[Ketogenic diet improves low temperature tolerance in mice by up-regulating PPARα in the liver and brown adipose tissue].

The aim of the present study was to investigate the effects of short-term ketogenic diet on the low temperature tolerance of mice and the involvement of peroxisome proliferator-activated receptor α (PPARα). C57BL/6J mice were divided into two groups: normal diet (WT+ND) group and ketogenic diet (WT+KD) group. After being fed with normal or ketogenic diet at room temperature for 2 d, the mice were exposed to 4 °C low temperature for 12 h. The changes in core temperature, blood glucose, blood pressure of mice under low temperature condition were detected, and the protein expression levels of PPARα and mitochondrial uncoupling protein 1 (UCP1) were detected by Western blot. PPARα knockout mice were divided into normal diet (PPARα-/-+ND) group and ketogenic diet (PPARα-/-+KD) group. After being fed with the normal or ketogenic diet at room temperature for 2 d, the mice were exposed to 4 °C low temperature for 12 h. The above indicators were also detected. The results showed that, at room temperature, the protein expression levels of PPARα and UCP1 in liver and brown adipose tissue of WT+KD group were significantly up-regulated, compared with those of WT+ND group. Under low temperature condition, compared with WT+ND, the core temperature and blood glucose of WT+KD group were increased, while mean arterial pressure was decreased; The ketogenic diet up-regulated PPARα protein expression in brown adipose tissue, as well as UCP1 protein expression in liver and brown adipose tissue of WT+KD group. Under low temperature condition, compared to WT+ND group, PPARα-/-+ND group exhibited decreased core temperature and down-regulated PPARα and UCP1 protein expression levels in liver, skeletal muscle, white and brown adipose tissue. Compared to the PPARα-/-+ND group, the PPARα-/-+KD group exhibited decreased core temperature and did not show any difference in the protein expression of UCP1 in liver, skeletal muscle, white and brown adipose tissue. These results suggest that the ketogenic diet promotes UCP1 expression by up-regulating PPARα, thus improving low temperature tolerance of mice. Therefore, short-term ketogenic diet can be used as a potential intervention to improve the low temperature tolerance.

Consensus of MASs With Input and Communication Delays by Predictor-Based Protocol.

In this article, the consensus problem of multiagent systems (MASs) affected by input and communication delays is investigated. A predictor-based state feedback protocol is used to reach the consensus of linear MASs by delay compensation. In order to analyze the maximum delay under the predictor-based protocol, the overall MASs are equivalent to the feedback interconnection system, including a linear time-invariant system and a time-delay operator, in view of the characteristic of the Laplacian matrix. Then, the maximum delay corresponding to the predictor-based protocol is evaluated by using the small gain theorem (SGT). Finally, two numerical examples are given to verify the effectiveness of the obtained consensus condition.

[Comparison of Plasma Components between Frozen Plasma and Fresh Frozen Plasma].

OBJECTIVE: To compare the plasma components of frozen plasma (FP) and fresh frozen plasma (FFP). METHODS: Twenty samples of FP and 20 samples of FFP from Beijing Red Cross Blood Center were randomly selected. Immediately after plasma melting, 12 plasma components including coagulation factor, fibrinolytic system and anticoagulation protein were detected, including activated partial thromboplastin time (APTT), prothrombin time (PT), coagulation factor Ⅷ (FⅧ) activity, coagulation factor Ⅴ (FⅤ) activity, fibrinogen(FIB) level, ADAMTS-13 activity, von Willebrand factor(vWF) activity, D-dimer (D-dimer, DD), fibrin degradation products (FDP), antithrombin (AT), protein C (PC), and protein S (PS). All these coagulation components between the two types of plasma were compared and analyzed. RESULTS: Compared with FFP, APTT in FP was significantly prolonged(t=3.428, P<0.01), and PT was also significantly prolonged(z=-2.140, P<0.05), and FⅧ activity was decreased (t=-3.372, P<0.01), but all in the reference range, and PS activity was decreased(t=-2.458，P＜0.05), there was no statistical difference in the other part between two types of plasma(P>0.05). CONCLUSION: FP can substitute FFP in the treatment of some diseases, although it is lack of some coagulation factors and anticoagulation protein.

Prognosis of Neurological Improvement in Inpatient Acute Ischemic Stroke Survivors: A Propensity Score Matching Analysis.

OBJECTIVES: Stroke has become a national concern in China. Early prediction of stroke benefits patients and aids medical professionals in clinical decision making and rehabilitation plans to improve successful outcomes. To identify prediction factors influencing short-term outcomes in patients with acute ischemic stroke (AIS). MATERIALS AND METHODS: This was a hospital-based prospective observational study. Recovery of neurological improvement was represented by a percent reduction in the National Institutes of Health Stroke Scale (NIHSS) at discharge. We performed propensity score matching (PSM) to balance the NIHSS at admission and compared NIHSS scores before and after matching with PSM criteria. Finally, we assessed the prognosis of neurological improvement and patient-related variables. RESULTS: In the matched cohort, 92 pairs were matched by NIHSS admission after PSM. Modified Barthel Index, modified Rankin scale, NIHSS on admission, hypertension, sleep time, and Montreal Cognitive Assessment (MoCA) were statistically different between the two groups (P<0.05) before matching. Multivariable analysis identified two factors independently associated with neurological improvement: diabetes (P=0.030; adjusted odds ratio, 2.129; 95% confidence interval [CI] 1.078-4.026) and MoCA (P<0.001; adjusted odds ratio, 5.385; 95% CI 2.278-12.730). CONCLUSION: Consistent with previous studies, diabetes affected the short-term outcomes of AIS, while cognitive impairment had a negative effect on long-term AIS prognosis.Diabetes and early cognitive impairment have adverse effects on short-term prognosis after AIS.

Time-restricted feeding alleviates cardiac dysfunction induced by simulated microgravity via restoring cardiac FGF21 signaling.

Dietary restriction has been well-described to improve health metrics, but whether it could benefit pathophysiological adaptation to extreme environment, for example, microgravity, remains unknown. Here, we investigated the effects of a daily rhythm of fasting and feeding without reducing caloric intake on cardiac function and metabolism against simulated microgravity. Male rats under ad libitum feeding or time-restricted feeding (TRF; food access limited to 8 hours every day) were subjected to hindlimb unloading (HU) to simulate microgravity. HU for 6 weeks led to left ventricular dyssynchrony and declined cardiac function. HU also lowered pyruvate dehydrogenase (PDH) activity and impaired glucose utilization in the heart. All these were largely preserved by TRF. TRF showed no effects on HU-induced loss of cardiac mass, but significantly improved contractile function of cardiomyocytes. Interestingly, TRF raised liver-derived fibroblast growth factor 21 (FGF21) level and enhanced cardiac FGF21 signaling as manifested by upregulation of FGF receptor-1 (FGFR1) expression and its downstream markers in HU rats. In isolated cardiomyocytes, FGF21 treatment improved PDH activity and glucose utilization, consequently enhancing cell contractile function. Finally, both liver-specific knockdown (KD) of FGF21 and cardiac-specific FGFR1 KD abrogated the cardioprotective effects of TRF in HU rats. These data demonstrate that TRF improves cardiac glucose utilization and ameliorates cardiac dysfunction induced by simulated microgravity, at least partially, through restoring cardiac FGF21 signaling, suggesting TRF as a potential countermeasure for cardioprotection in long-term spaceflight.

[Analysis of the Irregular Antibodies of the Patients and Its Clinical Significance].

OBJECTIVE: To retrospectively analyze the identification results of irregular antibodies, to clarify the distribution features and to explore the relation of alloantibodies and autoantibodies with the immunized history of patients and disease kinds. METHODS: 49 820 patients who applied for red blood transfusion during Sep 1st 2017 to Sep 1st 2018 were selected. All the specimens were screened for the antibody by microcolumn gel antiglobulin technique, which then were identified for irregular antibody. RESULTS: Antibodies were found in 861 (1.73%) of all 49 820 transfused samples. The alloimmunization history of the patients with antibodies was significantly different between male and female (χ2=18.54，P＜0.01). The alloantibody was the most common, accounting for 59.50% in all of the antibodies. Warm autoantibody, anti-E, anti-M, anti-cE and anti-Ce accounted for 68.5% of the antibodies. The blood group of Rh, MNS and Lewis were responsible for 92.40% of alloantibody, especially anti-E accounted for the largest percentage(38.60%) of alloantibody. Patients with alloantiboies experienced much more the alloimmunization and transfusion history (χ2=20.13，P＜0.01；χ2=5.40，P＜0.05) . The distribution of auto and alloantibody was very significantly different among the ddifferent isease (χ2=51.8，P＜0.01), Hematopathy, solid tumor and osteoarthropathy were often associated with alloantibody, otherwise, autoantibodies often occurred in hematopathy and autoimmune disease. CONCLUSION: The most important factor that results in antibody-screening positive is alloantibody, in which anti-E antibody from Rh blood group system in most common.

[Effect of HIF-1α on Angiogenesis-Related Factors in K562 Cells].

OBJECTIVE: To explore the mechanisms of angiogenesis in chronic myeloid leukemia (CML) through detecting the levels of angiogenesis-related factors secreted from K562 cells after overexpression and interference of HIF-1α gene in K562 cells. METHODS: The K562 cells were transfected by lentiviruses carried and interfered HIF-1α gene, then the transtected K562 cells with carried and interfered with HIF-1α gene were enrolled in overexpression and interference groups respectively, at the same time the K562 cells transfected by the empty virus were enrolled in control group. The cells were harvested after culture for 72 hours under normoxid condition. The transfection efficient in 3 groups was detected by fluorescence microscopy; the mRNA expression of HIF-1α gene and angiogenesis-related factors was detected by RT-PCR; the concentration of angiogenesis-related factors in the caltured supernatant was detected by ELISA. RESULTS: The optimal MOI of K562 cells transfected with lentivirus was 10 and the transfection efficiency was about 50%. The positive rate of transfection after screening by puromycin was more than 90%. The mRNA expression of ANG-I, ANG-II, TGF-α and VEGF in the interference group was lower than that in the over-expression group, and the TGF-ß1 mRNA expression in the interference group was higher than in the over-expression group. The mRNA expression of ANG-I and VEGF in the interference group was lower than that in the control group. TGF-αdid not could be detected, and the culture supernatant concentration of ANG-I and TNF-α in the interference group was lower than in the over-expression group, while the VEGF concentration in the interference group was higher than that in the over-expression group. All of the above-mentioned differences were statistically significant (P＜0.05). CONCLUSION: The positive K562 cells transfected with leutivirus have been harvested by screening with puromycin. The HIF-1α mRNA positively regulates the mRNA expression of ANG-1, ANG-2, TGF-α, VEGF in K562 cells, promotes the antocrine ability of ANG-1 and TNF-α, moreover not stimulates the autocrine of TGF-α, the up-regulation of HIF-1α expression can inhibit the expression TGF-ß1 in K562 cells and the autocrine of TGF-ß1.

[A modified protocol for generating the simulated weightlessness rat model].

OBJECTIVE: To introduce a modified protocol for generating the simulated weightlessness rat model by hindlimb unloading. METHODS: Ninety male adult SD rats were randomly divided into three groups: the control group, classical suspension group and modified suspension group (n=30/group). In the classical suspension group, a strip of medical adhesive tape was attached to the tail, with horizontal filament tape wrapping. A piece of gauze was wrapped around the tail at the outermost layer and the tail was suspended for hindlimb unloading. In the modified suspension group, a layer of plastic net was added between the horizontal filament tape and the gauze to reduce the squeeze on the tail as a buffer zone and ensure proper circulation of the tail. After 4 weeks of suspension, damage to the tail and sheath detachment were observed. Meanwhile the body weight and right soleus wet weight of rats were measured. RESULTS: The ratio of right soleus wet weight to body weight was decreased significantly in both the classical suspension group and the modified suspension group compared with the control group, while there was no difference in body weight among the three different groups. Importantly, the incidence of tail ischemia and necrosis (13.3% vs 40.0% in the classical suspension group) and the incidence of sheath detachment from tail (3.3% vs 26.7% in the classical suspension group) were significantly lower whereas the success rates of model (33.3% vs 83.3% in classical suspension group) was significantly higher in the modified suspension group. CONCLUSION: The modified protocol decreases the incidence of tail necrosis and sheath detachment in the rat tail suspension and increases the success rate of the hindlimb unloading rat model, with improved simplicity and practicability.

[miR-494-3p reduces insulin sensitivity in diabetic cardiomyocytes by down-regulation of insulin receptor substrate 1].

More and more evidence suggests that microRNA is widely involved in the regulation of cardiovascular function. Our preliminary experiment showed that miR-494-3p was increased in heart of diabetic rats, and miR-494-3p was reported to be related to metabolism such as obesity and exercise. Therefore, this study was aimed to explore the role of miR-494-3p in diabetic myocardial insulin sensitivity and the related mechanism. The diabetic rat model was induced by high fat diet (45 kcal% fat, 12 weeks) combined with streptozotocin (STZ, 30 mg/kg), and cardiac tissue RNA was extracted for qPCR. The results showed that the level of miR-494-3p was significantly up-regulated in the myocardium of diabetic rats compared with the control (P < 0.05). The level of miR-494-3p in H9c2 cells cultured in high glucose and high fat medium (HGHF) was significantly increased (P < 0.01) with the increase of sodium palmitate concentration, whereas down-regulation of miR-494-3p in HGHF treated cells led to an increase in insulin-stimulated glucose uptake (P < 0.01) and the ratio of p-Akt/Akt (P < 0.05). Over-expression of miR-494-3p in H9c2 cell line significantly inhibited insulin-stimulated glucose uptake and phosphorylation of Akt (P < 0.01). Bioinformatics combined with Western blotting experiments confirmed insulin receptor substrate 1 (IRS1) as a target molecule of miR-494-3p. These results suggest that miR-494-3p reduces insulin sensitivity in diabetic cardiomyocytes by down-regulating IRS1.

[Genomics-based Biomarkers for Diseases:A Bibliometric Analysis Based on Web of Science].

Objective To analyze the research hotspots and trends of biomarkers for diseases based on genomics and thus provide basis for the future studies in this field. Method Based on the Web of Science,we analyzed the genomics-based biomarkers for diseases in literature published between 2006 and 2018 in terms of country and institutions,knowledge base,research hotspots,and trends by using bibliometric methods and CiteSpace software. Results A total of 998 articles were retrieved.The total number of articles has shown an upward trend and reached a peak of 112 in 2017 and 2018.Most articles(n=477)were from the United States,follwed by China(n=93).Nature,P Natl Acad Sci USA,PLoS One,Science,and New Engl J Med are core journals in this field.Keywords co-occurrence analysis identified four research hotspots:disease research,research method and technology,research level,and application purpose. Conclusion Research in functional genomics,cancer immunotherapy,genome-wide association and multi-omics techniques,personalized medicine,and precision medicine are research hotspots and frontiers in this field.

Paradoxical Elevation of High Density Lipoprotein Cholesterol in Association with Lacunar-Type Cerebral Infarction.

BACKGROUND: The aim of this study was to evaluate the association between high-density lipoprotein cholesterol (HDLC) levels and the risk of lacunar infarction (LI) in a retrospective cohort study in China. MATERIAL AND METHODS: We recruited 229 patients with obsolete brain infarctions single side (SOBI), 218 with obsolete brain infarctions bilateral sides (BOBI), 193 with both acute stroke and obsolete lacunar infarctions single side (AI&SOBI), 113 with both acute stroke and obsolete lacunar infarctions bilateral sides (AI&BOBI), and 203 without any infarctions (Control). RESULTS: 1) The plasma levels of HDLC in group BOBI, AI&SOBI, and AI&BOBI were higher than in the control group, and lower in group SOBI than in the control group (p<0.01). 2) The plasma levels of HDLC in group AI&SOBI were significantly higher than in group SOBI (p<0.01). 3) The plasma levels of HLDL were similar between group AI&SOBI and AI&BOBI. 4) There were significant relationships between HDLC and acute lacunar stroke, even after adjusting for these factors such as age, sex, triglyceride, total cholesterol, low-density lipoprotein cholesterol, and history of diabetes (p=0.001). 4) Compared with the controls, the calculation of odds ratios indicated relative risk estimates of higher HDLC for acute lacunar stroke with obsolete lacunar infarction. CONCLUSIONS: Elevated HDLC may be an independent predictor of recurrent stroke with obsolete lacunar infarctions single side in Chinese people, justifying clinical trials for secondary prevention of stroke by generally increasing HLDL level. According to the difference between single and bilateral side multiple silent lacunar infarcts, it is inferred that HDLC may increase the risk of atherothrombotic infarction but reduce the risk of cardioembolic infarction in the general Chinese population.

Prognostic factors in older patients with advanced non-small cell lung cancer in China.

AIMS AND BACKGROUND: The human life expectancy and the incidence of lung cancer have increased dramatically in recent years. As a result, there is a high demand for the management of older patients with advanced non-small cell lung cancer (NSCLC) in clinical practice. The purpose of this study is to evaluate the prognostic factors in ≥65-year-old patients with advanced NSCLC in China. METHOD: This study involved a retrospective review of 78 ≥65-year-old patients with a diagnosis of NSCLC and at an advanced stage of disease, defined as stage IIIB or IV. All patients were followed up for a 3-year interval to determine the survival rates. Clinical data including gender, smoking history, comorbidities, performance status (PS), histological differentiation, disease stage, treatment and overall survival were recorded. The log-rank test was used to calculate survival rates. Multivariate Cox regression analysis was performed to determine independent prognostic factors. RESULTS: The 1-year, 2-year and 3-year survival rates of the 78 patients were 44.9%, 23.1% and 9.0%, respectively. In univariate analysis by the log-rank test, the 3-year survival rate was significantly associated with PS (P <0.01), disease stage (P <0.01) and chemotherapy treatment (P <0.01). The results of multivariate Cox regression analysis confirmed that PS and disease stage were independent prognostic factors. CONCLUSION: The 3-year survival rate in ≥65-year-old patients with advanced NSCLC was significantly associated with PS, disease stage and chemotherapy. PS and disease stage were independent prognostic factors. Older patients with advanced NSCLC in China might benefit from chemotherapy.

Economic design in a long-distance migrating molluscivore: how fast-fuelling red knots in Bohai Bay, China, get away with small gizzards.

We carried out an observational and experimental study to decipher how resource characteristics, in interaction with the predator's phenotype, constrain a fitness-determining performance measure, i.e. refuelling in a migrant bird. Two subspecies of red knot (Calidris canutus rogersi and C. c. piersmai) use northern Bohai Bay, Yellow Sea, China, for the final prebreeding stopover, during their 10,000-15,000 km long migrations between wintering and breeding areas. Here, they feed on small bivalves, especially 2-7 mm long Potamocorbula laevis. With an average stay of 29 days, and the need to store 80 g of fat for the onward flights to high-Arctic breeding grounds, red knots need to refuel fast. Using existing knowledge, we expected them to achieve this on the basis of (1) prey with high flesh to shell mass ratios, (2) large gizzards to crush the ingested molluscs, or (3) a combination of the two. Rejecting all three predictions, we found that red knots staging in Bohai Bay had the smallest gizzards on record (4.9 ± 0.8 g, mean ± s.e.m., N = 27), and also found that prey quality of P. laevis is much lower than predicted for the measured gizzard size (i.e. 1.3 rather than the predicted 4.5 kJ g(-1) dry shell mass, DM(shell)). The estimated handling time of P. laevis (0.2 s) is much shorter than the observed time between two prey ingestions (0.7 s), indicating that prey handling time is no constraint. Based on field observations of dropping rates and on indoor digestion trails, the shell processing rate was estimated at 3.9 mg DM(shell) s(-1), i.e. three times higher the rate previously predicted for red knots eating as fast as they can with the measured gizzard size. This is explained by the small and easily crushed P. laevis enabling high processing rates. As P. laevis also occurred in high densities, the metabolizable energy intake rate of red knots with small gizzards at 5 J s(-1) was as high as at northward staging sites elsewhere in the world. Currently, therefore, food characteristics in Bohai Bay are such that red knots can refuel fast whilst economizing on the size of their gizzard. These time-stressed migrants thus provide an elegant example of symmorphosis.

Diversity of endophytic fungi from roots of Panax ginseng and their saponin yield capacities.

Endophytes of medicinal plants have the capacity to synthesis same or similar active substances with their hosts. To investigate the diversity and capacity to produce saponins of endophytic fungi of Panax ginseng, thirty-eight strains of were isolated. Polymerase chain reaction (PCR) and sequencing were used to identify the isolates, and saponins concentrations in the cultures were measured. Agar diffusion method was used to test antimicrobial activity. High-performance liquid chromatography (HPLC) was used to analyze ginsenosides produced by representative strains. Nectria, Aspergillus, Fusarium, Verticillium, Engyodontium, Plectosphaerella, Penicillium, Cladosporium, and Ascomycete species were isolated. Overall, 18.4% of the isolates belonged to Nectria (Nectria haematococca), 13.2% belonged to Aspergillus, and 10.5% belonged to Penicillium. The highest concentration of triterpenoid saponin was 0.181 mg/ml (Pg27), followed by 0.144 mg/ml (Pg30 and Pg42-1). According to the results of the phylogenetic results, these isolates were species of Fusarium, Aspergillus and Verticillium, respectively. The culture filtrate of Pg30 exhibited its antibacterial activity Staphylococcus aureus. Pg 27 and Pg30 could excrete the substances to inhibit the growth of Rhizoctonia solani. Pg42-1 showed strong inhibition against Klebsiella pneumoniae. From HPLC results, the ginsenoside Rb2 was detected in both Pg27 and Pg30 cultures. The ginsenoside Rc was found in Pg42-1 cultures. In conclusion, thirty-eight endophytic fungal strains were isolated and Pg27 (Fusarium sp.) has a potential application value in saponins production.

[Reconstruction of facial soft tissue defects with free flaps by microsurgery].

OBJECTIVE: To investigate the method and indications for reconstruction of facial complicated soft tissue defects with free flaps by microsurgery. METHODS: 37 patients (16 males and 21 females, aged from 1 to 54 years) with different size of facial soft tissue defects were reconstructed with free flaps, including 10 latissimus dorsi myocutaneous flaps, 3 thoracodorsal artery perforator flaps, 9 scapular flaps, forearm flaps and 9 postauricular flaps. The defects size ranged from 1 cm x 2 cm to 25 cm x 12 cm. RESULTS: Venous obstruction happened in 3 postauricular flaps, resulting partial necrosis in 2 flaps. All the other flaps survived completely. The cosmetic and functional results were both satisfactory. CONCLUSIONS: The facial complicated soft tissue defects can be treated successfully with free flaps by microsurgery. The wounds can be healed primarily with short recovery time and reliable cosmetic and functional result.

Chinese medicine for menopausal syndrome: current status, problems and strategies.

The use of Chinese medicine (CM) for the management of: menopausal syndrome is considered effective both at home and abroad, and more and more clinical studies are confirming its efficacy. However, many problems still exit in current studies, such as the standard of CM syndrome differentiation, the design methodology and criteria to assess the quality of clinical trials and the efficacy of interventions. In this paper, the authors present the CM research and treatment strategies for menopausal syndrome with concepts explaining the CM understanding of the mechanism of the disorder. It is concluded that CM is effective for menopausal syndrome, but improvement in both study methodology and treatment strategy is needed. In detail, it is firstly necessary to conduct clinical studies to evaluate the difference of various CM treatments for menopausal syndrome manifesting different symptoms, so as to establish a comprehensive treatment protocol of CM. Secondly, an acknowledged evaluation system needs to be founded, which embodies the characteristics of CM, and covers appropriate endpoint indices and parameters to objectively evaluate the effect and study quality of CM. Finally, an epidemiological survey with large sample size should be implemented with robust statistical design and CM expertise to collect data for establishing diagnostic criteria for menopause in different stages and with different symptoms.

[Study on the specific immunity induced by dendritic cell vaccine loading allogenic microvascular endothelial cell bEnd.3 antigen against U14 cervical cancer cell in mice].

OBJECTIVE: To explore the specific cellular and humoral immunity induced by dendritic cells (DC) vaccine loading allogenic microvascular endothelial cell bEnd.3 antigen against U14 cervical cancer cell of mice. METHODS: Mouse brain microvascular endothelial cell bEnd.3 was cultured and identified for preparation endothelial cell bEnd.3 antigen. The level of mRNA expression of vascular endothelial growth factor receptor 2 (VEGF-R2) and integrin αV was detected by reverse transcription (RT)-PCR. The BALB/c mice were immuned with DC loading bEnd.3 antigen 4 times in 4 weeks (bEnd.3-DC group), while the mice only were immuned with DC or injected with phosphate buffer saline (PBS group) as control group. One week after last vaccination, U14 cervical cancer cells were injected subcutaneously into the mice. The tumor size, cytotoxic T lymphocyte (CTL) response of spleen lymphocytes in vitro, the percentage of CD3+CD8+ surface markers of spleen lymphocytes, and the titer of serum antibody were detected. The specific immunity was examined by immunocytochemistry and western blot. RESULTS: The expression of VEGF-R2 and integrin αV gene in bEnd.3 cells were expressed highly. After the vaccine was injected, the tumors of mice in PBS group grew faster than those in other groups, while the tumors in bEnd.3-DC group grew slowly and disappeared after 2 weeks. The volume of tumors in DC group grew slower than those in PBS group [(0.11 ± 0.13) cm³ versus (3.38 ± 0.34) cm³]. The CTL response of spleen lymphocytes in vitro showed that bEnd.3-DC cells could kill bEnd.3 cells, the special lysis rate was more than 60%. The percentage of CD3+CD8+ spleen lymphocytes in bEnd.3-DC group [(38.6 ± 0.7)%] was higher than those in other groups (P < 0.05). The titer of serum antibody of bEnd.3-DC group was 1:3200, while it was 1:800 in DC group and there were not any in PBS group. Immunocytochemistry analysis indicated there were specific antigen-antibody reaction to bEnd.3 cell in bEnd.3-DC group. Western blot analysis revealed that there were specific bands at 220,000 (VEGF-R2). CONCLUSIONS: bEnd.3-DC vaccine can inhibit the tumor growth of U14 cervical cancer cell of mice, which indicates that the special cellular and humoral immunity are induced by bEnd.3-DC antigen which maybe have some antigens in bEnd.3 cells that reacts with endothelial cell proliferation-related antigens.

[Study on the specific immunity of dendritic cell vaccine loading human umbilical vein endothelial cell antigen against U14 cervical cancer].

AIM: To explore the specific immunity of dendritic cell (DC) vaccine loading human umbilical vein endothelial cell (HUVEC) antigen.against U14 cervical cancer of mice. METHODS: Primary HUVECs were cultured, identified and made into antigen. The BALB/c mice were immunized with DCs loading HUVEC antigen 4 times a week. One week after last vaccination, U14 cervical cancer cells were injected subcutaneously into the mice. The tumor size, CTL response of spleen lymphocytes in vitro, the percentage of CD3(+)CD8(+) surface markers of spleen lymphocytes, and the titer of serum antibody were detected. The specific immunity was examined by immunocytochemistry and Western blot. RESULTS: HUVECs with high purity were successfully cultured and by identified by immunocytochemistry and RT-PCR. After the vaccine was injected into mice, the tumors of mice in PBS group grew faster than the those in other groups. The tumors of mice in HUVEC-DC groups grew slowly and disappeared after 2 weeks and The tumors of mice in DC group disappeared after 3 weeks. The CTL response of spleen lymphocytes in vitro showed that HUVEC-DC-T cells could kill HUVEC cells. The percentage of CD3(+)CD8(+) surface markers of spleen lymphocytes in HUVEC-DC group was higher than that in other groups. The titer of serum antibody was 1:800. immunocytochemistry analysis indicated HUVEC-DC group had specific antigen-antibody reaction to HUVECs through and Western blot analysis revealed there were specific bands at 130 KDa and 220 KDa. CONCLUSION: HUVEC-DC vaccine and DC vaccine can inhibit the tumor growth of U14 cervical cancer of mice. The special cellular and humoral immunity are induced by HUVEC-DC vaccine. Furthermore, some antigens in HUVECs maybe have special immune reaction with integrin alphav and VEGF-R2.

[Effect of the microenvironment of esophageal carcinoma on dendritic cells].

AIM: To study the effect of microenvironment simulated by esophageal carcinoma homogenate supernatant on the differentiation and development of human dendritic cells (DCs) and to investigate the mechanisms of tumor immune escape for the clinical application of DC vaccines. METHODS: Fresh esophageal carcinoma and peri-cancer tissues were collected to prepare homogenate supernatant and the content of VEGF-A was detected by ELISA. The peripheral blood monouclear cells were isolated by density gradient centrifugation and cultured with RPMI1640 medium including rhGM-CSF and rhIL-4 to induce to DCs. Then the esophageal carcinoma homogenate supernatant, peri-carcinoma homogenate supernatant and VEGF-A were added on the second day and half of the medium was changed every other day. Antigen of esophageal carcinoma cell line EC9706 was added on day 4 and lipopolysaccharide (LPS) was added on day 6. DCs were collected on day 8 for further study. Checked the morphology of DCs by microscope, the immunophenotype by flow cytometry, the gene of CD1a by RT-PCR and the proliferation and killing rate of T cell by CCK-8. RESULTS: The content of VEGF-A in the homogenate supernatant of esophageal carcinoma was significantly higher than that of the peri-carcinoma (0.987+/-0.319 microg/L, 0.152+/-0.105 microg/L, P<0.05). The cell morphology in esophageal carcinoma homogenate supernatant group was inhibited. Besides, compared with normal DCs, the positive expression rate of CD86 decreased from 69+/-8 to 42+/-11, CD1a decreased from 56+/-12 to 27+/-12 and CD11c decreased from 21+/-13 to 18+/-13 (P<0.01). CD1a gene almost showed no expression. The proliferation capacity of T cells decreased from 112.53+/-7.16 to 70.18+/-3.47 (P<0.01), and their killing capacity of T cells decreased from 62.42+/-0.57 to 46.81+/-1.62 (P<0.01). However, the cells had no difference among peri-carcinoma homogenate supernatant group, VEGF-A group and normal DC group. CONCLUSION: The tumour microenvironment stimulated by the esophageal carcinoma homogenate supernatant obviously has inhibitory effect on the differentiation and function of DCs.VEGF-A may not be the key factor in the process.