Immune response and protective efficacy of mannosylated polyethylenimine (PEI) as an antigen delivery vector, administered with a Streptococcus agalactiae DNA vaccine in Nile tilapia (Oreochromis niloticus).

This study examined the effectiveness of a DNA vaccine for S. agalactiae that was delivered by mannose-based polyethyleneimine (Man-PEI). The results showed that Man-PEI/pcDNA-Sip stimulated a higher serum antibody titer compared to control or other vaccine groups (p < 0.05). Additionally, it induced higher expression of immune-related genes, and increased activities of superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Furthermore, the Man-PEI/pcDNA-Sip group showed an improved relative percent survival (RPS) of 85.71%. These results demonstrate the potential value of Man-PEI as a vaccine delivery vehicle, and suggest that it can be effective in boosting the immune protective rate induced by pcDNA-Sip vaccines.

Tumor-Associated Antigens (TAAs) for the Serological Diagnosis of Osteosarcoma.

Osteosarcoma (OS) is the most common form of malignant bone tumor found in childhood and adolescence. Although its incidence rate is low among cancers, the prognosis of OS is usually poor. Although some biomarkers, such as p53, have been identified in OS, the association between the biomarkers and clinical outcome is not well understood. Thus, it is necessary to establish a method to identify patients diagnosed with OS at an early stage. It is becoming obvious that anti-tumor-associated antigens (TAAs) autoantibodies (TAAbs) in sera could be used as serological biomarkers in the detection of many different types of cancers. This notion indicates that TAAbs are considered as immunological "sentinels" associated with tumorigenesis underlying molecular events. It provides new insights into the molecular and cellular biology of the differential diagnosis of cancers. What's more, it is reported that a customized TAA array could significantly increase the sensitivity/specificity. TAA arrays also have great application prospects in detecting cancer at an early stage, monitoring cancer progression, discovering new therapeutic targets, and designing personalized treatment. In this review, we provide an overview of the TAAs identified in OS as well as the possibility that TAAs and TAAbs system be used as biomarkers in the immunodiagnosis and prognosis of OS.

Extracellular vesicle-associated repetitive element DNAs as candidate osteosarcoma biomarkers.

Osteosarcoma (OS) is the most common malignant bone tumor in children and young adults. Despite that high-risk factors have been identified, no test for early detection is available. This study aimed to identify circulating nucleic acid sequences associated with serum extracellular vesicle (EV) preparations at the time of OS diagnosis, as a step towards an OS early detection assay. Sequencing of small nucleic acids extracted from serum EV preparations revealed increased representation of diverse repetitive element sequences in OS patient versus control sera. Analysis of a validation cohort using qPCR of PEG-precipitated EV preparations revealed the over-representation of HSATI, HSATII, LINE1-P1, and Charlie 3 at the DNA but not RNA level, with receiver operating characteristic (ROC) area under the curve (AUC) ≥ 0.90. HSATI and HSATII DNAs co-purified with EVs prepared by precipitation and size exclusion chromatography but not by exosome immunocapture, indicative of packaging in a non-exosomal complex. The consistent over-representation of EV-associated repetitive element DNA sequences suggests their potential utility as biomarkers for OS and perhaps other cancers.

Immunoseroproteomic profiling in autoantibody to ENO1 as potential biomarker in immunodiagnosis of osteosarcoma by serological proteome analysis (SERPA) approach.

Osteosarcoma (OS) is the most common highly malignant primary solid bone tumor. Despite its relatively low incidence among cancers, it remains one of the most harmful primary malignant tumors in childhood and adolescence. It is now evident that serum autoantibodies against tumor-associated antigens (TAAs) could be used as serological cancer biomarkers in types of cancers. Serological proteome analysis (SERPA) approach was applied to profile anti-TAA autoantibody response in sera from patients with OS and normal human, as well as explore difference between this response. This approach can detect autoantibodies that could serve as clinical biomarkers and immunotherapeutic agents. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were further used to validate the level of identified TAAs. ENO1 as a 47kD TAA in OS was identified and characterized by SERPA. Analysis of 172 serum samples with OS, osteochondroma (OC), and normal human sera (NHS) by ELISA showed higher frequency of anti-ENO1 autoantibodies in OS sera compared to others. Interestingly, decrease of ENO1 immunoreactivity was observed in most patients after treatments, which may imply a potential association between anti-ENO1 autoantibody titers and disease progression. Nine of twelve sera reacted strongly against purified ENO1, but three reacted weakly against purified ENO1, which indicated 75.0% sera with positive optimal density values from ELISA were consistently positive in Western blotting. The expression of ENO1 in OS tissues was evaluated by immunohistochemistry in tumor microarray. ENO1 was one of the autoantibodies that elicit autoimmune responses in OS and can be used as biomarkers in immunodiagnosis and progression of OS.

Association between sheath blight resistance and chitinase activity in transgenic rice plants expressing McCHIT1 from bitter melon.

No usable resources with high-level resistance to sheath blight (SB) have yet been found in rice germplasm resources worldwide. Therefore, creating and breeding new disease-resistant rice resources with sheath blight resistance (SBR) are imperative. In this study, we inoculated rice plants with hyphae of the highly pathogenic strain RH-9 of rice SB fungus Rhizoctonia solani to obtain eight stable transgenic rice lines harbouring the chitinase gene (McCHIT1) of bitter melon with good SBR in the T5 generation. The mean disease index for SB of wild-type plants was 92% and 37-44% in transgenic lines. From 24 h before until 120 h after inoculation with R. solani, chitinase activity in stable transgenic plants with increased SBR was 2.0-5.5 and 1.8-2.7 times that of wild-type plants and plants of a disease-susceptible stable transgenic line, respectively. The correlation between SBR and chitinase activity in McCHIT1-transgenic rice line plants was significant. This work stresses how McCHIT1 from bitter melon can be used to protect rice plants from SB infection.

Matrine-induced autophagy counteracts cell apoptosis via the ERK signaling pathway in osteosarcoma cells.

The aim of the present study was to observe whether autophagy was induced by matrine, and to investigate the role of autophagy in the antitumor effects of matrine on human osteosarcoma MG-63 cells and its underlying mechanism. MG-63 cells were cultured in vitro in matrine at a concentration of 0.6, 0.8, 1.0 and 1.2 g/l for 0, 24, 48 and 72 h. A MTT assay was used to evaluate the proliferation inhibition of MG-63 cells by matrine, and Annexin V-fluorescein isothiocyanate/propidum iodide (PI) staining flow cytometry was used to analyze the apoptotic rate. Alterations in cell morphology was assessed by PI and Hoechst 33258 cell staining. Matrine-induced autophagy in MG-63 cells was confirmed by green fluorescent protein-microtubule-associated protein 1-light chain 3 (LC3) b transfection and fluorescence microscopy, and cell viability was investigated by MTT assay following inhibition of autophagy by chloroquine (CQ) pretreatment. The expression level of apoptosis-associated proteins B-cell lymphoma-2 (Bcl-2) and Bcl-2-like protein 4 (Bax), autophagy-associated LC3II protein, and the activation of extracellular signal-regulated kinase (ERK) was detected by western blotting. Cell proliferation was clearly inhibited by matrine in a dose- and time-dependent manner. Flow cytometry and Hoechst 33258/PI staining verified that matrine induced apoptosis in a time-dependent manner when cells were exposed to 1.1 g/l matrine; fluorescence microscopy demonstrated that green fluorescence puncta were enhanced with prolonged time of matrine incubation. Western blotting confirmed that the expression of pro-apoptosis-associated proteins Bax and LC3II, and phosphorylated-ERK were upregulated, and anti-apoptosis protein Bcl-2 was downregulated in a time-dependent manner following treatment with matrine. The cell viability of the matrine + CQ group was increased compared with the matrine group alone, which revealed that matrine treatment alone induced protective autophagy in MG-63 cells. In addiiton, expression of LC3II/LC3I decreased and the expression of BAX/Bcl-2 increased in the matrine + U0126 group compared with the matrine alone group. The present study demonstrated, to the best of our knowledge, for the first time that matrine induced protective autophagy via ERK activation in MG-63 cells, and matrine combined treatment with CQ or U0126 led to an increase in apoptosis in osteosarcoma cells.

Crosstalk between Beclin-1-dependent autophagy and caspase­dependent apoptosis induced by tanshinone IIA in human osteosarcoma MG-63 cells.

The aim of the present study was to ascertain whether or not autophagy is induced by tanshinone IIA (TanIIA), and to explore the crosstalk between autophagy and apoptosis in regards to the antitumor effects of TanIIA on MG-63 cells and the potential mechanism. MG-63 cells were cultured in vitro with various concentrations of TanIIA (0, 2.5, 5, 10 and 20 mg/l) for 0, 24, 48 and 72 h, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay was used to evaluate the inhibition of the proliferation of osteosarcoma MG-63 cells by TanIIA or in the presence/absence of chloroquine (CQ). Autophagic vacuoles and characteristic autophagosomes were observed by transmission electron microscopy (TEM). TanIIA-induced autophagy in MG-63 cells was confirmed by GFP-LC3 punctate fluorescence. The expression levels of apoptosis-related proteins caspase-3, caspase-8, caspase-9 and cleaved-PARP and autophagy-related proteins LC3II/LC3I and Beclin-1 were detected by western blotting. FITC-Annexin V/propidium iodide (PI) staining, flow cytometry and Hoechst 33258 staining were used to analyze the apoptotic rate. Fluorescence intensity of reactive oxygen species (ROS) was examined under a fluorescence microscope using an analysis software system. Cell proliferation was obviously inhibited by TanIIA in a dose- and time-dependent manner. Generation of autophagy was triggered by TanIIA (0-20 mg/l) treatment, and in a Beclin-1-dependent manner. Compared with the control group, the apoptosis ratio following treatment with 2.5 mg/l TanIIA failed to achieve statistical significance. Expression of caspase-3, -8 and -9, and cleaved-PARP in the other groups was gradually enhanced in dose-dependent manner. Our analysis also suggested that the influence of autophagy on TanIIA cytotoxicity had a phase effect; with low­dose drugs and shorter treatment periods, autophagy functioned as a damage repair mechanism. In conrast, when the cells were treated with higher doses of TanIIA for longer treatment periods, autophagic cell death contributed to apoptosis. Furthermore, generation of ROS occurred in a dose-dependent manner and pretreatment with NAC, a selective ROS scavenger, blocked the coexistence of Beclin-1 autophagy and caspase-dependent apoptosis. In conclusion, our findings provide strong evidence that TanIIA may be a potential therapeutic drug against osteosarcoma. Moreover, its cytotoxity can be enhanced with ROS agonists.

Cinobufagin induces autophagy-mediated cell death in human osteosarcoma U2OS cells through the ROS/JNK/p38 signaling pathway.

The main objective of this study was to explore whether autophagy could be triggered by cinobufagin, and to clarify the role of autophagy in the antitumor effects of cinobufagin on U2OS cells and the underlying mechanisms. U2OS cells were exposed to 15, 30, 60 and 120 mg/l cinobufagin for 0, 12, 24 and 48 h. An MTT assay was used to measure cell viability. FITC-Annexin â…¤/PI staining and flow cytometry were used to analyze the apoptotic ratio, while apoptotic morphological changes were assessed by PI and Hoechst 33258 viable cell staining. The effects of autophagy on the cells were investigated with GFP-LC3b green fluorescence plasmid transfection and transmission electron microscopy. The levels of caspase-3, -8, - 9, cleaved PARP, LC3-II/LC3-I, p62 and the activation of JNK/p-38 were detected by western blot analysis. Reactive oxygen species (ROS) fluorescence intensity was examined under fluorescence microscopy with an analysis software system. Cell proliferation was obviously inhibited by cinobufagin in a dose- and time-dependent manner. The apoptosis ratio was gradually increased with treatment time as evidenced by flow cytometric analysis and Hoechst 33258 staining. Exposure to cinobufagin resulted in the activation of caspase-3, -8, -9, as well as cleaved PARP which indicated that cinobufagin induced caspase-dependent apoptosis. Autophagy was confirmed in the cinobufagin-treated cells as evidenced by formation of autophagosomes, accumulation of GFP-LC3 fluorescence particles as well as the upregulation of LC3-II/LC3-I levels. Inhibition of autophagy diminished apoptosis as detected by the MTT assays. Moreover the percentage of apoptotic cells decreased following pretreatment with 3-MA, CQ and si-beclin-1. Cinobufagin also induced phosphorylation of the JNK and p38 signaling pathway as well as ROS generation. The JNK and p38 inhibitors significantly attenuated coexistence of apoptosis and autophagy-related proteins. The ROS scavenger also prevented phosphorylation of the JNK and p38 signaling pathway. Our research proved that cinobufagin triggered apoptosis and autophagic cell death via activation of the ROS/JNK/p-38 axis.

Identification of osteosarcoma-related specific proteins in serum samples using surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry.

Osteosarcoma (OS) is the most common malignant bone tumor. To identify OS-related specific proteins for early diagnosis of OS, a novel approach, surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry (SELDI-TOF-MS) to serum samples from 25 OS patients, 16 osteochondroma, and 26 age-matched normal human volunteers as controls, was performed. Two proteins showed a significantly different expression in OS serum samples from control groups. Proteomic profiles and external leave-one-out cross-validation analysis showed that the correct rate of allocation, the sensitivity, and the specificity of diagnosis were 100%. These two proteins were further identified by searching the EPO-KB database, and one of the proteins identified as Serine rich region profile is involved in various cellular signaling cascades and tumor genesis. The presence of these two proteins in OS patients but absence from premalignant and normal human controls implied that they can be potential biomarkers for early diagnosis of OS.

[Reconstruction of tumor-induced bone defects with vascularized fibula graft].

OBJECTIVE: To study the methods and effects of the reconstruction of tumor-induced bone defects with vascularized fibula graft. METHODS: From Oct. 1996 to Jan. 2005, 89 patients with bone defects were treated with fibula graft using different methods. Among the patients, 48 patients were male and 41 patients were female, ranging in age from 12 to 67 years, with an average of 35 years. Thirty-five patients were treated with inlay bone grafting after excision of focus, 15 patients were treated with single or double-strut fibula graft after tumor resection, 16 patients were treated with fibular head graft for juxta articular tumor resection, and 23 patients were treated with double-strut fibula combined with iliac graft after marginally resection. RESULTS: Enneking evaluation system was used to evaluate therapeutic effects. Among 35 patients treated with inlay bone graft after excision, 29 patients were followed up, and 26 patients got an excellent result, 1 good and 2 poor. Among 15 patients treated with single or double-strut fibula graft after tumor resection, 12 patients were followed up, and 8 patients got an excellent result, 1 good, 1 poor and 2 bad. Among 16 patients treated with fibular head graft for juxta articular tumor resection, 12 patients were followed up, and 7 patients got an excellent result, 3 good,1 poor and 1 bad. Among 23 patients treated with double-strut fibula combined with iliac graft after marginally resection, 17 patients were followed up, and 11 got an excellent result, 3 good, 1 fair and 2 bad. CONCLUSION: These treatment methods can greatly enrich the treatment methods for bone tumor, and satisfy the reconstruction after bone tumor excision in different position of the four limbs. These methods are reliable and can be chosen according to disease types.

[Prosthesis replacement of proximal humerus after resection of malignant tumor].

OBJECTIVE: To study the operative effect and complication of the prosthesis replacement of the proximal humerus with malignant tumor. METHODS: From October 1998 to August 2003, the prosthesis replacement was performed to treat the proximal femur with malignant tumor in 4 patients, including 2 patients with osteosarcoma (Enneking staging, I A) and 2 patients with giant cell tumor of the bone (II A, II B). By the International Society of Limb Salvage (ISOLS) criterion, the 2 cases of osteosarcoma were preoperatively scored as 4 and 5 points, and 2 cases of giant cell tumor of the bone were scored as 9 and 11 points. The patients' psychological conditions as well as their limb pain, shape, locality, activity, and function were also observed. RESULTS: The follow-up for 24-58 months (mean, 44 months) showed that there was no local recurrence or infection in all the patients except one patient who had the loosening of the screws for the fixation 17 months after operation and had no treatment. After operation, all the patients had a better postoperative extention angle from 22 degrees to 41 degrees (mean, 25 degrees), bending angle from 29 degrees to 80 degrees (mean, 35 degrees), abduction angle from 5 degrees to 28 degrees, and circumgyrate angle from 15 degrees to 22 degrees in their shoulder joints. However, the shoulder joint function was still unstable to some extent and the joints had a decreased strength. By the criterion formulated by the ISOLS, the postoperative score for assessing the 2 patients with osteosarcoma was increased by 16 points when compared with the preoperative score; the score for the 2 patients with giant cell tumor of the bone was increased by 9.5 points. CONCLUSION: The prosthesis replacement to treat the malignant tumor of the proximal humerus is the good method of choice and has a good therapeutic result; however, there are more complications and so the method should be cautiously employed in the clinical practice.