Effects of trunk training using motor imagery on trunk control ability and balance function in patients with stroke.

OBJECTIVE: To explore the effects of trunk training using motor imagery on trunk control and balance function in patients with stroke. METHODS: One hundred eligible stroke patients were randomly divided into a control group and trial group. The control group was given routine rehabilitation therapy, while the trial group was given routine rehabilitation therapy and trunk training using motor imagery. RESULTS: Prior to treatment, there was no significant difference between the two groups (P > 0.05) in Sheikh's trunk control ability, Berg rating scale (BBS), Fugl-Meyer assessment (FMA), movement length, movement area, average front-rear movement speed, average left-right movement speed, and surface electromyography (sEMG) signal of the bilateral erector spinae and rectus abdominis. After treatment, Sheikh's trunk control ability, FMA, and BBS in the two groups were significantly higher than those before treatment (P < 0.05). The movement length, movement area, the average front-rear movement speed, and the average left-right movement speed in the two groups decreased significantly (P < 0.05). The differences of these indicators between the two groups were statistically significant (P < 0.05). After treatment, the rectus abdominis and erector spinae on the affected side of the two groups improved when compared with those before treatment (P < 0.05). The rectus abdominis and erector spinae on the healthy side of the trial group descended after treatment (P < 0.05), while little changes were observed on the healthy side of the control group after treatment (P > 0.05). The rectus abdominis and erector spinae on the affected side of the trial group improved when compared with those in the control group (P < 0.05). There was no significant difference between the two groups in the decline of abdominalis rectus and erector spinal muscle on the healthy side. CONCLUSION: Trunk training using motor imagery can significantly improve the trunk control ability and balance function of stroke patients and is conducive to promoting the recovery of motor function.

Imaging signs and the qualitative diagnosis of solitary rib lesions using 99mtechnetium-methylene diphosphonate whole-body bone imaging in patients with a malignant tumor.

Background: The aim of this study was to summarize the valuable information for qualitative diagnosis by investigating the imaging signs from the whole-body bone imaging of solitary rib lesions. Methods: A retrospective analysis was conducted of the data from 313 patients with malignant tumors and solitary rib lesions identified using whole-body bone imaging in Department of Nuclear Medicine of Central South University Xiangya School Affiliated Haikou Hospital between January 2015 and December 2017. Based on the final comprehensive diagnosis of the rib lesions, the patients were divided into a bone metastasis group, fracture group, other benign lesions group, and an uncertain group, and the characteristic imaging changes in rib lesions in each group were explored. Results: (I) Significant differences were identified among the 4 groups (P<0.001) in the distribution of lesions in the anterior, posterior, and lateral ribs and proximal costal cartilage. The fracture group had the highest proportion of lesions in the anterior ribs (99/121, 81.8%) and proximal costal cartilage (74.4%, 90/121). (II) Significant differences were detected in morphology, concentration, boundaries, and radioactivity distribution among the 4 groups of patients (P<0.001). The bone metastasis group had the highest proportion of lesions appearing as stripes (35/67, 52.2%), and the fracture group had the highest proportion of lesions appearing as spots (94.2%, 114/121) and the lowest proportion appearing as stripes (3/121, 2.5%). (III) Significant differences were found in the longitudinal diameter, transverse diameter, aspect ratio, and tumor-to-normal tissue ratio between the 4 groups (P<0.001). The longitudinal diameter (27.8±16.0 mm) and aspect ratio (1.9±1.0) of the bone metastasis group were the highest, whereas the longitudinal diameter (15.2±3.9 mm) and aspect ratio (1.0±0.2) of the fracture group were the smallest. Conclusions: This study revealed that different types of solitary rib lesions had relatively characteristic imaging signs in whole-body bone imaging.

Revitalizing quality of life: a case report on the beneficial impact of comprehensive rehabilitation therapy in treating upper-limb lymphedema following breast cancer surgery.

Objective: To underscore the paramount significance of incorporating comprehensive rehabilitation therapy as a crucial aspect of managing lymphedema caused by breast cancer surgery, and to illuminate our first-hand experience and insights gained in utilizing this approach. Methods: We present a case report of a breast cancer survivor who had been suffering from persistent left upper-limb edema for over 15 years, who was effectively treated with a combination of conventional rehabilitation (seven-step decongestion therapy) and a comprehensive rehabilitation program (seven-step decongestion therapy, along with core and respiratory function training, as well as functional brace wearing). The efficacy of the rehabilitation therapy was evaluated through a comprehensive assessment. Results: Although the patient underwent the conventional rehabilitation program for one month, only limited improvement was observed. However, after an additional month of comprehensive rehabilitation treatment, the patient exhibited significant improvement in both lymphedema and the overall function of the left upper limb. The patient's progress was quantified by measuring the reduction in arm circumference, which demonstrated a notable decrease. Furthermore, improvements in joint range of motion were observed, with forward flexion of the shoulder enhancing by 10°, forward flexion improving by 15°, and elbow flexion increasing by 10°. In addition, manual muscular strength tests revealed an increase in strength from Grade 4 to Grade 5. The patient's quality of life was also significantly improved, as evidenced by the increase in the Activities of Daily Living score from 95 to 100 points, the increase in the the Functional Assessment of Cancer Therapy: Breast score from 53 to 79 points, and the decrease in the Kessler Psychological Distress Scale score from 24 to 17 points. Conclusion: While seven-step decongestion therapy has been shown to be effective in reducing upper-limb lymphedema caused by breast cancer surgery, it has limitations in treating more chronic cases of the condition. However, when combined with core and respiratory function training and functional brace wearing, seven-step decongestion therapy has been shown to be even more effective in reducing lymphedema and improving limb function, ultimately leading to significant improvements in quality of life.

Effects of paired associated stimulation with different stimulation position on motor cortex excitability and upper limb motor function in patients with cerebral infarction.

OBJECTIVE: To investigate the effects of paired associated stimulation (PAS) with different stimulation position on motor cortex excitability and upper limb motor function in patients with cerebral infarction. METHOD: A total of 120 volunteers with cerebral infarction were randomly divided into four groups. Based on conventional rehabilitation treatment, the PAS stimulation group was given the corresponding position of PAS treatment once a day for 28 consecutive days. The MEP amplitude and RMT of both hemispheres were assessed before and after treatment, and a simple upper limb Function Examination Scale (STEF) score, simplified upper limb Fugl-Meyer score (FMA), and improved Barthel Index (MBI) were used to assess upper limb motor function in the four groups. RESULTS: Following PAS, the MEP amplitude decreased, and the RMT of abductor pollicis brevis (APB) increased on the contralesional side, while the MEP amplitude increased and the RMT of APB decreased on the ipsilesional side. After 28 consecutive days the scores of STEF, FMA, and MBI in the bilateral stimulation group were significantly better than those in the ipsilesional stimulation group and the contralesional stimulation group, but there was no significant difference in the scores of STEF, FMA, and MBI between the ipsilesional stimulation group and the contralesional stimulation group. CONCLUSION: The excitability of the motor cortex can be changed when the contralesional side or the ipsilesional side was given the corresponding PAS stimulation, while the bilateral PAS stimulation can more easily cause a change of excitability of the motor cortex, resulting in better recovery of the upper limb function.

The Association Between Lung Fluorodeoxyglucose Metabolism and Smoking History in 347 Healthy Adults.

OBJECTIVE: This study aimed to evaluate the relationship between fluorodeoxyglucose metabolism and smoking history in healthy adults by analyzing lung standardized uptake value (SUV). METHODS: The 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) studies of 347 patients who did not show signs of having malignant diseases or lung inflammation were retrospectively evaluated. Four circular regions of interest (ROI) were manually drawn on the upper and lower lung regions. The averages of maximum SUV (SUVmax-avr) and mean SUV (SUVmean-avr) were calculated, and the mean values of each parameter for non-smokers, ex-smokers, and current smokers were compared. The correlation between SUVmax-avr and smoking history (tobacco burden and the duration of smoking cessation) was assessed based on present smoking status. The ex-smokers and current smokers were divided into three groups according to their tobacco burden, and the SUVmax-avrs of the two groups were compared. RESULTS: Both the mean values of SUVmax-avr and SUVmean-avr increased based on smoking history, with non-smokers having the lowest values and current smokers the highest. Tobacco burden had a positive correlation with SUVmax-avr in current smokers (r = 0.474, P< 0.001). However, neither tobacco burden (r = 0171, P = 0.162) nor duration of smoking cessation (r = 0.212, P = 0.082) had a significant correlation with SUVmax-avr in ex-smokers. The mean SUVmax-avr of current smokers was significantly higher than that of ex-smokers in patients with a medium or large tobacco burden (P = 0.012, P< 0.001, respectively). Although there was no significant difference between the mean SUVmax-avrs of ex-smokers and current smokers in patients with a small tobacco burden (P = 0.888), the mean SUVmax-avrs of both ex-smokers and current smokers with a small tobacco burden were significantly higher than that of non-smokers (P< 0. 001, P< 0.001, respectively). CONCLUSION: The findings indicate that lung SUV increases in current heavy smokers and partially decreases after the cessation of smoking, which is in line with previous reports studied by analyzingfluorodeoxyglucose (FDG) metabolism of lung specimens.

Effect of Paired Associative Stimulation on Motor Cortex Excitability in Rats.

Paired associative stimulation (PAS), combining transcranial magnetic stimulation (TMS) with electrical peripheral nerve stimulation (PNS) in pairs with an optimal interstimulus interval (ISI) in between, has been shown to influence the excitability of the motor cortex (MC) in humans. However, the underlying mechanisms remain unclear. This study was designed to explore an optimal protocol of PAS, which can modulate the excitability of MC in rats, and to investigate the underlying mechanisms. The resting motor thresholds (RMTs) of TMS-elicited motor evoked potentials (MEPs) recorded from the gastrocnemius muscle and the latency of P1 component of somatosensory evoked potentials (SEPs) induced by electrical tibial nerve stimulation were determined in male Sprague-Dawley rats (n=10). Sixty rats were then randomly divided into 3 groups: a PAS group (further divided into 10 subgroups at various ISIs calculated by using the latency of P1, n=5, respectively), a TMS (only) group (n=5) and a PNS (only) group (n=5). Ninety repetitions of PAS, TMS and PNS were administered to the rats in the 3 groups, respectively, at the frequency of 0.05 Hz and the intensity of TMS at 120% RMT and that of PNS at 6 mA. RMTs and motor evoked potentials' amplitude (MEPamp) were recorded before and immediately after the interventions. It was found that the MEPamp significantly decreased after PAS at ISI of 5 ms (P<0.05), while the MEPamp significantly increased after PAS at ISI of 15 ms, as compared with those before the intervention (P<0.05). However, the RMT did not change significantly after PAS at ISI of 5 ms or 15 ms (P>0.05). PAS at other ISIs as well as the sole use of TMS and PNS induced no remarkable changes in MEPamp and RMT. In conclusion, PAS can influence motor cortex excitability in rats. Neither TMS alone nor PNS alone shows significant effect.

Effects of Repetitive Transcranial Magnetic Stimulation on Astrocytes Proliferation and nNOS Expression in Neuropathic Pain Rats.

This study investigated the effects of different frequencies of repetitive transcranial magnetic stimulation (rTMS) on chronic neuropathic pain in rats. The behavior of rats with experimental chronic neuropathic pain was observed, and the expression of neuronal nitric oxide synthase (nNOS) in the ipsilateral dorsal root ganglions (DRGs) and the activation and proliferation of astrocytes in the ipsilateral spinal dorsal horn were detected. Thirty-two male Sprague-Dawley rats were randomly divided into four groups: sham-operated group, sham-rTMS group, 1 Hz group and 20 Hz group (8 rats in each group). Chronic constriction nerve injury induced by sciatic nerve ligation was made to establish the models of the chronic neuropathic pain in rats except those in the sham-operated group. Then we applied different frequencies of rTMS to the primary motor cortex (Ml) contralateral to the pain side once daily for 10 consecutive days. Pain behavior scores were observed before and after treatment. Western blot analysis was used to detect the expression of nNOS in ipsilateral L4-6 DRGs. Double immunofluorescent labeling for glial fibrillary acidic protein (GFAP) and 5-bromo-2- deoxyuridine (BrdU) was employed to observe the activation and proliferation of astrocytes in the ipsilateral L4-6 spinal dorsal horn. After rTMS treatment, the spontaneous pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS group (P<0.05). Moreover, the brush-evoked pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS and 1 Hz group (P<0.05), suggesting that the spontaneous pain and brush-evoked pain in the 20 Hz group were significantly alleviated. Western blot analysis revealed that the expression of nNOS in ipsilateral L4-6 DRGs was significantly decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group (P<0.01) after rTMS treatment. Double immunofluorescence suggested that the expression of GFAP and the co-localization with BrdU in astrocytes were less in the sham-operated group than those in the sham-rTMS group and the 1 Hz group in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain. After rTMS treatment, the expression of GFAP and the co-localization with BrdU decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group (P<0.05). In addition, the alleviation degree of spontaneous pain and brush-evoked pain in the 20 Hz group was negatively correlated with the expression of nNOS in ipsilateral DRGs and the number of GFAP/BrdU co-labelled astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain (P<0.05). It was suggested that high-frequency rTMS may relieve neuropathic pain through down-regulating the overexpression of nNOS in ipsilateral DRGs and inhibiting the activity and proliferation of astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain.

Prediction and analysis of HLA-A2/A24-restricted cytotoxic T-lymphocyte epitopes of the tumor antigen MAGE-n using the artificial neural networks method on NetCTL1.2 Server.

Cancer immunotherapy has become one of the most important therapeutic approaches to cancer in the past two decades. Tumor antigen-derived peptides have been widely used to elicit tumor-specific cytotoxic T lymphocytes (CTLs). Antigen-specific CTLs induced by MAGE-derived peptides have proven to be highly efficacious in the prevention and treatment of various types of tumor. MAGE-n is a new member of the MAGE gene family and has been shown to be closely associated with hepatocellular carcinoma. It is highly homologous to the MAGE-A gene subfamily, particularly to MAGE-3 (93%). MAGE-n-derived peptide QLVFGIEVV is a novel HLA-A2.1-restricted CTL epitope that induces MAGE-n-specific CTLs in vitro. Identification of these CTL epitopes may lead to clinical applications of these peptides as cancer vaccines for patients with MAGE-n(+)/HLA-A2(+) tumors. In the present study, HLA-A/A24-restricted CTL epitopes of antigen MAGE-n were predicted using the NetCTL1.2 Server on the web, COMB >0.85. The results showed that the NetCTL1.2 Server prediction method improved prediction efficacy and accuracy. Additionally, 8 HLA-A2- and 9 HLA-A24-restricted CTL epitope candidates (nonamers) derived from the tumor antigen MAGE-n were predicted. These nonamers, following identification via experimentation, may contribute to the development of potential antigen peptide tumor vaccines.

The antitumor immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70/SEA complex protein vaccine following different administration routes.

Our previous study showed that nanoemulsion-encapsulated MAGE1-HSP70/SEA (MHS) complex protein vaccine elicited MAGE-1 specific immune response and antitumor effects against MAGE-1-expressing tumor and nanoemulsion is a useful vehicle with possible important implications for cancer biotherapy. The purpose of this study was to compare the immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70 and SEA as NE(MHS) vaccine following different administration routes and to find out the new and effective immune routes. Nanoemulsion vaccine was prepared using magnetic ultrasound methods. C57BL/6 mice were immunized with NE(MHS) via po., i.v., s.c. or i.p., besides mice s.c. injected with PBS or NE(-) as control. The cellular immunocompetence was detected by ELISpot assay and LDH release assay. The therapeutic and tumor challenge assay were also examined. The results showed that the immune responses against MAGE-1 expressing murine tumors elicited by NE(MHS) via 4 different routes were approximately similar and were all stronger than that elicited by PBS or NE(-), suggesting that this novel nanoemulsion carrier can exert potent antitumor immunity against antigens encapsulated in it. Especially, the present results indicated that nanoemulsion vaccine adapted to administration via different routes including peroral, and may have broader applications in the future.

The antitumor immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70/SEA complex protein vaccine following peroral administration route.

Previous studies have shown that there are profuse lymphatic tissues under the intestinal mucous membrane. Moreover, vaccine administered orally can elicit both mucous membrane and system immune response simultaneously, accordingly induce tumor-specific cytotoxic T lymphocyte. As a result, the oral route is constituted the preferred immune route for vaccine delivery theoretically. However, numerous vaccines especially protein/peptide vaccines remain poorly available when administered by this route. Nanoemulsion has been shown as a useful vehicle can be developed to enhance the antitumor immune response against antigens encapsulated in it and it is good for the different administration routes. Of particular interest is whether the protein vaccine following peroral route using nanoemulsion as delivery carrier can induce the same, so much as stronger antitumor immune response to following conventional ways such as subcutaneous (sc.) or not. Hence, in the present study, we encapsulated the MAGE1-HSP70 and SEA complex protein in nanoemulsion as nanovaccine NE (MHS) using magnetic ultrasound method. We then immuned C57BL/6 mice with NE (MHS), MHS alone or NE (-) via po. or sc. route and detected the cellular immunocompetence by using ELISpot assay and LDH release assay. The therapeutic and tumor challenge assay were examined then. The results showed that compared with vaccination with MHS or NE (-), the cellular immune responses against MAGE-1 could be elicited fiercely by vaccination with NE (MHS) nanoemulsion. Furthermore, encapsulating MHS in nanoemulsion could delay tumor growth and defer tumor occurrence of mice challenged with B16-MAGE-1 tumor cells. Especially, the peroral administration of NE (MHS) could induce approximately similar antitumor immune responses to the sc. administration, but the MHS unencapsulated with nanoemulsion via po. could induce significantly weaker antitumor immune responses than that via sc., suggesting nanoemulsion as a promising carrier can exert potent antitumor immunity against antigen encapsulated in it and make the tumor protein vaccine immunizing via po. route feasible and effective. It may have a broad application in tumor protein vaccine.

The anti-tumor immune response induced by a combination of MAGE-3/MAGE-n-derived peptides.

Tumor antigen-derived peptides have been widely used to elicit tumor-specific cytotoxic T lymphocytes (CTLs). MAGE gene products are of particular interest owing to their wide expression in many tumors and their potential to induce tumor-specific CTL responses. Antigen-specific CTLs induced by MAGE gene-derived peptides have proven to be highly efficacious in the prevention and treatment of various types of tumors. MAGE-3 has been used as a target for tumor immunotherapy. MAGE-n is a new member of the MAGE gene family and has been shown to be closely associated with hepatocellular carcinoma (HCC). However, the majority of previous investigations focused on the single MAGE antigen-derived peptides as a cancer vaccine which has many limitations. The tumor antigen expression is known to be heterogeneous and tumor cells can express multiple tumor antigens. Thus, vaccines incorporating single antigen-derived epitopes may be inadequate in generating a complete immune response against the tumor. Instead, a polyvalent vaccine incorporating epitopes derived from several tumor antigens may be more effective. Our study combined the MAGE-3 and MAGE-n-derived peptides as a cancer vaccine. The results showed that the combination of MAGE-3 and MAGE-n epitopes induced more effective anti-tumor immune responses than either of the peptides alone. In addition, the peptide-specific activity was observed to be in an MHC-restricted manner. Our study indicated that the combination of several tumor antigen-derived peptides may present a better peptide-based cancer immunotherapy.

[Anti-tumor immune responses of nanoemulsion-encapsulated MHS vaccine].

AIM: To prepare nanoemulsion-encapsulated MAGE1-Hsp70/SEA and to evaluate its anti-tumor effects in mouse. METHODS: Nanoemulsion vaccine NE(MHS) was prepared using magnetic ultrasound methods and used to immunize C57BL/6 mice. The cellular immune responses were detected by IFN-gamma ELISPOT and LDH release assay. The tumor challenge assay was performed too. RESULTS: (1) The mean size of NE(MHS) was (20+/-5) nm. The encapsulation rate was 87% and the nanoemulsion vaccine had a good stability. (2) The frequency of MAGE-1 specific CTL and cytotoxicity of CTL to B16-MAGE-1 cells were both greatly enhanced in immunization group than those in control group (P<0.05). NE(MHS) could significantly delay the appearance of tumors and increase the percentage of tumor-free mice. CONCLUSION: The nanoemulsion had excellent physical and chemical characteristics. It could elicit MAGE-1-specific cellular immune response and anti-tumor effects against the MAGE-1-expressing tumor.

[Adenovirus mediated targeted genetherapy of Staphylococcal enterotoxin A and CD80 for hepatoma and its primary immune mechanisms].

AIM: To observe the effects of hepatoma-targeting recombinant adenovirus vectors of staphylococcal enterotoxin A (SEA) and/or CD80 gene on hepatoma and to study its immunological mechanisms. METHODS: Using AdEasy adenovirus system, we constructed recombinant adenovirus vectors of SEA and/or CD80 gene driven by alpha-fetoprotein (AFP) enhancer I and promoter. After intratumoral therapy for the mice bearing subcutaneous xenograft hepatoma with the recombinant adenoviruses, SEA and/or CD80 mRNA and protein were detected by RT-PCR and Western blot. IFN-gamma-producing cell frequency and specific cytotoxicity of T lymphocytes to Hepa1-6 cells were detected by ELISpot and LDH-released assay in the splenocytes. Effects of recombinant adenoviruses on hepatoma were assessed by changes of tumor volumes and survival time in the treated mice. RESULTS: The recombinant adenoviruses constructed by us made SEA and/or CD80 mRNA and protein targetedly express in hepatoma tissues. When compared with the empty vector and PBS groups, the IFN-gamma-producing cell frequency and specific cytotoxicity of T lymphocytes increased, the tumor volumes of mice decreased and the survival time increased in the double-gene and single-gene groups. Double genes elicited better antitumor effects and stronger immune responses. There were no significant differences in the effects between CD80 group and SEA group or between empty vector group and PBS group. CONCLUSION: The hepatoma-targeting recombinant adenovirus vectors constructed in this study can elicit effective antitumor effects on hepatoma and the effects of double genes are better than that of single gene.

Immunization with truncated sequence of Telomerase Reverse Transcriptase induces a specific antitumor response in vivo.

To select the MHC-I-binding epitope-rich sequence of mice telomerase reverse transcriptase (mTERT) and study the antitumor immune response induced by truncated TERT through mRNA-transfected dendritic cells (DCs) immunization in mice. The MHC-I-binding epitopes of TERT were predicted using bioinformatics software. The selected sequence of TERT (Truncated mTERT, TERT(t), mTERT cDNA 1776 bp-2942 bp encoding 584 aa-969 aa) was cloned from B16 mouse melanoma cells and inserted into pBluescriptIIKS(+) plasmid downstream of the T7 promoter. TERT(t) RNA was prepared through in vitro transcription. Bone marrow-derived DCs were electroporated with TERT(t) RNA and used to immunize syngeneic naïve mice. The quantity and cytotoxic activity of TERT-specific cytotoxic T lymphocytes (CTLs) in mice spleen were evaluated using IFN-gamma enzyme-linked immunospot (ELISPOT) and Lactate dehydrogenase release assay. The immunoprophylactic effects against TERT positive tumor induced by TERT(t) RNA transfected DC in vivo were evaluated through an immunized-challenged mouse model. TERT(t) was cloned and in vitro transcribed into TERT(t) mRNA. As shown in FCM analysis, the efficiency of DC electroporation is 35.1% (29.7-41.2%). After electroporation, a subtle increase of costimulator and MHC-II molecules were expressed on the cell surface. Immunization of TERT(t) mRNA transfected DCs induced IFN-gamma-secreting CTLs which manifested specific cytotoxic activity against TERT-positive target cells. In a cancer mouse model, vaccination of TERT(t) mRNA-transfected DCs suppressed the growth of TERT positive tumors (p=0.001) and prolong the survival time of tumor-bearing animals (p=0.029). TERT(t) evokes an antitumor immune response in vivo which is targeted to TERT. TERT(t) can be used as an antigeneic sequence to produce anti-TERT tumor vaccine.

Truncated TERT mRNA transfected dendritic cells evoke TERT specific antitumor response in vivo.

BACKGROUND/AIMS: To evaluate the antitumor immune response induced by truncated TERT (TERTt) mRNA transfected dendritic cells (DCs) in METHODOLOGY: Truncated mouse TERT sequence (according to mice telomerase reverse transcriptase mRNA 1776bp-2942bp) was cloned from B16 mice melanoma cells and inserted into pBluescript II KS(+) plasmid downstreaming of T7 promoter. The in vitro transcription was performed to prepare TERTt mRNA. The bone marrow-derived DCs isolated from BALB/c or C57B/L mice were electroporated with TERTt mRNA and recruited to immunize syngeneic naive mice respectively. The quantity and cytotoxic activity of tumor specific cytotoxic T lymphocytes (CTLs) in mice spleen were evaluated by using IFN-gamma enzyme-linked immunospot (ELIspot) and LDH release assay. The immunoprophylactic effects induced by TERTt mRNA transfected DC were evaluated in immunized-challenged mouse model. RESULTS: TERTt was cloned and transcripted into TERTt mRNA in vitro. TERTt mRNA transfected bone marrow-derived DCs were prepared. As shown by transfecting with EGFP mRNA, the DC transfected efficiency is 35.1% and there was a subtle increase of costimulator and MHC-II molecule expression after electroporation. Immunization with TERTt mRNA transfected DCs can induce TERTt and TERT-specific IFN-gamma secreting CTLs in the spleen of immunized mice. The splenocytes isolated from mice immunized with TERTt mRNA transfected DCs showed specific cytotoxic activity against TERTt and TERT-positive target cells. Using a syngeneic cancer mouse model, it was shown that TERTt mRNA transfected DCs vaccination can suppress the growth of TERT-positive tumor inoculation. CONCLUSIONS: TERTt mRNA transfected bone marrow-derived DCs can evoke antitumor immune response in vivo effectively and TERTt can serve as a universal tumor associated antigen to produce DC-based tumor vaccine.

[Construction of hepatoma-targeting recombinant co-expression adenovirus vector of SEA and CD80 and identification of its expression].

AIM: To construct hepatoma-targeting recombinant co-expression adenovirus vector of Staphylococcal enterotoxin A (SEA) and CD80 gene. METHODS: Us-ing the adenovirus transfer plasmids pShuttle and pShuttle-CMV, we constructed a new transfer plasmid pShuttle2 with polyA signal sequence instead of CMV enhancer/promoter. AFP enhancer, promoter, SEA or CD80 gene was subcloned into pShuttle2 from the vectors pKS-EP or pMD18-T-BIS respectively, and then the constructed plasmid pShuttle2-BIS containing AFP enhancer, promoter, SEA or CD80 gene was cotransformed into E.coli BJ5183 with backbone vector pAdEasy-1 to obtain recombinant adenovirus DNA. The recombinant adenovirus DNA was transfected into 293 cells to prepare adenovirus. After AFP-producing cell line Hepa1-6 and AFP-nonproducing cell lines B16 and NIH3T3 were infected by recombinant adenovirus, the expression of SEA and CD80 on the surface of cells was detected by indirect immunofluorescent staining, laser confocal microscope and flow cytometry (FCM). RESULTS: SEA and CD80 was specifically co-expressed on the surface of infected Hepa1-6 cells but not on B16 and NIH3T3 cells. CONCLUSION: Hepatoma-targeting recombinant co-expression adenovirus vector of SEA and CD80 gene was successfully constructed, which lays the foundation for further research on application of SEA and CD80 in targeted gene therapy for hepatoma and the underlying immunological mechanisms.

Tumor cells with B7.1 and transmembrane anchored staphylococcal enterotoxin A generate effective antitumor immunity.

Staphylococcus enterotoxin A (SEA) stimulates T cells bearing certain TCR beta-chain variable regions, when bound to MHC-II molecules, and is a potent inducer of CTL activity and cytokines production. To decrease toxicity of SEA to the normal MHC-II(+) cells and to localize the immune response induced by SEA to the tumor site, my colleague previously genetically fused SEA with B7.1 transmembrane region (named as SEAtm) to make SEA express on the surface of tumor cells and tumor cells modified with SEAtm could induce efficient antitumor immunity in vitro. The tumor cell vaccines modified with multiple immune activators frequently elicited stronger antitumor immune responses than single-modified vaccines. In this study, we modified the tumor cell vaccine with B7.1 and SEAtm to improve efficiency in the application of SEA. First, SEAtm gene was subcloned from recombinant plasmid pLXSNSEP by PCR and murine B7.1 gene was cloned from splenocytes derived from C57BL/6 mice by RT-PCR. Then, the eukaryotic co-expression vector of SEA and murine B7.1 gene was constructed and named as pcDNA-BIS. B16 cell lines stably expressing SEA and/or B7.1 were established by screening with G418 after transfection and inactivated for the preparation of tumor cell vaccines to treat mice bearing established B16 tumors. The results indicated that the dual-modified tumor cell vaccine B16/B7.1+SEAtm (B16-BIS) elicited significantly stronger antitumor immune responses in vivo when compared with the single-modified tumor cell vaccines B16/B7.1 (B16-B7.1) and B16/SEAtm (B16-SEAtm), and supported the feasibility and effectiveness of the dual-modified tumor cell vaccine with superantigen and co-stimulatory molecule.

[Establishment of an HHCC cell line stably transfected by tumor antigen MAGE-3 gene and its mRNA expression].

AIM: To construct the eukaryotic expression vector of tumor antigen MAGE-3 and establish human hepatocellular carcinoma cell line (HHCC) expressing MAGE-3. METHODS: The MAGE-3 gene was amplified by PCR and cloned into the eukaryotic expression vector pIRES2-EGFP to construct the pIRES2-EGFP-MAGE-3 plasmid. The recombinant plasmid pIRES2-EGFP-MAGE-3 was transfected into HHCC cells by lipofectamine, and then the positive clones were screened by G418. The expression of enhanced green fluorescent protein (EGFP) and MAGE-3 mRNA in positive clones were detected by fluorescence microscope and RT-PCR, respectively. RESULTS: The eukaryotic expression vector pIRES2-EGFP-MAGE-3 was successfully constructed. The expression of EGFP was found by fluorescence microscope detection and MAGE-3 mRNA transcription was detected by RT-PCR in the positive clones. CONCLUSION: The stable MAGE-3-transfected HHCC cell line is successfully established, which will provide experimental basis for further study on immunotherapy for hepatocellular carcinoma using MAGE-3 as target antigen.

Heat-shocked tumor cell lysate-pulsed dendritic cells induce effective anti-tumor immune response in vivo.

AIM: To study whether heat-shocked tumor cells could enhance the effect of tumor cell lysate-pulsed dendritic cells (DCs) in evoking anti-tumor immune response in vivo. METHODS: Mouse undifferentiated colon cancer cells (CT-26) were heated at 42 degrees Celsius for 1 h and then frozen-thawed. The bone marrow-derived DCs pulsed with heat-shocked CT-26 cell lysate (HSCT-26 DCs) were recruited to immunize syngeneic naive BALB/c mice. The cytotoxic activity of tumor specific cytotoxic T lymphocytes (CTLs) in mouse spleen was evaluated by IFN-enzyme-linked immunospot (ELISpot) and LDH release assay. The immunoprophylactic effects induced by HSCT-26 DCs in mouse colon cancer model were compared to those induced by single CT-26 cell lysate-pulsed DCs (CT-26 DCs) on tumor volume, peritoneal metastasis and survival time of the mice. RESULTS: Heat-treated CT-26 cells showed a higher hsp70 protein expression. Heat-shocked CT-26 cell lysate pulsing elevated the co-stimulatory and MHC-II molecule expression of bone marrow-derived DCs as well as interleukin-12 p70 secretion. The IFN-gamma secreting CTLs induced by HSCT-26 DCs were significantly more than those induced by CT-26 DCs (P=0.002). The former CTLs' specific cytotoxic activity was higher than the latter CTLs' at a serial E/T ratio of 10:1, 20:1, and 40:1. Mouse colon cancer model showed that the tumor volume of HSCT-26 DC vaccination group was smaller than that of CT-26 DC vaccination group on tumor volume though there was no statistical difference between them (24 mm3 vs 8 mm3, P=0.480). The median survival time of mice immunized with HSCT-26 DCs was longer than that of those immunized with CT-26 DCs (57 d vs 43 d, P=0.0384). CONCLUSION: Heat-shocked tumor cell lysate-pulsed DCs can evoke anti-tumor immune response in vivo effectively and serve as a novel DC-based tumor vaccine.