NK cells through the TLR4-p38-MICA/B pathway. Figure S7. The frequency of CD4+ T cells in splenocytes (n=3). Figure S8. There was no significant antitumour effect in the LA alone- or YL alone-treated group. Figure S9. No significant difference was detected in pathological examination of vital organs in xenograft mouse model. Figure S10. The KIR3DS1-activating pathway is not involved in the activation of NK cells by LA-pulsed DCs. Table S1. HLA-A alleles of tumour (or epithelium) cell lines. Table S2. Significant association of ECM1 mRNA expression with the malignant phenotype of various tumours. Table S3. Significant association of ECM1 protein expression with the malignant phenotype of various tumours. Table S4. Routine blood indexes were examined in different immune groups of HLA-A2.1 transgenic mice. Table S5. Main organ indexes are evaluated in different groups of HLA-A2.1 transgenic mice. Supplementary materials and methods.

CD8The immune Epitope database, Molecular Operating Environment software, and enzyme-linked immunosorbent assay were used for epitope identification. Flow cytometry, confocal microscopy, UPLC-QTOF-MS, and RNA-seq were utilized for evaluating immunity of PBMC-derived DCs, CD8We identified novel HLA-A2.1-restricted extracellular matrix protein 1(ECM1)-derived immunodominant epitopes in which LA induced a potent immune response. We also found that LA-loaded DCs upregulated the frequency of CD3LA-induced immune antitumour activity through DC cross-activation with CD8

Multiple sclerosis (MS) is a chronic demyelinating disease caused by central nervous system (CNS) inflammation and immune dysfunction, which often leaves patients with severe physical disabilities. Microglia function in the surveillance of the CNS, and an imbalance in the M1/M2 phenotypes of microglia contribute to the progression of MS. Recent studies indicate that exosomes secreted by bone marrow mesenchymal stem cells (BMSCs) play therapeutic roles in many autoimmune diseases and aid in tissue repair. However, it is not clear whether BMSC-derived exosomes can attenuate MS-associated inflammation and immune dysfunction, or how BMSC exosomes protect neurons. The experimental autoimmune encephalomyelitis (EAE) rat model was used to investigate the effect of exosomes on microglia polarization and inflammation in CNS. The results showed that exosome treatment significantly decreased neural behavioral scores, reduced the infiltration of inflammatory cells into the CNS, and decreased demyelination in comparison to untreated EAE rats. In addition, exosome treatment resulted in significant increases in the levels of M2-related cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β, whereas M1-related tumor necrosis factor (TNF)-α and IL-12 levels decreased significantly. Moreover, compared with the untreated EAE group, the exosome group displayed significantly increased protein and mRNA expression levels of M2 phenotype markers, whereas M1 marker expression decreased. Our findings were further confirmed in an in vitro HAPI microglia cell line model. In conclusion, these findings indicate that BMSC-derived exosomes can attenuate inflammation and demyelination of the CNS in the EAE rat model by regulating the polarization of microglia. Therefore, the use of BMSC-derived exosomes may be a potential therapeutic approach for the treatment of autoimmune and inflammatory diseases.

African swine fever (ASF) is a major threat to domestic pigs and wild boars. Since 2018, ASF outbreaks have been ongoing in China. As of August 3, 2019, a total of 151 ASF clusters of outbreaks reported in China have caused severe economic losses for the industry, the pig farmers and pork producers, due to the lack of an efficacious vaccine. The present study aims to analyze the epidemiologic characteristics of ASF outbreak that occurred in several regions across China during the period August 2018- August 2019. Particular focus was on the epidemic distribution, main transmission routes, incidence/fatality, impact on pig production capacity, and the main preventive measures adopted to mitigate the risk of ASF spread in pig farming systems by Chinese government. Results show that anthropogenic factors, spatial distribution, efficient measures taken by China,and good response timely in implementation of preventive measures are important on the transmission of ASF and these suggest that effective ASF risk management in China will require a comprehensive and integrated approach linking science and implemented by all relevant stakeholders. This provides an empirical basis to optimize current interventions as well as develop new tools and strategies to reduce the risk transmission of African swine fever virus (ASFV) to domestic pigs and wild boars.

The incidence of colorectal cancer (CRC) is increasing annually worldwide. However, traditional chemotherapy has obvious side effects. Low-dose naltrexone (LDN) has been reported to delay tumor progression, but the mechanism remains unclear. Therefore, the aim of this study was to explore the mechanisms underlying the inhibitory effect of LDN on CRC progression in vivo and in vitro. We found that expression of macrophage markers (F4/80, CD68) was increased in nude mice treated with LDN compared with the control group (p < 0.05). Additionally, levels of M1 macrophage phenotypic markers (CD80) and cytokines (tumor necrosis factor-α, TNF-α) were higher than in the control group (p < 0.05). LDN was able to upregulate expression of the opioid growth factor receptor (OGFr) and apoptosis-related factors Bax, caspase-9, caspase-3, and PARP and downregulate expression of Bcl-2, Survivin, and Ki67 to promote tumor cell apoptosis. Therefore, we speculate that LDN reduces tumor size by increasing levels of M1-like macrophages and activating the Bax/Bcl-2/caspase-3/PARP signaling pathway to induce apoptosis. We suggest that LDN has potential for the treatment of CRC.

The goal of this work was to investigate how MENK could regulate the functions of CD8+T cells and to explore the relationship between this regulation and opioid receptor expression. Our results showed that the opioid receptors presented on the cell menbrane of CD8+T cells were MOR and DOR. MENK promoted the expression of opioid receptors as well as the elevation of the surface molecules such as CD28, PD-1, CTLA-4 and FasL and intracellular granzyme B. Selectively blocking the MOR by CTAP or DOR by NTI could result in inhibition of the corresponding CD8+T cells proliferation and the expressions of surface molecules. In addition, non-selectively blocking both MOR and DOR by NTX could further impair the functions and proliferation of CD8+T cells. Our currently data indicated that MENK could play a vital role in immune functions via precise regulation to subunits of opioid receptors.

Xiaonan Wang,1 Jing Tian,1 Xue Jiao,2 Jin Geng,3 Reizhe Wang,4 Ning Liu,5 Xinghua Gao,6 Noreen Griffin,7 Yuan Gao,8 Fengping Shan1 1Department of Immunology, College of Basic Medical Science, China Medical University, Shenyang, China; 2Department of Translational Medicine, No. 4 Teaching Hospital, China Medical University, Shenyang, China; 3Department of Ophthalmology, China Medical University, Shenyang, China; 4Department of Gynecology, No. 1 Teaching Hospital, China Medical University, Shenyang, China; 5Department of Gynecologic Oncology, Shengjing Hospital, 6Department of Dermatology, No.1 Teaching Hospital, China Medical University, Shenyang, China; 7Immune Therapeutics, Inc., Orlando, FL, USA; 8Faculty of Information and Engineering, Northeastern University, Shenyang, China Background: Gastric cancer (GC) is the second cause of cancer-related deaths. Methionine enkephalin (MENK), an endogenous opioid peptide, has immunological and antitumor activity. Purpose: The aim of this work was to investigate whether MENK could exhibit activity against human GC in vitro and in vivo. Materials and methods: Human GC cells were treated with MENK. Cell viability, colony formation, cell morphology, cell cycle, and apoptosis were assessed. The effects of MENK on gene expression of OGFr, Bax, BCL-2, caspase-3, PARP, Ki67, cyclin D1, c-myc, survivin were quantifed by qRT-PCR. Western blot was used to analyze the effects of MENK on protein expression of OGFr, Bax, BCL-2, caspase-3, PARP. The anti-tumor activity of MENK in gastic carcinoma was also investigated with animal experiments. Results: The results indicate that MENK could significantly inhibit the growth of human GC cells SGC7901 and HGC27 in a concentration- and time-dependent manner, decrease the number of cell colonies, and arrest cell cycle in the G0/G1 phase by causing a decrease in Ki67, cyclin D1, and c-myc mRNA. Furthermore, MENK could induce tumor cell apoptosis associated with the upregulation of Bax, a corresponding downregulation of BCL-2 and survivin, and activation of caspase-3 and PARP. Moreover, MENK upregulated the expression of opioid receptors (OGFr) in SGC7901 and HGC27 cells. The interaction between MENK and OGFr in SGC7901 and HGC27 cells appears to be essential for the antitumor activity of MENK. Conclusion: We conclude that MENK may be a potential drug for the treatment of GC. Keywords: MENK, GC cells, OGFr, cell cycle, apoptosis

The aim of the work was to investigate the effect and possible mechanism of MENK on the growth of rat C6 glioma in vivo or in vitro. Our findings showed that MENK could inhibit the growth of rat C6 glioma, prolong median survival times in tumor-bearing rats, and induce glioma cell apoptosis. Moreover, MENK could increase the activities of caspase-3, caspase-8 and caspase-9. It also increased the expression of Fas, FasL, Bax, while decreased the expression of Bcl-2. We further confirmed that MENK could increase opioid receptors MOR and DOR expressions, Ca2+ influx into the cytoplasm, and a substantial increase of NFAT1accumulation in the nuclei in C6 glioma cell. When we specifically knocked down NFAT1, there was no effect of MENK on the cell viability and FasL up-regulation in NFAT1 knocked-down cell. These results demonstrate that MENK could bind to opioid receptors MOR and DOR on C6 glioma cells and trigger a Ca2+ influx into the cytoplasm, resulting in translocation of NFAT1 into the nucleus. The hyper-activation of NFAT1 may regulate transcription of downstream gene, such as FasL, and induce apoptosis of rat C6 glioma cells.

Cervical cancer is one of the most common in the female genital tract and remains a leading cause that threatens the health and lives of women worldwide, although preventive vaccines and early diagnosis have reduced mortality. While treatment by operation and chemoradiotherapy for early-stage patients achieve good outcomes, the great majority of cervical cancers caused by the human papilloma virus (HPV) make immunotherapy realizable for patients with advanced and recurrent cervical cancer. To date, some clinical trials of checkpoint immunotherapy in cervical cancer have indicated significant benefits of programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) inhibitors, providing strong evidence for PD-1/PD-L1 as a therapeutic target. In this review article, we discuss the role of PD-L1 and the application of PD-L1 inhibitors in cervical cancer, with the aim of providing direction for future research.

The morbidity and mortality associated with influenza A virus infections, have stimulated the search for novel prophylactic and therapeutic drugs. The purpose of this study was to investigate the prophylactic and therapeutic effect of synthetic methionine enkephalin (MENK) on mice infected by A/PR/8/34 influenza virus (H1N1) in vivo. The results showed that MENK could exert both prophylactic and therapeutic influences on infected mice, significantly improve the survival rate, relieve acute lung injury and decrease cytokine (IFN-α, IFN-β, TNF-α, IL-6, and IL-1β) levels. MENK also inhibited virus replication on day 4 post infection (p.i.) through upregulating opioid receptors (MOR, DOR) and suppressing TLR7-MyD88-TRAF6-NF-κB p65 signaling pathways. These results suggest that MENK, given via intranasal administration, could provide a novel drug with a new mode of action as a nonspecific anti-influenza agent or vaccine adjuvant.

This study examined the antitumor effect of methionine enkephalin (MENK) against lung cancer in vivo and in vitro and explored the underlying mechanisms. Changes in the immune status of the tumor microenvironment (TME) in response to MENK administration were examined in mice. MENK significantly inhibited the proliferation of lung cancer cells in vivo and in vitro by regulating the Wnt/β-catenin pathway and causing cell cycle arrest at the G0/G1 phase. Knockdown of opioid growth factor receptor abolished the effect of MENK on lung cancer cells. The immune status of the TME of mice differed between the MENK and control groups. MENK increased the infiltration of M1-type macrophages, natural killer cells, CD8+ T cells, CD4+ T cells, and dendritic cells into the TME, and decreased the proportion of myeloid inhibitory cells and M2-type macrophages. Immunohistochemical analysis of the expression of cytokines in the TME showed that MENK upregulated IL-15, IL-21, IFN-γ, and granzyme B and downregulated IL-10 and TGF-β1 in mice. Taken together, these finding indicate that MENK may be a potential agent for lung cancer treatment in the future, especially for overcoming immune escape and immune resistance.

Colorectal cancer (CRC), known as a frequently fatal disease, ranking as the third most common malignancy, is the second leading cause of cancer related mortality worldwide. Metastases are common in CRC patients which account for approximately 25% of the patients at diagnosis, 50% of patients during treatment which is associated closely with CRC mortality. Conventional therapies such as surgery, chemotherapy, and radiotherapy are standards of care for the treatment of CRC patients. However, primary tumor recurrence and secondary disease in patients receiving standard of care treatment modalities occur in 50% of patients so that new treatment modalities are needed. Immune checkpoint inhibition (ICI) has transformed the management of patients suffered from metastatic CRC (mCRC) with mismatch repair deficiency (dMMR) and microsatellite instability (MSI) -high (MSI-H) while manifests ineffectiveness in preserved mismatch repair (pMMR) or microsatellite stable (MSS) "cold" tumors which makes up the majority (95%) of mCRC. In this review, we mainly lay emphasis on the development of combinations in therapy strategies with ICIs with other immune based treatment approaches to increase the intra-tumoral immune response and render tumors 'immune-reactive', thereby increasing the efficacy of tumor immunotherapy.

The aim of this study was to investigate how methionine enkephalin (MENK) regulates the biological behavior of lung cancer cells and to further explore its anti-lung cancer mechanisms in vitro and in vivo. The results showed that MENK enhanced the expression of opioid receptor (OGFr) and induced apoptosis of lung cancer cells by activating the Bcl-1/Bax/caspase-3 signaling pathway in vitro and in vivo. However, the regulatory effects of MENK disappeared after blockade of the OGFr. This confirmed that a prerequisite for the anti-tumor action of MENK is binding to OGFr. Additionally, we observed that MENK treatment enhanced the immunogenicity of lung cancer by enhancing the exposure of calreticulin and high mobility group box 1, and increasing the expression of NKG2D ligands. Further studies showed that MENK treatment increased the expression of natural killer (NK) cell-related cytokines such as granzyme B and interferon-γ and NK cell activation. Thus, we concluded that MENK might inhibit the proliferation of lung cancer cells by activating the Bcl-2/Bax/caspase-3 signaling pathway and enhancing immunogenicity and NK cell-driven tumor immunity.

Several lines of evidences have shown that Nocardia rubra cell wall skeleton (Nr-CWS) has immunoregulatory and anti-tumor activities. However, there is no information about the effect of Nr-CWS on CD4+ T cells. The aim of this study was to explore the effect of Nr-CWS on the phenotype and function of CD4+ T cells. Our results of in vitro experiments showed that Nr-CWS could significantly up-regulate the expression of CD69 and CD25 on CD4+ T cells, promote the proliferation of CD4+ T cells, increase the production of IFN-γ, TNF-α and IL-2 in the supernatants, but has no significant effect on the apoptosis and death of CD4+ T cells. Results of in vivo experiments showed that Nr-CWS could promote the proliferation of CD4+ T cells, and increase the production of IL-2, IFN-γ and TNF-α (Th1 type cytokines). These data suggest that Nr-CWS can enhance the activation of CD4+ T cells, promote the proliferation of CD4+ T cells and the differentiation of CD4+ T cells to Th1 cells.

Melanoma is a common cutaneous malignancy, that is also found in specific mucosal sites, and is associated with a poor prognosis. The aim of the present study was to investigate the cytotoxicity of methionine enkephalin (MENK) for B16 melanoma cells in vivo and in vitro. The results of the present study allowed our conclusion that MENK regulates the proliferation of B16 cells, causing cell cycle arrest in the G0/G1 phase and a decrease in the percentage of cells in the S and G2/M phases. Reverse transcription-quantitative polymerase chain reaction demonstrated that MENK increased opioid receptor expression in the B16 cells. Furthermore, the tumor volume and weight in the MENK-treated group were lower than those in the control group (NS) and MENK and naltrexone (NTX)-treated groups. MENK exerted both significant antitumor activity on the growth of B16 cells and a longer survival time in mice. The mice treated with MENK exhibited an increased ratio of CD4+ to CD8+ T cells as tested by flow cytometry (FCM), resulting in a ratio of 2.03 in the control group, 3.69 in the MENK-treated group, and 2.65 in the MENK and NTX group. Furthermore, a significant increase in plasma levels of IL-2, IFN-γ and TNF-α was revealed as assessed by ELISA. In conclusion, the results of the present study indicate that MENK has a cytotoxic effect on B16 melanoma cells in vitro and in vivo, and suggest a potential mechanism for these bioactivities. Therefore, we posit that MENK should be investigated, not only as a primary therapy for melanoma, but also as an adjuvant therapy in combination with chemotherapies.

MENK, an endogenous opioid peptide has been reported to have many immunological and antitumor activities. So far the detailed mechanisms of antitumor through regulating DCs by MENK have not been elucidated yet. The aim of this work was to investigate the antitumor mechanisms of MENK via regulating DC. The monitoring methods, such as ELISA, MTS assay, CFSE, Real-time PCR and Western blot were included in our research. We found bone marrow derived dendritic cells (BMDCs) in 36 female C57BL/6 mice treated with MENK enhanced expression of key surface molecules, increased production of critical cytokines reduced endocytosis of FITC-dextran, upregulated TLR4 through MyD88/NF-κB signaling pathway and mounted higher antitumor activity. These observations were further supported by an enhancement of nuclear translocation of the p65NF-κB subunit involved in this process. Surprisingly, mu-opioid receptors were the main participants of this kind of activation, not delta-opioid receptors nor kappa-opioid receptors, and these interactions could be partly blocked by Naltrexone (a kind of opioid antagonist). In vivo study the activated CD4 + , CD8 + T cells and decreased ability to induce differentiation of Foxp3 + regulatory T cells were detected post treatment of MENK. Thus, it is concluded that MENK could exert antitumor effect through precisely regulating opioid receptor mediated functions of DCs. In addition, MENK treated DCs may serve as a new immunotherapy approach against tumor.

Natural killer (NK) cells are innate lymphocytes that aid in the protection of the host from infectious diseases and cancer. In vitro studies of NK cells have provided a foundation for developing clinical adoptive NK-cell transferred immunotherapy against human tumors. To elucidate the functions and mechanisms of NK cell populations, it is important to develop an optimal, highly reproducible and reliable isolation method. The present comparative study was performed with four different NK cell isolation kits of magnetic bead labeling made by Miltenyi and Stemcell companies, including positive selection kits [cluster of differentiation (CD)-49b, using the monoclonal antibody DX5) MicroBeads] and negative selection kits. In addition, the viability of NK cells isinterleukin-2 (IL-2)-dependent in vitro and thus the concentration of IL-2 is critical for maintaining longer cell viability of NK cells. NK cell purity and viability after culturing, for 24, 48 or 72 h, with or without IL-2 (0, 100, 300 or 500 U/ml) was investigated in the present study. Purity of NK cells varied depending on the purification kit used, despite the same method being applied. Furthermore, more granulocytes were present in purified NK cells using Miltenyi sorting kits, particularly when using the negative selection kit. The main disadvantage of DX5-positive selection using the Stemcell and Miltenyi kits was that a high percentage of CD3e+ cells were mixed into the isolated NK cells. Additionally, a significant difference of NK cell purity (P=0.003) was observed while purification was performed using different surface markers. As a consequence, the use of the positive selection kit was modified and subsequently a significantly higher purity (P=0.002) and yield (P=0.004) of NK cells was obtained. Moreover, the purity of NK cells and viability with or without a range of concentrations of IL-2 was compared. Results indicated that with a higher IL-2 concentration, the NK cell purity and viability were significantly higher (P

Recently, immunotherapy has evolved into a true treatment modality with the approval of PD-1 and PD-L1 inhibitors as the standard care for first-line treatment in patients with non-small cell lung cancer (NSCLC). Until now, for patients with advanced NSCLC, treatment of targeting immune checkpoints reveals a promising survival benefit, and some patients even get long term survive, which creates a paradigm shift in NSCLC treatment. However, many issues or problems are also appearing in clinical practice, such as the lower overall efficacy rate (20-40%), treatment modes, populations choice of immunotherapy, drug resistance, and safety, etc. Thus, in this review, we will mainly summarize and discuss the recent development and confusion of PD-1/PD-L1 inhibitors for advanced NSCLC patients based on current clinical studies.

The standard treatments for neoplasia include surgery, chemotherapy, hormone antagonists and radiotherapy, which can prolong survival, but rarely cure the tumors of gynecological cancer patients. OGF - OGFr expression, in various gynecologic cells and tissues, is an intersection point between cell development, neuroendocrine function and immune modulation. It has been identified that OGF and OGFr expression differs between gynecological tumor and normal cells. Further, exogenous or endogenous OGF and OGFr antagonists have been known to have a role in regulating cell viability and apoptosis. Moreover, the expression of proteins in the OGF - OGFr axis modulate differentiation and membrane expression of immune cells, which can enhance the immune response. In vivo and in vitro assays have shown that OGF and OGFr antagonists inhibit mitosis as well as induce apoptosis in gynecologic cancer cells. Although immune augmentation combination therapies can intensify cytotoxic activity, OGF or OGFr antagonists do not increase toxicities associated with dual-immune regulation. In conclusion, the OGF - OGFr axis provides significant strategies for antitumor efficiency in gynecological cancer.

Cutaneous squamous cell carcinoma (CSCC) is a common skin tumour. Due to weak immunogenicity, recurrence is frequent after treatment. In this study, we explored the effects and mechanisms of methionine enkephalin (MENK), an endogenous opioid peptide and negative growth regulator, in CSCC. MENK inhibited A431 cell proliferation and promoted apoptosis through the opioid growth factor receptor (OGFr). Importantly, MENK also induced autophagy in CSCC and stimulated the emission of DAMPs in A431 cells, which resulted in enhanced activation of dendritic cells (DC).In conclusion, MENK provides an effective method with therapeutic potential to modulate the CSCC microenvironment by utilizing autophagy in the cancer cells.

Naltrexone (NTX) is a nonspecific opioid antagonist that exerts pharmacological effects on the opioid axis by blocking opioid receptors distributed in cytoplastic and nuclear regions. NTX has been used in opioid use disorder (OUD), immune-associated diseases, alcoholism, obesity, and chronic pain for decades. However, low-dose naltrexone (LDN) also exhibits remarkable inhibition of DNA synthesis, viability, and other functions in numerous cancers and is involved in immune remodeling against tumor invasion and chemical toxicity. The potential anticancer activity of LDN is a focus of basic research. Herein, we summarize the associated studies on LDN oncotherapy to highlight the potential mechanisms and prospective clinical applications.

The aim of this study was to characterize the immune response of dendritic cells derived from monocytes (Mo-DCs) in the porcine peripheral blood following infection with porcine reproductive and respiratory syndrome virus (PRRSV). Viral load assays indicated that PRRSV efficiently infected Mo-DCs but failed to replicate, whereas PRRSV infection of Mo-DCs decreased the expression of SLA-I, SLA-II, CD80 and CD40 compared with those of mock Mo-DCs. Furthermore, we analyzed the cytokine profiles using quantitative RT-PCR and ELISA. Results indicated apparent changes in IL-10 and IL-12 p40 expression but not in IFN-γ and TNF-α among Mo-DCs infected with PRRSV and uninfected Mo-DCs. Additionally, flow cytometry analysis of the altered Mo-DCs together with IL-4 and GM-CSF induction for 7days revealed the typical morphology and phenotype with 91.73% purity before infection with PRRSV. Overall, our data demonstrate that PRRSV impaired the normal antigen presentation of Mo-DCs and led to inadequate adaptive immune response by down-regulating the expression of SLA-I,SLA-II, CD80 and CD40. Enhanced Th2 -type cytokine IL-10 secretion and reduced Th1-type cytokines IL-12p40,IFN-γ and TNF-α secretion results in Th1/Th2 imbalance.

Methionine enkephalin (MENK), an endogenous neuropeptide has a crucial role in both neuroendocrine and immune systems. MENK is believed to have an immunoregulatory activity to have cancer biotherapy activity by binding to the opioid receptors on immune and cancer cells. Clinical trial studies in cancer patients have shown that MENK activates immune cells directly and by inhibiting regulatory T-cells (Tregs). MENK may also change the tumor microenvironment by binding to opioid receptor on or in cancer cells. All of these mechanisms of action have biologic significance and potential for use in cancer immunotherapy. Furthermore, they reveal a relationship between the endocrine and immune systems. Due to the apparent role of MENK in cancer therapy we reviewed herein, the research undertaken with MENK in recent years; which has advanced our understanding of the role MENK has in cancer progression and its relationship to immunity, supporting MENK as a new strategy for cancer immunotherapy.

The metastasis of cervical cancer has always been a clinical challenge. We investigated the effects of low-dose naltrexone (LDN) on the epithelial mesenchymal transition of cervical cancer cells in vitro as well as its influence on macrophage polarization and associated cytokines in vivo. The results suggested that LDN supressed the proliferation, migration and invasion abilities and promote their apoptosis in Hela cells, whereas the opioid growth factor receptor (OGFr) silenced significantly reversed these effects in vitro. Knockdown the expression of OGFr, the inhibitory of LDN on EMT was weakened. LDN could inhibit cervical cancer progression in nude mice. In additon, LDN indirectly reduced the number of tumor-associated macrophages (TAMs), mainly M2 macrophages, and decreased expression of anti-inflammatory factor IL-10 in the serum of nude mice. These findings demonstrate that LDN could be a potential treatment for cervical cancer.

This study was to explore the effect and mechanisms of anti- human gastric cancer by MENK in vitro and in vivo. The results showed in MENK-treated xenograft tissue, the percentage of M2-type macrophages decreased while M1-type macrophages increased. MENK increased the expression of M1-related cytokine TNF-α and attenuated the expression of M2-related cytokine IL-10 expression. MENK upregulated the expression of opioid receptor (OGFr), while it inhibited HGC27 and SGC7901 cells through blocking PI3K/AKT/mTOR signal pathway in vitro and in vivo. These effects of MENK could be cancelled when OGFr was knockdown. This indicates that binding to OGFr by MENK appears to be essential for the anti- GC cells. Therefore, it is concluded that MENK might skew macrophage toward M2 phenotype from M1 phenotype within tumor and induce cells apoptosis though blocking OGFr/PI3K/AKT/mTOR signaling pathway.

The goal of this work was to investigate how MENK could regulate the functions of CD8

Naltrexone, a non-selective antagonist of opioid receptors, is mainly used as rehabilitation therapy for discharged opiate addicts to eliminate addiction in order to maintain a normal life and prevent or reduce relapse. In recent years, there have been some novel and significant findings on the off-label usage of naltrexone. Within a specific dosage window, LDN can act as an immunomodulator in multiple autoimmune diseases and malignant tumors as well as alleviate the symptoms of some mental disorders. The results of increasing studies indicate that LDN exerts its immunoregulatory activity by binding to opioid receptors in or on immune cells and tumor cells. These new discoveries indicate that LDN may become a promising immunomodulatory agent in the therapy for cancer and many immune-related diseases. In this article, we review the pharmacological functions and mechanisms of LDN as well as its clinical therapeutic potential as revealed by our team and other researchers.

Owing to its unrevealed etiology, multiple sclerosis lacks specific therapies up to now. Experiential administration of methionine enkephalin (MENK) on mouse model improved disease manifestations to some extent. In order to gain more insight on the significance of MENK application, a capillary electrophoresis-mass spectrometry (CE-MS) technique was employed to profile intracellular metabolite fluctuation in 5 astrocytoma cell lines challenged by MENK. The processed data were first evaluated through a bioinformatic process to ensure their compatibility with the study aims and then subjected to multivariate analysis. The results indicated that MENK administration increased intracellular tyrosine, phenylalanine, methionine and glycylglycine. Exemplified by U87 cells, glycylglycine inhibited cell proliferation as well as MENK but it also decreased cell nitric oxide excretion which could not be evoked by MENK. The neuron protective effects were also mirrored by the increased expression of some genes related to remyelination. This study demonstrated CE-MS to be a promising tool for cell metabolomic analysis and benefited the therapeutic exploring of multiple sclerosis with respect to metabolism intervention.

To characterize different tissue MSCs as sources of cell immunomodulatory therapy. Examined the effects of IFN-γ on WJ-MSC and their immunomodulatory function characteristics. We compared human fetal bone marrow (F-BM), adipose tissue (AT), and Warton's Jelly-derived MSCs (WJ-MSCs) for surface antigen expression, differentiation ability, proliferation capacity, clonality, tolerance for aging, gene expression, and whether IFN-γ affected WJ-MSC gene expression, as determined by real time quantitative PCR. Fifteen geneswere examined. We further assess WJ-MSCs-mediated immunomodulatory on peripheral blood mononuclear, stimulated by PHA, IL-2 and CD3Ab after 5 days of co-cultured in a 5:1 ratio (PBMC:MSCs). Examined the effect of WJ-MSCs on the Th1, Th2, Th17 cytokines production and Treg augument. MSCs from different tissues have similar levels of cell surface antigen expression and differentiation ability, while F-BM-MSCs and WJ-MSC had higher rates of cell proliferation and clonality than AD-MSCs. All 15 genes were expressed at similar levels in WJ-MSCs and AD-MSCs (P > 0.05). 9 genes were upregulated in WJ-MSCFor F-MSC, including IL-6, CXCL9, CXCL10, CXCL11, ICAM-1, IDO1, HLA-G5, SDF1A, and NOTCH were down expression, but VCAM-1 was lower expressionin WJ-MSCS. After IFN-γ treatment, 7 genes were upregulated in WJ-MSC, including chemokine ligands CXCL9, CXCL10 and CXCL11, and the adhesion protein VCAM1and ICAM1. Additionally, immunosuppressive factors, such as HLA-G and IDO were both increased. When cocultured with peripheral blood mononuclear, WJ-MSCs showed an immunosuppressive function by inhibit the proliferative response of Th1 and Th17 but augment Th2 and Treg. Primed WJ-MSCs by IFNγ caused a greater reduction in IFNγ and TNFα than untreated WJ-MSCs, also the effect on augument in Treg and inhibit Th17 (P < 0.01). Our results demonstrate that primitive F-BM-MSCs and WJ-MSCs have biological advantages as compared to adult cells, WJ-MSCs have a gene expression pattern similar to AT-MSCs but not F-BM MSCs, and that inflammatory stimuli regulate gene expression in WJ-MSCs. WJ-MSC showed the immunosuppressive function in co-cultured system with PBMC, and IFNγ can promoted the immunosuppressive function.