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Antibodies (Abs) have been widely used in both immunodiagnostics and immunotherapy for the treatment of various diseases and, in recent years, scientific research applications. With the increasing use of Abs, there has been an urgent demand for low-cost and highly efficient purification methods. In this study, we present a novel formulation based on a β-d-glucan particle loaded protein A/G (GP-protein A/G conjugates) by the carbodiimide method for the purification of immunoglobulin (IgG) antibodies. The prepared GP-protein A/G conjugates exhibit high stability and isolation efficiency. The microspheres also constitute an essential specialty reagent useful for isolating IgG from mammalian species such as goat, mouse and rabbit. Recovery of IgG showed that up to a purity of 92% was reached in the elution step. In addition, they has been shown to be important tools for molecular purification methods such as immunoprecipitation and co-immunoprecipitation. Taken together, these results suggest that the GP-protein A/G system has the potential to be used as a platform for purification techniques.

Methyltransferase-like 3 (METTL3) is an RNA methyltransferase that mediates modification of N6-methyladenosine (m6A), which serves as an oncogene in various types of cancer. The role of m6A modification in the onset and progression of cancer has attracted growing attention. However, the functional and regulatory mechanisms of METTL3 in non-small cell lung cancer (NSCLC) progression are still poorly understood. In the present study, METTL3 expression in NSCLC tissue was analyzed using the Gene Expression Profiling Interactive Analysis database. Western blotting and reverse transcription-quantitative PCR were performed to evaluate the expression of METTL3 in NSCLC tissue and cell lines. Here, knockdown and overexpression of METTL3 notably decreased NSCLC cell viability, apoptosis and migration in vitro and, as well as tumorigenicity in vivo. Expression of METTL3 was upregulated in NSCLC tissue. METTL3 overexpression promoted cell viability and migration in NSCLC, while knockdown of METTL3 yielded the opposite result in vivo and in vitro. METTL3 increased Bcl-2 translation via m6A modification, which increased viability and enhanced migration of NSCLC cells. METTL3 served as an oncogene in NSCLC via METTL3-mediated Bcl-2 mRNA m6A modification, which indicated that targeting METTL3 may be an effective therapeutic strategy for clinical management of NSCLC.

Recent studies have claimed that iron overload was correlated with the risk of hepatocellular carcinoma (HCC), and our previous studies have also demonstrated that dandelion polysaccharide (DP) suppressed HCC cell line proliferation via causing cell cycle arrest and inhibiting the PI3K/AKT/mTOR pathway, but the effect of DP on metabolism is still not very clear. Here, we aim to clarify the effects of DP on iron metabolism and the underlying mechanism. In this study, we found that DP could reduce iron burden in hepatoma cells and grafted tumors. Hepcidin is a central regulator in iron metabolism. We confirmed that the expression of hepcidin in HCC tumor tissues was significantly higher than that in the adjacent nontumor tissues. The expression of hepcidin was downregulated in the liver of mouse model treatment with DP, as well as in hepatoma cells. Moreover, RNA sequencing and western blot data revealed that DP inhibited the IL-6-activated JAK-STAT signaling pathway. In summary, our results revealed that DP might be a new potential drug candidate for the regulation of iron burden and the treatment of HCC.

Melanoma is one of the most fatal and therapy-resistant types of cancer; therefore, identifying novel therapeutic candidates to improve patient survival is an ongoing effort. Previous studies have revealed that pimozide is not sufficient to treat melanoma; therefore, enhancing the treatment is necessary. Indoleamine 2, 3‑dioxygenase (IDO) is an immunosuppressive, intracellular rate-limiting enzyme, which contributes to immune tolerance in various tumours, including melanoma, and inhibition of IDO may be considered a novel therapeutic strategy when combined with pimozide. The present study aimed to assess the antitumour activities of pimozide in vitro, and to investigate the effects of pimozide combined with L‑methyl-tryptophan (L‑MT) in vivo. For in vitro analyses, the B16 melanoma cell line was used. Cell cytotoxicity assay, cell viability assay, wound‑healing assay and western blotting were conducted to analyse the effects of pimozide on B16 cells. Furthermore, B16 cell-bearing mice were established as the animal model. Haematoxylin and eosin staining, immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end-labelling staining, western blotting and flow cytometry were performed to determine the effects of monotherapy and pimozide and L‑MT cotreatment on melanoma. The results demonstrated that pimozide exhibited potent antitumour activity via the regulation of proliferation, apoptosis and migration. Furthermore, the antitumour effects of pimozide were enhanced when combined with L‑MT, not only via regulation of proliferation, apoptosis and migration, but also via immune modulation. Notably, pimozide may regulate tumour immunity through inhibiting the activities of signal transducer and activator of transcription (Stat)3 and Stat5. In conclusion, the present study proposed the use of pimozide in combination with the IDO inhibitor, L‑MT, as a potential novel therapeutic strategy for the treatment of melanoma.

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with a poor prognosis and high mortality. At present, vaccination with tumor cell lysate (TCL) loaded dendritic cells (DC) has been shown to be an effective therapy against HCC. However, the ability of promoting the specific T cell immune response is rather weak, influencing the antitumor response. Thus, it is necessary to find a strategy to improve the antitumor effect of TCL-loaded DC. Activation of signal transducer and activator of transcription 3 (STAT3) significantly inhibits antitumor immune response and DC maturity. Nifuroxazide, an antidiarrheal agent, has been proved to directly inhibit STAT3 activation. Thus, we investigated whether nifuroxazide could improve the antitumor immune response in mice vaccinated with TCL-loaded DC. The study provides the theoretical and experimental basis for developing an effective adjuvant for DC vaccine to treat HCC. Our results showed that the administration of nifuroxazide and DC-loaded TCL could significantly improve the survival rate, inhibit the tumor growth, and prompt the antitumor immune responses in mice with orthotopically implanted hepatocarcinomas, thus, possibly providing a new combination strategy to treat HCC.

The progression of cancer through local expansion and metastasis is well recognized, but preventing these characteristic cancer processes is challenging. To this end, a new strategy is required. In this study, we presented a novel dual functional podophyllotoxin derivative, 2-pyridinealdehyde hydrazone dithiocarbamate S-propionate podophyllotoxin ester (PtoxPdp), which inhibited both matrix metalloproteinases and Topoisomerase II. This new podophyllotoxin derivative exhibited significant anti-proliferative, anti-metastatic that correlated with the downregulation of matrix metalloproteinase. In a xenograft animal local expansion model, PtoxPdp was superior to etoposide in tumor repression. A preliminary mechanistic study revealed that PtoxPdp induced apoptosis and autophagy via the PI3K/AKT/mTOR pathway. Furthermore, PtoxPdp could also inhibit epithelial-mesenchymal transition, which was achieved by downregulating both PI3K/AKT/mTOR and NF-κB/Snail pathways. Taken together, our results reveal that PtoxPdp is a promising antitumor drug candidate.

Diabetes mellitus (DM) complications affect patients and cause varying damage. Skin ulcers exhibit difficulties in wound healing, and the regulatory basis for this remains unclear. High glucose concentration (HG) was utilized to mimic DM in cultured cells. Reverse transcription-quantitative polymerase chain reaction, western blotting and fluorescence dye analyses were performed to analyze the effects of hedgehog signaling in regulation of HG or diabetes in fibroblasts. HG-stress suppressed hedgehog-signaling gene expression, whereas the apoptosis and inflammatory response markers, Caspase-3 and plasminogen activator inhibitor-1 (PAI1), respectively, were induced. In addition, HG-stress inhibited the fibroblast proliferation rate. In parallel, treatment with Sonic hedgehog (Shh), an activator of hedgehog signaling, together with HG eliminated effects of HG on expression of hedgehog-signaling genes, Caspase-3 and PAI1, and rescued the cell proliferation rate in fibroblasts. In addition, Shh application activated c-Jun N-terminal kinase (JNK), which was inhibited by HG stress. sp600125, a JNK specific inhibitor, treatment inhibited the effect of Shh on fibroblast proliferation and hedgehog-signaling marker gene expression. Furthermore, zinc finger protein Gli1 (Gli1) overexpression partially eliminated the effect of HG and sp600125 on fibroblast proliferation, and reduced HG-induced ROS generation in fibroblasts. Together, these results indicate that HG stress inhibits hedgehog signaling, and Shh-JNK-Gli1 pathway positively regulates HG-induced damage on fibroblasts.

Graft‑vs.‑host disease (GvHD) is a major and lethal complication of allogeneic bone marrow transplantation (allo‑BMT). Although great development has been made, the treatment progress of this disorder is slow. Research has illustrated that STAT3 was critical for T cell alloactivation in GvHD. In the present study, the authors hypothesized that nifuroxazide, as the STAT3 inhibitor, treatment may attenuate the development of acute GvHD (aGvHD). The results demonstrated that nifuroxazide suppressed the development of aGvHD and significantly delayed aGvHD‑induced lethality. Mice receiving nifuroxazide had mostly normal‑appearing skin with minimal focal ulceration, mild edema and congestion in the liver, and a less‑pronounced villus injury and less inflammatory infiltrate in the small intestine. Treatment with nifuroxazide inhibited the activation of STAT3, resulting in the regulation of the CD4+ T cells and CD4+CD25+ T cells and reduction of interferon‑γ and tumor necrosis factor‑α levels. In conclusion, nifuroxazide may be efficacious for post‑transplant of GvHD, providing a potent drug for use as a prophylactic or as a second‑line therapy for aGvHD in clinical trials.

A one-step purification process using flow-through mode ion-exchange chromatography (Ft-IEC) was evaluated for the purification of Immunoglobulin G (IgG) from rabbit and guinea pig serum. A simple protein precipitation with saturated ammonium sulfate was used to pretreated plasma samples. Chromatographic separation was carried out on DEAE Sepharose Fast Flow (F.F.) column at a flow rate of 1 mL min−1. Ft-IEC using Tris–HCl buffer at pH 7.0 allowed the recovery of 22% of the loaded IgG with purity of 95% existed in flowthrough and washing effluents but not in elution effluents. To be compared, bind-elute mode IEC using Tris–HCl buffer at pH 8.5 based on the routine protocol showed that the recovery of 15% of the loaded IgG with purity of 80% existed in elution effluents. These results indicate that the Ft-IEC is an effective method for purifying IgG from sera of rabbit and guinea pig and may also be suitable for other animal sera by adjusting pH at equilibrium buffer for chromatography column.

To distinguish the antibodies induced by Foot-and-mouth disease virus (FMDV) infection from those induced by vaccination, a recombinant N-terminal truncated FMDV non-structural protein (NSP) of 3AB, designated as r3aB, was constructed by deleting 80 amino acids displayed about 30% homology to transposase IS4 family protein of Escherichia coli, expressed in E. coli BL21 (DE3) and then purified. The r3aB was majorly expressed in soluble fraction and presented as homogeneous monomers after purification. Using r3aB as coating antigen, an indirect ELISA was established to specifically identify antibodies induced by FMDV infection but not those induced by vaccination. Compared with 3AB, r3aB was more specific to catch antibodies against NSP. The performance of this assay was validated by two commercial FMDV NSP ELISA kits. The result suggested that the r3aB coated ELISA could be developed into a kit to better distinguish between infected and vaccinated cattle.

Lung cancer is a high-grade malignancy with poor 5 year-survival rates that remains incurable with current therapies. Different cellular stresses, including antitumor agents, ionizing radiation and ultraviolet (UV) light, can induce apoptosis and activate signaling pathways. UV has multiple effects on tumor cells, including DNA damage, and increases the expression of some genes involved in tumor cell apoptosis and DNA repair. It has been reported that UV can also activate casein kinase 2 (CK2). CK2, a Ser/Thr protein kinase, has been reported to be frequently overexpressed in various types of human cancer, including lung cancer, and is associated with tumor development. Thus, combination of UV and CK2 inhibitors may be a new strategy for the treatment of lung cancer. Our results demonstrated that inhibition of CK2a through CK2 siRNA or a CK2 inhibitor [(4,5,6,7-tetrabromobenzotriazole (TBB)] enhances the decrease in cell viability of lung cancer cells (A549 and H2030) induced by UV. Western blot analysis demonstrated that the combination increased the expression of apoptotic protein markers cytochrome c and the cleavage of poly ADP-ribose polymerase (PARP) and caspase-3. Furthermore, our results indicated that UV decreased the expression of the tumor suppressor protein PML through activation of CK2. Inhibition of CK2 by CK2 siRNA and TBB can recover the reduction of PML induced by UV. Collectively, these results demonstrate the significant apoptosis of lung cancer cells induced by combination treatment of the CK2 inhibitor and UV radiation. CK2 enhanced cell apoptosis by UV radiation may due, at least partly, to recover the expression of PML. These findings warrant the clinical testing of CK2 inhibitors which, when used in conjunction with DNA-damaging agents such as radiation, may be an effective cancer therapeutic strategy.

Coxsackievirus B3 was one of the major pathogens causing viral myocarditis. Toll-like receptor 9 activation contributed to the innate immune response in the process of CVB3-induced myocarditis. In order to find out how CpG oligodeoxynucleotide, known as a TLR-9 agonist, would affect the CVB3-induced myocarditis, we chose a C-type CpG oligodeoxynucleotide (YW002) injected to the mice one day before CVB3 challenge. On day 4 post CVB3 infection, 3 mice in each group were randomly sacrificed and their hearts were isolated to detect CVB3 replication. On day 10, the CVB3 neutralizing antibody and inflammatory change of the hearts were detected. The results indicated that the CVB3-induced myocarditis was aggravated with the declining body weight of mice, decreasing neutralizing antibody, and uncontrolling virus replication by injecting 20 μg YW002 per mouse. When adjusted the amount at 10 μg YW002 per mouse, there were no signs of aggravation in myocarditis. Plus, the mortality of the infected mice was reduced, the neutralizing antibody level was raised and the replication of virus was restrained. These results suggested that a proper amount of CpG oligodeoxynucleotide application could help to inhibit CVB3 infection.

To develop a therapeutic vaccine against hepatitis B virus surface antigen (HBsAg)-expressing liver cancer, we tried to prepare a vaccine by formulating recombinant HBsAg with BW006, a B type CpG oligodeoxynucleotide (ODN) with Th1-biasing activity, and examined its potency of inducing therapeutic immunity against HBsAg-expressing liver cancer cells in mice. When applied therapeutically, BW006 could assist HBsAg to induce vigorous immune responses capable of inhibiting the growth of HBsAg-expressing liver cancer cells and prolonging the survival of mice bearing HBsAg-expressing liver cancer cells. In vivo and in vitro experiments showed that the BW006-adjuvanted HBsAg enhanced the production of IgG2a antibodies, interferon-γ, and interleukin-12 and facilitated the generation of specific cytotoxic T lymphocyte that killed the HBsAg-expressing liver cancer cells. These results suggest that the BW006-adjuvanted HBsAg might be developed into a candidate tumor vaccine for the treatment of HBsAg-expressing liver cancer.

Hepatocellular carcinoma (HCC) is one of the most aggressive carcinomas. Limited therapeutic options, mainly due to a fragmented genetic understanding of HCC, and major HCC resistance to conventional chemotherapy are the key reasons for a poor prognosis. Thus, new effective treatments are urgent and gene therapy may be a novel option. Signal transducer and activator of transcription 3 (Stat3) is a highly studied member of the STAT family. Inhibition of Stat3 signaling has been found to suppress tumor growth and improve survival, providing a molecular target for cancer therapy. Furthermore, HCC is a hypervascular tumor and angiogenesis plays a crucial role in tumor growth and metastasis. Thus, anti-angiogenic therapy, combined with inhibition of Stat3, may be an effective approach to combat HCC. We tested the effect that the combination therapy consisting of endostatin (a powerful angiogenesis inhibitor) and Stat3-specific small interfering RNA, using a DNA vector delivered by attenuated S. typhimurium, on an orthotopic HCC model in C57BL/6 mice. Although antitumor effects were observed with either single therapeutic treatment, the combination therapy provided superior antitumor effects. Correlated with this finding, the combination treatment resulted in significant alteration of Stat3 and endostatin levels and that of the downstream gene VEGF, decreased cell proliferation, induced cell apoptosis and inhibited angiogenesis. Importantly, combined treatment also elicited immune system regulation of various immune cells and cytokines. This study has provided a novel cancer gene therapeutic approach.

A synthesized single-stranded oligodeoxynucleotide (ODN), designed as SAT05f with the sequence of human microsatellite DNA, has been studied for its capacity of alleviating the lupus nephritis in the chronic graft versus host disease (cGVHD) induced lupus-prone mice. In cGVHD model mice, both of continuous and discontinuous treatment with SAT05f was effective on reducing anti-ssDNA antibody production, decreasing renal IgG deposition and delaying the onset of lupus nephritis. In addition, SAT05f could down-regulate TLR9 mRNA expression in splenocytes of cGVHD model mice. These results indicated that SAT05f could be developed as a new therapeutic agent for the treatment of lupus nephritis by inhibiting TLR9 signaling pathways.