Caspase-3/Gasdermin E-mediated pyroptosis contributes to Ricin toxin-induced inflammation.

Ricin toxin (RT) is highly cytotoxic and can release a considerable amount of pro-inflammatory factors due to depurination, causing excessive inflammation that may aggravate the harm to the body. Pyroptosis, a type of gasdermin-mediated cell death, is a contributor to the exacerbation of inflammation. Accumulating evidence indicate that pyroptosis plays a significant role in the pathogen infection and tissue injury, suggesting a potential correlation between pyroptosis and RT-induced inflammation. Here, we aim to demonstrate this correlation and explore its molecular mechanisms. Results showed that RT triggers mouse alveolar macrophage MH-S cells pyroptosis by activating caspase-3 and cleaving Gasgermin E (GSDME). In contrast, inhibition of caspase-3 with Z-DEVD-FMK (inhibitor of caspase-3) or knockdown of GSDME attenuates this process, suggesting the essential role of caspase-3/GSDME-mediated pyroptosis in contributing to RT-induced inflammation. Collectively, our study enhances our understanding of a novel mechanism of ricin cytotoxicity, which may emerge as a potential target in immunotherapy to control the RT-induced inflammation.

Self-assembling ferritin nanoparticles coupled with linear sequences from canine distemper virus haemagglutinin protein elicit robust immune responses.

BACKGROUND: Canine distemper virus (CDV), which is highly infectious, has caused outbreaks of varying scales in domestic and wild animals worldwide, so the development of a high-efficiency vaccine has broad application prospects. Currently, the commercial vaccine of CDV is an attenuated vaccine, which has the disadvantages of a complex preparation process, high cost and safety risk. It is necessary to develop a safe and effective CDV vaccine that is easy to produce on a large scale. In this study, sequences of CDV haemagglutinin (HA) from the Yanaka strain were aligned, and three potential linear sequences, termed YaH3, YaH4, and YaH5, were collected. To increase the immunogenicity of the epitopes, ferritin was employed as a self-assembling nanoparticle element. The ferritin-coupled forms were termed YaH3F, YaH4F, and YaH5F, respectively. A full-length HA sequence coupled with ferritin was also constructed as a DNA vaccine to compare the immunogenicity of nanoparticles in prokaryotic expression. RESULT: The self-assembly morphology of the proteins from prokaryotic expression was verified by transmission electron microscopy. All the proteins self-assembled into nanoparticles. The expression of the DNA vaccine YaHF in HEK-293T cells was also confirmed in vitro. After subcutaneous injection of epitope nanoparticles or intramuscular injection of DNA YaHF, all vaccines induced strong serum titres, and long-term potency of antibodies in serum could be detected after 84 days. Strong anti-CDV neutralizing activities were observed in both the YaH4F group and YaHF group. According to antibody typing and cytokine detection, YaH4F can induce both Th1 and Th2 immune responses. The results of flow cytometry detection indicated that compared with the control group, all the immunogens elicited an increase in CD3. Simultaneously, the serum antibodies induced by YaH4F and YaHF could significantly enhance the ADCC effect compared with the control group, indicating that the antibodies in the serum effectively recognized the antigens on the cell surface and induced NK cells to kill infected cells directly. CONCLUSIONS: YaH4F self-assembling nanoparticle obtained by prokaryotic expression has no less of an immune effect than YaHF, and H4 has great potential to become a key target for the easy and rapid preparation of epitope vaccines.

Self-assembled raccoon dog parvovirus VP2 protein confers immunity against RDPV disease in raccoon dogs: in vitro and in vivo studies.

BACKGROUND: Raccoon dog parvovirus (RDPV) causes acute infectious diseases in raccoon dogs and may cause death in severe cases. The current treatment strategy relies on the extensive usage of classical inactivated vaccine which is marred by large doses, short immunization cycles and safety concerns. METHODS: The present study aimed at optimization of RDPV VP2 gene, subcloning the gene into plasmid pET30a, and its subsequent transfer to Escherichia coli with trigger factor 16 for co-expression. The protein thus expressed was purified with ammonium sulfate precipitation, hydrophobic chromatography, and endotoxin extraction procedures. VLPs were examined by transmission electron microscopy, dynamic light scattering, and the efficacy of VLPs vaccine was tested in vivo. RESULTS: Results indicated that RDPV VP2 protein could be expressed soluble. Transmission electron microscopy and dynamic light scattering results indicated that RDPV VP2 self-assembled into VLPs. Hemagglutination inhibition antibody titers elicited by Al(OH)3 adjuvanted RDPV VLPs were comparable with RDPV inactivated vaccines, and the viral loads in the blood of the struck raccoon dogs were greatly reduced. Hematoxylin and eosin and Immunohistochemical results indicated that RDPV VLPs vaccine could protect raccoon dogs against RDPV infections. CONCLUSIONS: These results suggest that RDPV VLPs can become a potential vaccine candidate for RDPV therapy.

Embryonic Stem Cells-loaded Gelatin Microcryogels Slow Progression of Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) has become a public health problem. New interventions to slow or prevent disease progression are urgently needed. In this setting, cell therapies associated with regenerative effects are attracting increasing interest. We evaluated the effect of embryonic stem cells (ESCs) on the progression of CKD. METHODS: Adult male Sprague-Dawley rats were subjected to 5/6 nephrectomy. We used pedicled greater omentum flaps packing ESC-loaded gelatin microcryogels (GMs) on the 5/6 nephrectomized kidney. The viability of ESCs within the GMs was detected using in vitro two-photon fluorescence confocal imaging. Rats were sacrificed after 12 weeks. Renal injury was evaluated using serum creatinine, urea nitrogen, 24 h protein, renal pathology, and tubular injury score results. Structural damage was evaluated by periodic acid-Schiff and Masson trichrome staining. RESULTS: In vitro, ESCs could be automatically loaded into the GMs. Uniform cell distribution, good cell attachment, and viability were achieved from day 1 to 7 in vitro. After 12 weeks, in the pedicled greater omentum flaps packing ESC-loaded GMs on 5/6 nephrectomized rats group, the plasma urea nitrogen levels were 26% lower than in the right nephrectomy group, glomerulosclerosis index was 62% lower and tubular injury index was 40% lower than in the 5/6 nephrectomized rats group without GMs. CONCLUSIONS: In a rat model of established CKD, we demonstrated that the pedicled greater omentum flaps packing ESC-loaded GMs on the 5/6 nephrectomized kidney have a long-lasting therapeutic rescue function, as shown by the decreased progression of CKD and reduced glomerular injury.

Combined effects of radiotherapy and endostatin gene therapy in melanoma tumor model.

PEgr-Endostatin-EGFP plasmid was constructed to investigate its expression properties induced by ionizing irradiation and the effect of pEgr-Endostatin-EGFP gene-radiotherapy on melanoma tumor-bearing mice. The pEgr-Endostatin-EGFP plasmid was transfected into B16 cell line with liposome. The expression property of endostatin was investigated by RT-PCR and that of EGFP was detected by flow cytometry. Tumor-bearing mice were treated by the plasmid injection and 2 Gy X-irradiation of three fractions. Tumor growth was observed for 18 days after treatment. Change of tumor capillary formation was measured with histochemistry assay at the end of the experiment. The expression of GFP in B16 melanoma cells was detected after X-irradiation with 0.05-20 Gy. Time-course studies showed that the expression of GFP in B16 cells reached its peak at 8 h after irradiation with 2 Gy. The injection of pEgr-Endostatin-EGFP recombinant plasmid into the implanted B16 melanoma in C57BL/6J mice followed by local X-irradiation could significantly inhibit tumor growth with inhibition of intratumor micro-vessel density. The inhibitory effect of pEgr-Endostatin-EGFP gene-radiotherapy on the growth of B16 melanoma is correlated with the marked decrease of intratumoral vascularization. The present data point to the potential of an anti-angiogenic approach in gene-radiotherapy of cancer.

Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro.

AIM: To construct pcDNA3.1-Egr.1p-p16 recombinant plasmid and investigate the expression of p16 in pancreatic cancer JF305 cells induced by radiation and the feasibility of gene radiotherapy for pancreatic carcinoma. METHODS: Human p16 cDNA was ligated to the downstream of Egr-1 promotor to construct pcDNA3.1-Egr.1p-p16 plasmid by restriction enzyme digested. The recombined plasmids were transfected into pancreatic cancer JF305 cells with lipofectamine. p16 mRNA level was detected by RT-PCR. The expression of p16 after different doses of X-ray radiation was detected by Western blot technique. Cell survival was assessed by clonogenic assays and cell viability was analysed by trypen blue exclusion. Flow cytometry was performed to study the apoptosis of JF305 cells. RESULTS: Restriction enzyme digestion showed the correctly constructed pcDNA3.1-Egr.1p-p16. The p16 expression in cells transfected with pcDNA3.1-Egr.1p-p16 induced by different doses of radiation was higher than that in the control group (P < 0.05). Eight hours after 2 Gy X-ray radiation, the expression reached its peak (87.00 ng/L), and was significantly higher than that in the control group (P < 0.0.5). Clonogenic analysis and trypan blue extraction test showed that the pcDNA3.1-Egr.1p-p16 transfer enhanced radiation-induced cell killing in p16-null JF305 cell lines. The induction of apoptosis was lower in combined transfection and irradiation group than that in irradiation alone. CONCLUSION: X-ray can induce the recombinant plasmid pcDNA3.1-Egr.1p-p16 expression in JF305 cells. The detection of dose and time provides an experimental basis for in vivo study in future.

[Effect of irradiation-induced pcDNA3.1-Egr.1p-p16 on cell apoptosis and cell cycle of JF305 cells].

OBJECTIVE: To observe the effect of pcDNA3.1-Egr.1p-p16 plasmid on apoptosis and cell cycle of pancreatic carcinoma JF305 cell line. METHODS: JF305 cells were cultured and transfected with pcDNA3.1-Egr.1p-p16 plasmid via Lipofectamine(TM) 2000, followed by irradiation by 6MV-X at 4 Gy (dose rate 2.50 Gy/min). The cell cycle and cell apoptosis changes were analyzed by flow cytometry. RESULTS: In cells infected with pcDNA3.1-Egr.1p-p16 plasmid and those with pcDNA3.1-Egr.1p-p16 plasmid infection before 4 Gy irradiation, the percentages of viable apoptotic cells were 6.4% and 10.4%, and those of advanced stage apoptotic or dead cells were 16.8% and 33.8%, significantly higher than those in the control group (P<0.05). JF305 cell apoptosis in 4 Gy irradiation group was obviously increased in comparison with non-irradiated plasmid-infected cells (P<0.05). Irradiation resulted in a predominant G(2) arrest of the plasmid-infected JF305 cells. Both pcDNA3.1-p16 plasmid and pcDNA3.1-Egr.1p-p16 plasmid infections could induce G1 arrest of JF-305 cells, and irradiation did not produce significant changes in G(1)-arrested cells in the two plasmid infection groups, but cells arresting at G(2) phase increased. CONCLUSION: pcDNA3.1-Egr.1p-p16 can induce JF-305 cells apoptosis, which is enhanced by irradiation. pcDNA3.1-Egr.1p-p16 tranfection may result in G(1) arrest of the cells, and when combined with irradiation, the cells arrested at G(2) phase can increase.

[Anti-tumor effect of pcDNA3. 1-Egr. 1p-p16 gene combined with radiotherapy in tumor-bearing nude mice].

OBJECTIVE: To investigate the optimal doses of X-ray irradiation and plasmid injection in the anti-tumor effect of the pcDNA3. 1-Egr. 1p-p16 gene combined with radiotherapy in vivo. METHODS: We observed the anti-tumor effect of the pcDNA3. 1-Egr. 1p-p16 gene combined with radiotherapy with different doses of X-ray irradiation (2, 10, 20 Gy) and plasmid injection (10, 20, 30 microg) in nude mice with JF-305 pancreatic carcinoma, and detected the expression of p16 in tumor by RT-PCR. RESULTS: The tumor growth rate of the nude mice irradiated locally with 20 Gy X-rays after the plasmid injection was significantly lower (P < 0.05 ) than that of the nude mice irradiated locally with 2 Gy or 10 Gy X-ray 3 days after the irradiation. The tumor growth rate of the nude mice injected locally with 20 microg or 30 microg plasmid was significantly lower (P <0.05 ) than that of the nude mice injected locally with 10 microg plasmid. Both pcDNA3. 1-Egr. 1p-p16 group and pcDNA3. 1-Egr. 1p-p16 +20 Gy group had p16 mRNA expression, but the mRNA level of pcDNA3. 1-Egr. 1p-p16 +20 Gy group was higher than that of pcD- NA3. 1-Egr. 1p-p16 group. CONCLUSION: In the pcDNA3. 1-Egr. 1p-p16 gene combined with radiotherapy in vivo the optimal dose of X-ray irradiation was 20 Gy and the optimal dose of plasmid injection was 20 microg. The anti-tumor effect of pcDNA3. 1-Egr. 1p-p16 combined with radiotherapy is better than that of radiotherapy or gene therapy alone, which may be related with the enhanced p16 expression in tumor after the irradiation.

Effect of pEgr-TNFalpha gene radiotherapy on mice melanoma.

In the present study, we constructed a pEgr-tumour necrosis factor-alpha (TNFalpha) plasmid and investigated its expression properties in B16 cells on exposure to ionizing irradiation and, furthermore, the effect of gene radiotherapy on a melanoma model. Firstly, the recombinant pEgr-TNFalpha plasmid was constructed and transfected into B16 cells with liposomes to investigate its expression properties on exposure to X-irradiation. The melanoma-bearing model was then established and the tumour tissue was injected locally with the pEgr-TNFalpha plasmid and exposed to 20 Gy of X-irradiation. The tumour growth curve at different time points was described. At day 3, TNFalpha transcription in the tumour tissue was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that: (1) the eukaryotic expression vector pEgr-TNFalpha was successfully constructed and transfected into B16 cells; (2) TNFalpha expression was significantly increased in the transfected cells after X-irradiation, in contrast with that of the pCMV-TNFalpha group or the pEgr-TNFalpha group that received 0 Gy of irradiation; (3) after pEgr-TNFalpha gene radiotherapy, tumour growth was significantly slower than in those groups receiving irradiation or gene transfer alone. The TNFalpha concentration in the peripheral blood of tumour-bearing mice that received an injection of pEgr-TNFalpha and 20 Gy of irradiation was higher than that of the control group. Only in pEgr-TNFalpha and pEgr-TNFalpha + 20 Gy groups was TNFalpha messenger RNA (mRNA) detected in the tumour tissue. We conclude that pEgr-TNFalpha gene radiotherapy may significantly inhibit tumour growth, and that the anti-melanoma effect is superior to that of either gene therapy or radiotherapy alone. Our work provides the theoretical basis for further study on the gene radiotherapy of this tumour.

Construction of pETNF-P16 plasmid and its expression properties in EC9706 cell line induced by X-ray irradiation.

AIM: Recombined plasmid pETNF-P16 was constructed to investigate its expression properties in esophageal squamous carcinoma cell line EC9706 induced by X-ray irradiation and the feasibility of gene-radiotherapy for esophageal carcinoma. METHODS: Recombined plasmid pETNF-P16 was constructed and transfected into EC9706 cells with lipofectamine. ELISA, Western blot, and immunocytochemistry were performed to determine the expression properties of pETNF-P16 in EC9706 after transfection induced by X-ray irradiation. RESULTS: Eukaryotic expression vector pETNF-P16 was successfully constructed and transfected into EC9706 cells. TNFalpha expressions were significantly increased in the transfected cells after different doses of X-ray irradiation than in those after 0Gy irradiation (1,192.330-2,026.518 pg/mL, P<0.05-0.01), and the TNFalpha expressions and P16 were significantly higher 6-48 h after 2 Gy X-ray irradiation (358.963-585.571 pg/mL, P<0.05-0.001). No P16 expression was detected in normal EC9706 cells. However, there was strong expression in the transfected and irradiation groups. CONCLUSION: X-ray irradiation induction could significantly enhance TNFalpha and P16 expression in EC9706 cells transfected with pETNF-P16 plasmid. These results may provide important experimental data and therapeutic potential for gene-radiotherapy of esophageal carcinoma.

Anti-tumor effect of pEgr-IFNgamma gene-radiotherapy in B16 melanoma-bearing mice.

AIM: To construct a pEgr-IFNgamma plasmid and to investigate its expression properties of interferon-gamma (INF-gamma) induced by irradiation and the effect of gene-radiotherapy on the growth of melanoma. METHODS: A recombined plasmid, pEgr-IFNgamma, was constructed and transfected into B16 cell line with lipofectamine. The expression properties of pEgr-IFNgamma were investigated by ELISA. Then, a B16 melanoma-bearing model was established in mice, and the plasmid was injected into the tumor tissue. The tumor received 20 Gy X-ray irradiation 36 h after injection, and IFN-gamma expression was detected from the tumor tissue. A tumor growth curve at different time points was determined. RESULTS: The eukaryotic expression vector, pEgr-IFNgamma, was successfully constructed and transfected into B16 cells. IFN-gamma expression was significantly increased in transfected cells after X-ray irradiation in comparison with 0 Gy group (77.73-94.60 pg/mL, P<0.05-0.001), and was significantly higher at 4 h and 6 h than that of control group after 2 Gy X-ray irradiation (78.90-90.00 pg/mL, P<0.01-0.001). When the transfected cells were given 2 Gy irradiation 5 times at an interval of 24 h, IFN-gamma expression decreased in a time-dependent manner. From d 3 to d 15 after IFNgamma gene-radiotherapy, the tumor growth was significantly slower than that after irradiation or gene therapy alone. CONCLUSION: The anti-tumor effect of pEgr-IFNgamma gene-radiotherapy is better than that of genetherapy or radiotherapy alone for melanoma. These results may establish an important experimental basis for gene-radiotherapy of cancer.

[Construction of pEgr-TNFalpha plasmid and experimental study on the effect of gene-radiotherapy on mouse melanoma].

OBJECTIVE: The pEgr-TNFalpha plasmid was constructed to investigate the effect of gene-radiotherapy on melanoma and host immune system. METHODS: pEgr-TNFalpha plasmids were constructed and injected into tumor tissue, 36 hours later, the tumors were given 20 Gy X-ray irradiation. Tumor growth at different timepoints was record and immunologic parameters were detected 15 days later. RESULTS: From 3 to 15 d after pEgr-TNFalpha gene-radiotherapy the tumor growth was significantly slower than irradiation or genetherapy alone. NK activity, IL-2, TNFalpha and IL-1beta secretion activities of pEgr-TNFalpha gene-radiotherapy group and pEgr-TNFalpha gene group were higher than those of irradiation alone group significantly. CONCLUSION: The anti-tumor effect of pEgr-TNFalpha gene-radiotherapy is better than that of either one applied solely, and it can alleviate the lesion caused by radiation therapy.

Expression properties of recombinant pEgr-P16 plasmid in esophageal squamous cell carcinoma induced by ionizing irradiation.

AIM: To construct the recombinant pEgr-P16 plasmid for the investigation of its expression properties in esophageal squamous cell carcinoma induced by ionizing irradiation and the feasibility of gene-radiotherapy for esophageal carcinoma. METHODS: The recombinant pEgr-P16 plasmid was constructed and transfected into EC9706 cells with lipofectamine. Western blot, quantitative RT-PCR and flow cytometry were performed to study the expression of pEgr-P16 in EC9706 cells and the biological characteristics of EC9706 cell line after transfection induced by ionizing irradiation. RESULTS: The eukaryotic expression vector pEgr-P16 was successfully constructed and transfected into EC9706 cells. The expression of P16 was significantly increased in the transfected cells after irradiation while the transfected cells were not induced by ionizing irradiation. The induction of apoptosis in transfection plus irradiation group was higher than that in plasmid alone or irradiation alone. CONCLUSION: The combination of pEgr-P16 and irradiation could significantly enhance the P16 expression property and markedly induce apoptosis in EC9706 cells. These results may lay an important experimental basis for gene radiotherapy for esophageal carcinoma.