CYP4B1 polymorphisms and the risk of breast cancer in Chinese women: a case-control study.

BACKGROUND: Breast cancer (BC) is one of the malignant diseases threatening the life and health of women worldwide. The CYP4B1 gene was abnormally expressed in BC and was associated with the prognosis of BC patients. This study aimed to explore the relationship between CYP4B1 single nucleotide polymorphisms (SNPs) and BC risk in Chinese women. METHODS: A case-control study of 1,143 women (571 patients and 572 healthy individuals) was conducted. Rs2297813 G/T, rs12142787 G/A, and rs3766197 C/T in CYP4B1 were selected and genotyped by MassARRAY system. The relationships between these SNPs and the risk of BC were assessed by logistic regression analysis. In addition, multi-factor dimensionality reduction (MDR) was used to analyze SNP-SNP interactions. RESULTS: CYP4B1 rs2297813 had a risk-increasing effect on BC in women with body mass index (BMI) ≤ 24 kg/m2 (OR = 1.72, p = 0.026). CYP4B1 rs12142787 was associated with an increased BC risk in smokers (AA: OR = 1.32, p = 0.045). Among non-drinkers, rs2297813 (OR = 1.69, p = 0.009) and rs12142787 (OR = 1.51, p = 0.020) were related to an increased incidence of BC. CYP4B1 rs3766197 (OR = 1.61p = 0.031) was associated with a higher risk of advanced stages (III/IV stage) of BC. Besides, the contributions of CYP4B1 rs2297813 (OR = 1.55, p = 0.021) and rs12142787 (OR = 1.53, p = 0.033) to BC risk might be associated with more than one birth in patients with BC. The three-locus model consisting of rs2297813, rs12142787, and rs3766197 was regarded as the best predictive model for BC risk. CONCLUSION: CYP4B1 SNPs were associated with BC risk in Chinese women, especially in patients with BMI ≤ 24 kg/m2, smokers, non-drinkers, patients in advanced stages (III/IV stage), and patients who reproduced once. These findings shed light on the relationship between CYP4B1 SNPs and BC risk in Chinese women.

Uncovering transcription factor and microRNA risk regulatory pathways associated with osteoarthritis by network analysis.

Osteoarthritis (OA) is the most common form of joint disease. The development of inflammation have been considered to play a key role during the progression of OA. Regulatory pathways are known to play crucial roles in many pathogenic processes. Thus, deciphering these risk regulatory pathways is critical for elucidating the mechanisms underlying OA. We constructed an OA-specific regulatory network by integrating comprehensive curated transcription and post-transcriptional resource involving transcription factor (TF) and microRNA (miRNA). To deepen our understanding of underlying molecular mechanisms of OA, we developed an integrated systems approach to identify OA-specific risk regulatory pathways. In this study, we identified 89 significantly differentially expressed genes between normal and inflamed areas of OA patients. We found the OA-specific regulatory network was a standard scale-free network with small-world properties. It significant enriched many immune response-related functions including leukocyte differentiation, myeloid differentiation and T cell activation. Finally, 141 risk regulatory pathways were identified based on OA-specific regulatory network, which contains some known regulator of OA. The risk regulatory pathways may provide clues for the etiology of OA and be a potential resource for the discovery of novel OA-associated disease genes.

[Effect of interactive scalp acupuncture on upper limb function in patients with hemiplegia after stroke].

OBJECTIVE: To explore the clinical effect of interactive scalp acupuncture combined with task-oriented mirror therapy on hemiplegia after stroke. METHODS: A total of 86 patients with hemiplegia after stroke were randomly divided into an observation group (43 cases, 2 cases dropped out) and a control group (43 cases, 2 cases dropped out). Both groups received routine treatment and rehabilitation treatment for stroke. The control group was treated with task-oriented mirror therapy, 40 min each time, once a day, 6 days a week. The observation group was treated with interactive scalp acupuncture at ipsilateral upper 1/5 and 2/5 of the parietal and temporal anterior oblique line and upper 1/5 and 2/5 of the parietal and temporal posterior oblique line on the basis of the treatment as the control group, 45 min each time, once a day, 6 days a week. Both groups were treated for 4 weeks. Before and after treatment and in follow-up of 8 weeks after treatment completion, the Fugl-Meyer assessment (FMA) score, modified Ashworth scale (MAS) score, shoulder abduction angle, wrist dorsiflexion angle and N20 latency and amplitude of somatosensory evoked potential were compared between the two groups. RESULTS: After treatment and in follow-up, the FMA scores were increased (P<0.01), the MAS scores were decreased (P<0.01) compared with those before treatment in the two groups; the FMA scores in the observation group were higher than those in the control group (P<0.01), the MAS scores were lower than those in the control group (P<0.01). After treatment and in follow-up, the shoulder abduction angle and wrist dorsiflexion angle was increased (P<0.01), the N20 latency was shortened and amplitude was increased (P<0.01) compared with that before treatment in both groups; the shoulder abduction angle and wrist dorsiflexion angle in the observation group was greater than that in the control group (P<0.01), the N20 latency was shorter than that in the control group (P<0.01), and the N20 amplitude was higher than that in the control group (P<0.01). CONCLUSION: Interactive scalp acupuncture combined with task-oriented mirror therapy can improve upper limb function in patients with hemiplegia after stroke, and reduce the muscular tone of the affected limb.

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.

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.

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.

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.

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 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 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.

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.

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.

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.

Prediction of HLA-A2-restricted CTL epitope specific to HCC by SYFPEITHI combined with polynomial method.

AIM: To predict the HLA-A2-restricted CTL epitopes of tumor antigens associated with hepatocellular carcinoma (HCC). METHODS: MAGE-1, MAGE-3, MAGE-8, P53 and AFP were selected as objective antigens in this study for the close association with HCC. The HLA-A*0201 restricted CTL epitopes of objective tumor antigens were predicted by SYFPEITHI prediction method combined with the polynomial quantitative motifs method. The threshold of polynomial scores was set to -24. RESULTS: The SYFPEITHI prediction values of all possible nonamers of a given protein sequence were added together and the ten high-scoring peptides of each protein were chosen for further analysis in primary prediction. Thirty-five candidates of CTL epitopes (nonamers) derived from the primary prediction results were selected by analyzing with the polynomial method and compared with reported CTL epitopes. CONCLUSION: The combination of SYFPEITHI prediction method and polynomial method can improve the prediction efficiency and accuracy. These nonamers may be useful in the design of therapeutic peptide vaccine for HCC and as immunotherapeutic strategies against HCC after identified by immunology experiment.

In vivo tumor co-transfection with superantigen and CD80 induces systemic immunity without tolerance and prolongs survival in mice with hepatocellular carcinoma.

BACKGROUND: Since transfection of established tumors with immunostimulatory genes can elicit antitumor immunity, we treat mouse HCC with in vivo transfection of superantigen SEA and/or costimulatory molecule CD80 and evaluated the safety and efficacy. METHODS: Mice with HCC were treated with lipid-complexed plasmid DNA encoding staphylococcal enterotoxin A and/or CD80. Then the mice were evaluated for tumor regression, systemic immunologic responses, survival times and treatment-associated toxicity. RESULTS: Of all treated mice, the overall response rates (complete or partial remission) for SEA, CD80 and SEA/CD80 treated mice in this study were 65%, 60% and 75% separately, and were significantly higher than that of untreated mice. Most of the treat mice completed the therapy without any significant reaction. CTL activity increased with time of treatment and correlated temporally with an objective tumor response. Also our results indicated that local intratumoral expression of SEA did not lead to detectable deletion or anergy of SEA-reactive spleen T cells. Survival times for hepatoma mice in this study treated by intratumoral injection of SEA, CD80 and SEA/CD80 were prolonged significantly (P < 0.01) compared with the control mice.

Identification of HLA-A2-restricted CTL epitope encoded by the MAGE-n gene of human hepatocellular carcinoma.

BACKGROUND: Identification of the cytotoxic T lymphocytes (CTL) restricted epitopes of tumor antigens opens up possibilities of developing a new cancer vaccine. For the MAGE-n has been demonstrated closely associated with hepatocellular carcinoma (HCC) and HLA-A2.1 is found in over 50% of HCC patients in China, we aim at identifying MAGE-n-encoded peptide presented by HLA-A2.1. MATERIALS: A HLA-A2.1-restricted CTL epitope was identified by using an improved "reverse immunology" strategy: (a) computer-based epitope prediction from the amino acid sequence of MAGE-n antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward HCC cells expressing MAGE-n antigen and HLA-A2.1. RESULTS: Of the five tested peptides, effectors induced by a peptide of MAGE-n at residue position 159-167(QLVFGIEVV) lysed HCC cells expressing both MAGE-n and HLA-A2.1. Our results indicated that peptide QLVFGIEVV was a new HLA-A2.1-restricted CTL epitope capable of inducing MAGE-n specific CTLs in vitro. CONCLUSIONS: Identification of the MAGE-n /HLA-A2.1 peptide QLVFGIEVV may facilitate peptide-based specific immunotherapy for HCC. The combination of epitope prediction, epitope reconstruction method and immunological methods can improve the efficiency and accuracy of CTL epitope studies.

Efficient induction of cytotoxic T lymphocytes specific to hepatocellular carcinoma using HLA-A2-restricted MAGE-n peptide in vitro.

MAGE-n is a new member of MAGE gene family and has been demonstrated closely associated with hepatocellular carcinoma (HCC). In this study, MAGE-n-derived peptide-specific cytotoxic T lymphocytes (CTL) were induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with HLA-A2-restricted MAGE-n peptide-pulsed T2 cells. The induced CTLs exhibited specific lysis against T2 cells pulsed with the peptide and HLA-A2+ HCC cells expressing MAGE-n, while HLA-A2+ HCC cell lines that did not express MAGE-n could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-MHC class I monoclonal antibody. These results suggested the MAGE-n peptide could be a potential target of specific immunotherapy for HLA-A2 patients with HCC.

Expression of gap junction genes connexin32 and connexin43 mRNAs and proteins, and their role in hepatocarcinogenesis.

AIM: To investigate the relationship between hepatocarcinogenesis and the expression of connexin32 (cx32), connexin43 (cx43) mRNAs and proteins in vitro. METHODS: Gap junction genes cx32 and cx43 mRNA in hepatocellular carcinoma cell lines HHCC, SMMC-7721 and normal liver cell line QZG were detected by in situ hybridization (ISH) with digoxin-labeled cx32, and cx43 cDNA probes. Expression of Cx32 and Cx43 proteins in the cell lines was revealed by indirect immuno-fluorescence and flow cytometry (FCM). RESULTS: Blue positive hybridization signals of cx32 and cx43 mRNAs detected by ISH with cx32 and cx43 cDNA probes respectively were located in cytoplasm of cells of HHCC, SMMC-7721 and QZG. No significant difference of either cx32 mRNA or cx43 mRNA was tested among HHCC, SMMC-7721 and QZG (P=2.673, HHCC vs QZG; P=1.375, SMMC-7721 vs QZG). FCM assay showed that the positive rates of Cx32 protein in HHCC, SMMC-7721 and QZG were 0.7%, 1.7% and 99.0%, and the positive rates of Cx43 protein in HHCC, SMMC-7721 and QZG were 7.3%, 26.5% and 99.1% respectively. Significant differences of both Cx32 and Cx43 protein expression existed between hepatocellular carcinoma cell lines and normal liver cell line (P=0.0069, HHCC vs QZG; P=0.0087, SMMC-7721 vs QZG). Moreover, the fluorescent intensities of Cx32 and Cx43 proteins in HHCC, SMMC-7721 were lower than that in QZG. CONCLUSIONS: Hepatocellular carcinoma cell lines HHCC and SMMC-7721 exhibited lower positive rates and fluorescent intensities of Cx32, Cx43 proteins compared with that of normal liver cell line QZG. It is suggested that lower expression of both Cx32 and Cx43 proteins in hepatocellular carcinoma cells could play pivotal roles in the hepatocarcinogenesis. Besides, genetic defects of cx32 and cx43 in post-translational processing should be considered.