Diabetes and aortic dissection: unraveling the role of 3-hydroxybutyrate through mendelian randomization.

BACKGROUND: In observational and experimental studies, diabetes has been reported as a protective factor for aortic dissection. 3-Hydroxybutyrate, a key constituent of ketone bodies, has been found to favor improvements in cardiovascular disease. However, whether the protective effect of diabetes on aortic dissection is mediated by 3-hydroxybutyrate is unclear. We aimed to investigate the causal effects of diabetes on the risk of aortic dissection and the mediating role of 3-hydroxybutyrate in them through two-step Mendelian randomization. MATERIALS AND METHODS: We performed a two-step Mendelian randomization to investigate the causal connections between diabetes, 3-hydroxybutyrate, and aortic dissection and calculate the mediating effect of 3-hydroxybutyrate. Publicly accessible data for Type 1 diabetes, Type 2 diabetes, dissection of aorta and 3-hydroxybutyrate were obtained from genome-wide association studies. The association between Type 1 diabetes and dissection of aorta, the association between Type 2 diabetes and dissection of aorta, and mediation effect of 3-hydroxybutyrate were carried out separately. RESULTS: The IVW method showed that Type 1 diabetes was negatively associated with the risk of aortic dissection (OR 0.912, 95% CI 0.836-0.995), The weighted median, simple mode and weighted mode method showed consistent results. The mediated proportion of 3-hydroxybutyrate on the relationship between Type 1 diabetes and dissection of aorta was 24.80% (95% CI 5.12-44.47%). The IVW method showed that Type 2 diabetes was negatively associated with the risk of aortic dissection (OR 0.763, 95% CI 0.607-0.960), The weighted median, simple mode and weighted mode method showed consistent results. 3-Hydroxybutyrate does not have causal mediation effect on the relationship between Type 2 diabetes and dissection of aorta. CONCLUSION: Mendelian randomization study revealed diabetes as a protective factor for dissection of aorta. The protective effect of type 1 diabetes on aortic dissection was partially mediated by 3-hydroxybutyrate, but type 2 diabetes was not 3-hydroxybutyrate mediated.

Proteomic Analysis of Peri-Wounding Tissue Expressions in Extracorporeal Shock Wave Enhanced Diabetic Wound Healing in a Streptozotocin-Induced Diabetes Model.

Our former studies have demonstrated that extracorporeal shock wave therapy (ESWT) could enhance diabetic wound healing but the bio-mechanisms remain elusive. This study investigated the changes of topical peri-wounding tissue expressions after ESWT in a rodent streptozotocin-induced diabetic wounding model by using the proteomic analysis and elucidated the molecular mechanism. Diabetic rats receiving ESWT, normal control, and diabetic rats receiving no therapy were analyzed. The spots of interest in proteome analysis were subjected to mass spectrometry to elucidate the peptide mass fingerprints. Protein expression was validated using immunohistochemical staining and related expression of genes were analyzed using real-time RT-PCR. The proteomic data showed a significantly higher abundance of hemopexin at day 3 of therapy but down-regulation at day 10 as compared to diabetic control. In contrast, the level of serine proteinase inhibitor (serpin) A3N expression was significantly decreased at day 3 therapy but expression was upregulated at day 10. Using real-time RT-PCR revealed that serpin-related EGFR-MAPK pathway was involved in ESWT enhanced diabetic wound healing. In summary, proteome analyses demonstrated the expression change of hemopexin and serpin with related MAPK signaling involved in ESWT-enhanced diabetic wound healing. Modulation of hemopexin and serpin related pathways are good strategies to promote wound healing.

Modulation of vascular endothelial growth factor and mitogen-activated protein kinase-related pathway involved in extracorporeal shockwave therapy accelerate diabetic wound healing.

Extracorporeal shockwave therapy (ESWT) has a significant positive effect to accelerate chronic wound healing. This study investigated whether the vascular endothelial growth factor (VEGF)-related pathway has involved in ESWT enhancement of diabetic wound healing. A dorsal skin defect (area, 6 × 5 cm) in a streptozotocin-induced diabetes rodent model was used. Thirty-two male Wistar rats were divided into four groups. Group I consisted of nondiabetic control; group II, diabetic control without treatment; group III, diabetic rats received ESWT; and group IV, rats received Avastin (a VEGF monoclonal antibody) on day 0 (post-wounding immediately) to day 7 and ESWT on day 3 and day 7. The wound healing was assessed clinically. The VEGF, endothelial nitric oxide synthase (eNOS), and Ki-67 were analyzed with immunohistochemical staining. The mRNA expression of mitogen-activated protein kinase-related genes was measured by real-time quantitative real-time polymerase chain reaction. The results revealed wound size was significantly reduced in the ESWT-treated rats as compared to the diabetic control (p < 0.01). The positive effect of ESWT-increasing wound healing was significantly suppressed in pretreatment of the Avastin group. Histological findings revealed significant increase in neo-vessels in the ESWT group as compared to the control. In immunohistochemical stain, significant increases in VEGF, eNOS, and Ki-67 expressions were noted in the ESWT group as compared to that in controls. However, Avastin suppressed the shockwave effect and down-regulation of VEGF, eNOS, and Ki-67 expressions in the Avastin-ESWT group as compared to that in the ESWT alone group. We found that highly mRNA expression of Kras, Raf1, Mek1, Jnkk, Jnk, and Jun at early stage in the ESWT group, as compared to the diabetic control. These evidences indicated treatment with multiple sessions of ESWT significantly enhanced diabetic wound healing associated with increased neovascularization and tissue regeneration. The bio-mechanism of ESWT-enhanced wound healing is correlated with VEGF and mitogen-activated protein kinase-mediated pathway.

Suppression of Oxygen Radicals Protects Diabetic Endothelium Damage and Tissue Perfusion in a Streptozotocin-Induced Diabetes Rodent Model.

BACKGROUND: Oxygen free radicals play a central role in diabetic angiopathy. This study investigated whether suppression of oxygen radicals could decrease endothelial damage and increase peripheral tissue circulation in a diabetic rodent model. METHODS: Sprague-Dawley rats were treated using streptozotocin to induce diabetes. The experiments were performed 4 weeks after diabetes induction: group 1: control, consisted of normal rats; group 2: diabetes, did not receive treatment; groups III (SOD10) and IV (SOD50): diabetes, received polyethylene glycol-conjugated superoxide dismutase (SOD), an antioxidant, 10 and 50 U/kg per day intraperitoneally for 4 weeks. Each subgroup consisted of 10 rats. Oxygen radicals in blood mononuclear cells were detected by flow cytometry. The blood lipid peroxidation byproduct malondialdehyde was measured. Tissue perfusion of hind limb was examined by laser Doppler. The expressions of oxygen radicals, as demonstrated by 8-hydroxyguanosine (8-OG), and constitutive endothelial nitric oxide synthase in distal femoral vessels were examined by immunohistochemical staining. RESULTS: Oxygen radicals, as demonstrated by H2O2 with 2',7'-dichlorofluorescin diacetate-conjugated expression, were significantly increased in diabetic rats. However, the SOD treatment groups significantly suppressed the H2O2 reaction. Diabetic-induced high malondialdehyde levels were significantly suppressed in the SOD50 group. The topical tissue blood perfusion was significantly increased as detected by laser Doppler in SOD10 and SOD50 groups, as compared with that in diabetes without treatment group (P < 0.05). The expression of 8-OG was markedly increased in the diabetic endothelium and subintima compared with that in normal vessels. Polyethylene glycol-conjugated SOD significantly suppressed 8-OG expression and protected endothelial nitric oxide synthase expression. CONCLUSIONS: Suppression of oxygen radicals, particularly with the higher dosage of polyethylene glycol-conjugated SOD at 50 U/kg per day, could have a positive effect to protect against endothelial damage and enhance peripheral perfusion in diabetes.

VEGF-D-induced draining lymphatic enlargement and tumor lymphangiogenesis promote lymph node metastasis in a xenograft model of ovarian carcinoma.

BACKGROUND: Vascular endothelial growth factor (VEGF)-D has been shown to promote lymph node metastasis in several cancers. Although generally overexpressed in ovarian carcinoma, its role in nodal dissemination of this cancer is unclear. To clarify the role of VEGF-D and the underlying molecular mechanisms, we investigated the function of VEGF-D using a mouse xenograft model of ovarian cancer. METHODS: Human ovarian serous adenocarcinoma SKOV3 cells were transfected with VEGF-D recombinant plasmid DNA, or with control vectors. The cells were injected subcutaneously into the footpads of nude mice. Tumor growth was evaluated weekly. Draining lymphatics were observed grossly with Evan's blue lymphangiography. Tumoral lymphatics were delineated with both Evan's blue and LYVE-1 immunostaining. Tumor metastases to lymph nodes were evaluated by H&E and CA125/CD40 staining. Expression of VEGF-D in primary tumors and levels of CA125 in involved lymph nodes were examined by immunohistochemistry. Tumor cell apoptosis was analyzed by Hoechst dyeing. RESULTS: Mice bearing VEGF-D overexpressing xenografts showed a significantly higher rate of lymph node metastasis and markedly greater tumor volume compared with the controls. The functional lymphatic vessels were denser and enlarged in marginal and central tumor portions. Additionally, higher CA125 expression was observed in the involved lymph nodes. Mice bearing VEGF-D overexpressing xenografts also exhibited a markedly lower apoptotic index compared with the controls. CONCLUSIONS: Our data demonstrate the important role of VEGF-D in promoting lymph node metastasis by increasing tumor lymphangiogenesis, stimulating draining lymphatic vessel formation, and enhancing tumor invasiveness. Our findings show that VEGF-D can be a promising therapeutic target for ovarian cancer.

Combining Serum Procalcitonin Level, Thromboelastography, and Platelet Count to Predict Short-term Development of Septic Shock in Intensive Care Unit.

OBJECTIVE: Despite the recent advances in diagnosis and treatment, sepsis continues to lead to high morbidity and mortality. Early diagnosis and prompt treatment are essential to save lives. However, most biomarkers can only help to diagnose sepsis, but cannot predict the development of septic shock in high-risk patients. The present study determined whether the combined measurement of procalcitonin (PCT), thromboelastography (TEG) and platelet (PLT) count can predict the development of septic shock. METHODS: A retrospective study was conducted on 175 septic patients who were admitted to the intensive care unit between January 2017 and February 2021. These patients were divided into two groups: 73 patients who developed septic shock were assigned to the septic shock group, while the remaining 102 patients were assigned to the sepsis group. Then, the demographic, clinical and laboratory data were recorded, and the predictive values of PCT, TEG and PLT count for the development of septic shock were analyzed. RESULTS: Compared to the sepsis group, the septic shock group had statistically lower PLT count and TEG measurements in the R value, K value, α angle, maximum amplitude, and coagulation index, but had longer prothrombin time (DT), longer activated partial thromboplastin time (APTT), and higher PCT levels. Furthermore, the Sequential Organ Failure Assessment (SOFA) score was higher in the septic shock group. The multivariate logistic regression analysis revealed that PCT, TEG and PLT count were associated with the development of septic shock. The area under the curve analysis revealed that the combined measurement of PCT, TEG and PLT count can be used to predict the development of septic shock with higher accuracy, when compared to individual measurements. CONCLUSION: The combined measurement of PCT, TEG and PLT count is a novel approach to predict the development of septic shock in high-risk patients.

Compare the effectiveness of extracorporeal shockwave and hyperbaric oxygen therapy on enhancing wound healing in a streptozotocin-induced diabetic rodent model.

Studies have revealed that both extracorporeal shock-wave therapy (ESWT) and hyperbaric oxygen therapy (HBOT) can accelerate wound healing. This study aimed to compare the effectiveness of ESWT and HBOT in enhancing diabetic wound healing. A dorsal skin defect in a streptozotocin-induced diabetes rodent model was used. Postoperative wound healing was assessed once every 3 days. Histologic examination was performed with hematoxylin and eosin staining. Proliferation marker protein Ki-67 (Ki-67), endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), and 8-hydroxy-2-deoxyguanosine (8-OHdG) were evaluated with immunohistochemical (IHC) staining. The wound area was significantly reduced in the ESWT and HBOT groups compared to that in the diabetic controls. However, the wound healing time was significantly increased in the HBOT group compared to the ESWT group. Histological findings showed a statistical increase in neovascularization and suppression of the inflammatory response by both HBOT and ESWT compared to the controls. IHC staining revealed a significant increase in Ki-67, VEGF, and eNOS but suppressed 8-OHdG expression in the ESWT group compared to the HBOT group. ESWT facilitated diabetic wound healing more effectively than HBOT by suppressing the inflammatory response and enhancing cellular proliferation and neovascularization and tissue regeneration.

Effectiveness of combining plasma exchange with continuous hemodiafiltration on acute Fatty liver of pregnancy complicated by multiple organ dysfunction.

Acute fatty liver of pregnancy (AFLP) is a rare disease of progressive hepatic insufficiency and secondary systemic complications that induce significant maternal risk. The application of combining plasma exchange (PE) and continuous hemodiafiltration (CHDF) is a novel concept for patients with AFLP. Since 2002, we have utilized the combination of PE with CHDF as adjunctive medical therapy for 11 AFLP patients with multiple organ dysfunction. Before PE and CHDF initiation, four patients had signs and symptoms of encephalopathy, four required ventilatory support, and all 11 were developing liver failure, significant renal compromise, and coagulopathy. PE combined with CHDF for patients was initiated a mean of 2 days postpartum (range, days 0-3). Daily or every other day PE combined with CHDF was undertaken on two to eight occasions for each of the 11 patients. Ten patients responded with composite clinical and laboratory improvement and were discharged to the ward, then cured and discharged from hospital; one patient died of septic shock. Average duration of hospitalization was 17 days (range, days 9-38) from time of admission to discharge; the average duration of intensive care unit was 10 days (range, days 4-23). No significant PE- and CHDF-related complications occurred. These results indicate that combing PE and CHDF in a series-parallel circuit is an effective and safe treatment for patients with severe AFLP. This finding may have important implications for the development of an effective treatment for patients with AFLP suffering multiple organ dysfunction.

[The experience of extracorporeal membrane oxygenation for severe acute respiratory failure in adults].

OBJECTIVE: To summarize the experience of extracorporeal membrane oxygenation (ECMO) for patients with severe acute respiratory failure in adults and to investigate the factors associated with death. METHODS: The clinical data of patients with severe acute respiratory failure supported with ECMO in respiratory intensive care unit of Beijing Chaoyang Hospital from November 2009 to December 2011 were prospectively collected and analyzed. The data included general condition before EMCO, blood gas analysis, hemodynamics, ventilator settings of mechanical ventilation and complications during ECMO. The primary outcome was death or severe disability within 3 months. Statistical software of SPSS (version 16.0) was used for data analysis. RESULTS: Twenty-five patients with severe respiratory failure received ECMO treatment, of which 16 patients were analyzed. The mean age was (45 ± 14) years old (range, 22 - 64 years old). Thirteen patients were male. Before ECMO, all of the patients were treated with invasive positive pressure ventilation for (72 ± 64) hours. Eight patients had been treated with noninvasive ventilation for a median of 55(10-114) hours. Patients had severe respiratory failure despite advanced mechanical ventilator support. The mean PaO2/fraction of inspired oxygenation (FiO2) ratio was (54 ± 18) mm Hg (1 mm Hg = 0.133 kPa), positive end-expiratory pressure (PEEP) was (11 ± 6) cm H2O(1 cm H2O = 0.098 kPa), Murray lung injury score was 3.6 ± 0.5, serum lactate was (2.5 ± 2.0) mmol/L, serum white blood cell count was (16 ± 6)×10(9)/L, and APACHEII score was 17 ± 8. All of the patients were treated with venous-venous ECMO (VV-ECMO). The change of mechanical ventilation settings were (pre-ECMO vs 2 hours post-ECMO): FiO2 1.0 vs 0.55 ± 0.21, PEEP (11 ± 6) vs (9 ± 6) cm H2O, V(T) (6.8 ± 2.2) vs (4.4 ± 2.0) ml/kg PBW, peak airway pressure (27 ± 8) vs (24 ± 7) cm H2O, respiratory rate (37 ± 10) vs (23 ± 10) breaths/min. Arterial blood gas, including pH, PaO2 and PaCO2 were significantly improved after ECMO running 24 and 48 hours (P < 0.05). The mean VV-ECMO support interval was (9.7 ± 9.6) days (range, 2 - 41 days). Ten patients were successfully weaned from ECMO, of whom 2 died in ICU. Three patients died during ECMO, while another 3 patients died after withdrawal of further treatment. Eight patients survived to 3 months without severe disability. In a multi-variate Cox regression model, pre-ECMO factors including lower PaO2/FiO2 and increasing white blood cell count were associated with increased risks of death (RR was 0.733, 1.701 respectively, both P values < 0.05). CONCLUSIONS: VV-ECMO is a potentially effective approach for severe acute respiratory failure. PaO2/FiO2 and white blood cell count pre-ECMO may be the risk factors for poor outcome.

[Adenovirus mediated N19RhoA gene transfer attenuates neointimal formation in rats after carotid artery balloon injury].

OBJECTIVE: To investigate the role of small G-protein RhoA in neointimal formation following rat carotid artery balloon injury and related mechanisms. METHODS: Male 3-4-month-old Sprague-Dawley rats were used in the present study (10 rats per group). Group A: control; Group B: carotid artery balloon injury; Group C: injury + Ad-CMV-eGFP + Pluronic F-127; Group D: injury + Ad-CMV-N19RhoA-eGFP + Pluronic F-127; Group E: non injury + Ad-CMV-eGFP + Pluronic F-127. Perivascular gene transfer of an adenovirus co-expressing N19RhoA was performed to rat carotid artery following balloon injury and the effect on neointimal formation and the expressions of PCNA and α-SM-actin examined. Rats were killed after 14 days. RESULTS: The protein expression of RhoA in group B was significantly higher than in group A (P = 0.001), and the positive cells rate of PCNA and α-SM-actin which were assessed by immunohistochemistry in group C (45.2% and 75.6%) was significantly higher than in group D (28.4% and 51.9%, all P < 0.01). The area of neointima was significantly smaller [(0.14 ± 0.08) mm(2) vs. (0.23 ± 0.10) mm(2), P < 0.01], the luminal area was significantly larger [(0.47 ± 0.11) mm(2) vs. (0.31 ± 0.06) mm(2), P < 0.01] in group D than in group C. CONCLUSION: Gene transfer of N19RhoA attenuates neointimal formation after balloon injury in rat carotid arteries possibly related to the modulating capacities of small G-protein RhoA on the proliferation, phenotypic differentiation and migration of vascular adventitial fibroblasts.

[The effects of joint administration of 6% hydroxyethyl starch 130/0.4 and high-volume hemofiltration on patients with acute lung injury and acute kidney injury].

OBJECTIVE: To evaluate the effects of administration of 6% hydroxyethyl starch (6% HES 130/0.4, voluven) in combination with high volume hemofiltration (HVHF) in patients with ALI and AKI. METHODS: One hundred and eight patients with acute lung injury (ALI) and acute kidney injury (AKI) were enrolled from Department of Intensive Care Unit (ICU) of the provincial Hospital Affiliated to Shandong University between August 2006 and May 2011. The patients were randomly divided into two groups A (n = 68) and B (n = 40) to receive voluven (i.v., for volume resuscitation) and voluven+HVHF for 72 hours. The arterial blood lactate concentration (Lac), high sensitivity C-reactive protein (hs-CRP) serum concentration, pulmonary function index alveolar-arterial oxygen pressure difference [P(A-a)DO2] and oxygenation index (OI), as well as kidney function index serum cystatin C (Cyst C) and serum creatinine clearance rate (CCr) were measured at the time of admission and 72 hours after the treatment for statistical analysis. RESULTS: In comparison with group A, group B had significantly (all P < 0.01) lower mean value in the level of arterial Lac (mmol/L: 1.7 ± 0.7 vs. 2.7 ± 1.5), serum hs-CRP (mg/L: 35.8 ± 18.8 vs. 99.5 ± 20.4), P(A-a)DO2 (mm Hg, 1 mm Hg=0.133 kPa: 115.5 ± 23.1 vs. 155.4 ± 27.4), Cyst C (mg/L: 2.06 ± 1.12 vs. 3.95 ± 2.06) and significantly higher (both P < 0.01) mean value of OI (mm Hg: 295.2 ± 38.8 vs. 239.5 ± 32.7) and CCr (ml/min: 108.71 ± 31.33 vs. 90.21 ± 30.35) 72 hours after treatment. The mortality rate of group B was significantly lower than group A [10.00%(4/40) vs. 29.41%(20/68), P < 0.05] 7 days after the admission. CONCLUSION: 6% HES 130/0.4 in combination with HVHF could improve the lung and kidney function of the patients with ALI and AKI, prevent the development of multiple organ dysfunction syndrome (MODS), therefore improve the survival rate of these patients.

[Therapeutic effects of survivinT34A mutants on pulmonary metastases of melanoma B16 in mice].

OBJECTIVE: To study the antitumor effects and mechanism of cationic liposome-mediated SurvivinT34A mutants in mice with pulmonary metastases of melanoma B16. METHODS: The pulmonary metastases model of B16, a mouse melanoma cell line, was established by the injection of B16 cell suspension via tail vein of C57BL/6 mouse. 3 days later, 18 tumor-bearing mice were randomly divided into 3 groups, which were intravenously administrated with normal saline (100 microL), vector (5 microg DNA, 100 microL) and recombinant plasmid SurvivinT34A mutants (5 microg DNA, 100 microL) every other day for five doses, respectively. After 28 days, the mice were sacrificed, the lung tissue was weighted, and the number of metastasis foci on lung surface was counted and measured. Then, pathological change of lung tissue was studied with HE stainning. The number of apoptotic cells in tumor tissues was assessed by TUNEL assay. RESULTS: Compared with the control, mice treated with mSurvivnT34A had an intact structure of lung, with significant reduction in the number of metastasis foci on lung surface (P < 0.05), and high level of apoptosis in tumor tissue (P < 0. 05). CONCLUSION: Recombinant plasmid SurvivinT34A mutants (pORF9-mSurvivinT34A) may inhibit the formation of pulmonary metastases of melanoma.

Far-Infrared Therapy Accelerates Diabetic Wound Healing via Recruitment of Tissue Angiogenesis in a Full-Thickness Wound Healing Model in Rats.

Far-infrared ray (FIR) therapy has been applied in the tissue regeneration field. Studies have revealed that FIR could enhance wound healing. However, the biological effects of FIR on diabetic wounds remain unclear. Our study aims to investigate whether FIR could accelerate diabetic wound healing and analyze the biomechanisms. A dorsal skin defect (area, 6 × 5 cm2) in a streptozotocin (STZ)-induced diabetes rodent model was designed. Thirty-two male Wistar rats were divided into 4 groups (n = 8 each subgroup). Group 1 consisted of sham, non-diabetic control; group 2, diabetic control without treatment; group 3, diabetic rats received 20 min FIR (FIR-20, 20 min per session, triplicate/weekly for 4 weeks) and group 4, diabetic rats received 40 min FIR (FIR-40, 40 min per session, triplicate in one week for 4 weeks). The wound healing was assessed clinically. Skin blood flow was measured by laser Doppler. The vascular endothelial growth factor (VEGF), 8-hydroxy-2-deoxyguanosine (8-OHdG), eNOS, and Ki-67, were analyzed with immunohistochemical (IHC) staining. Laser Doppler flowmetry analysis of the blood flow of wounding area revealed the blood flow was higher in diabetic rats who received 40 min FIR (FIR-40) as compared to that in FIR-20 group. The wounding area was significantly reduced in the FIR-40 group than in the diabetic control groups. Histological findings of peri-wounding tissue revealed a significant increase in the neo-vessels in the FIR-treated groups as compared to the controls. IHC staining of periwounding biopsy tissue showed significant increases in angiogenesis expressions (VEGF, eNOS, and EGF), cell proliferation (Ki-67), and suppressed inflammatory response and oxygen radicles (CD45, 8-OHdG) expressions in the FIR-treated groups as compared to that in controls. Treatment with the optimal dosage of FIR significantly facilitated diabetic wound healing and associated with suppressed pro-inflammatory response and increased neovascularization and tissue regeneration.

Immunotherapy targeting fibroblast activation protein inhibits tumor growth and increases survival in a murine colon cancer model.

Murine studies have shown that immunological targeting of fibroblast activation protein (FAP) can elicit protective immunity in the absence of significant pathology. Fibroblast activation protein is a product overexpressed by tumor-associated fibroblasts (TAF) and is the predominant component of the stoma in most types of cancer. Tumor-associated fibroblasts differ from normal adult tissue fibroblasts, and instead resemble transient fetal and wound healing-associated fibroblasts. Tumor-associated fibroblasts are critical regulators of tumorigenesis, but differ from tumor cells by being more genetically stable. Therefore, in comparison to tumor cells, TAF may represent more viable therapeutic targets for cancer immunotherapy. To specifically target TAF, we constructed a DNA vaccine directed against FAP. This vaccine significantly suppressed primary tumor and pulmonary metastases primarily through CD8(+) T-cell-mediated killing in tumor-bearing mice. Most importantly, tumor-bearing mice vaccinated against FAP exhibited a 1.5-fold increase in lifespan and no significant pathology. These results suggest that FAP, a product preferentially expressed by TAF, could function as an effective tumor rejection antigen.

[The role of p38 mitogen-activated protein kinase in interleukin-6 induction by lipopolysaccharide in vascular smooth muscle cells].

OBJECTIVE: To investigate the regulation mechanism of p38 mitogen-activated protein kinase (p38MAPK) in interleukin-6 (IL-6) expression of vascular smooth muscle cell (VSMC) induced by lipopolysaccharide (LPS). METHODS: Rat VSMCs were divided into LPS group, SB203580+LPS group, SB203580 group and control group. LPS group was treated with 100 microg/L LPS for 0, 3, 6, 12, 24 hours, SB203580+LPS group was first treated with 10 micromol/L SB203580 for 2 hours and then exposed to 100 microg/L LPS for 0, 3, 6, 12, 24 hours, SB203580 group was pretreated with 10 micromol/L SB203580 for 2 hours. The level of IL-6 mRNA was determined by real-time polymerase chain reaction (PCR) and IL-6 secretion in the culture medium was measured by enzyme linked immunosorbent assay (ELISA) at different time points. RESULTS: The expression of IL-6 mRNA and the release of IL-6 were increased significantly in VSMC as early as 3 hours after being treated with LPS [mRNA: (21.3+/-3.2)x10(4), protein: (296.2+/-19.6) ng/L], peaked in 12 hours [mRNA: (131.4+/-11.2)x10(4), protein: (897.7+/-34.0) ng/L], and the elevation persisted up to 24 hours after treatment [mRNA: (15.3+/-4.7)x10(4), protein: (194.3+/-24.0) ng/L] compared with control group [mRNA: (9.4+/-1.9)x10(4), protein: (29.4+/-4.4) ng/L, all P<0.05]. On the other hand, the expression of IL-6 was significantly suppressed by p38MAPK inhibitor SB203580 at 3, 6, 12 hours [mRNA: (15.4+/-3.6)x10(4), (43.2+/-6.6)x10(4), (56.2+/-5.5)x10(4), protein: (180.3+/-23.6), (432.2+/-56.8), (546.2+/-57.9) ng/L, all P<0.05]. CONCLUSION: The release of IL-6 and the expression of IL-6 mRNA was increased significantly in LPS-challenged VSMC; however, the induction of IL-6 was significantly suppressed by p38MAPK inhibitor. p38MAPK may play an important role in the release of IL-6 induced by LPS.

[Effect of high volume hemofiltration on expression of Toll-like receptor 4 in myocardium in endotoxin induced shock in dogs].

OBJECTIVE: To investigate the effects of high volume hemofiltration (HVHF) on the expression of Toll-like receptor 4 (TLR4) mRNA in myocardium in endotoxin (lipopolysaccharide, LPS) induced shock in dogs. METHODS: Sixteen healthy male dogs were injected with LPS 650 microg/kg via central vein to reproduce the model of endotoxin shock. All dogs were divided randomly into two groups: control group and therapy group, with 8 dogs in each group. Contents of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), IL-10 in circulation were measured by radioimmunological method. The expression levels of TLR4 mRNA in all group were measured by reverse transcription-polymerase chain reaction (RT-PCR). Change in myocardial histopathology was observed and analyzed with the aid of electron microscope. RESULTS: The contents of TNF-alpha (microg/L: 0.59+/-0.15, 0.51+/-0.12, 0.41+/-0.10), IL-6 (ng/L: 11.08+/-2.83, 9.82+/-2.58, 8.25+/-2.05), IL-10 (microg/L: 57.28+/-5.93, 53.81+/-5.83, 50.67+/-6.33) in therapy group were found to have decreased significantly at 1, 2, and 4 hours after HVHF compared with those when the model was completed [(0.84+/-0.16) microg/L, (16.97+/-2.50) ng/L, (70.86+/-5.43) microg/L], showing a continuous trend of lowering (all P<0.01). The contents of TNF-alpha, IL-6, IL-10 in therapy group were lower than those in control group significantly at any time point [TNF-alpha (microg/L): 0.75+/-0.14, 0.74+/-0.11, 0.72+/-0.11, IL-6 (ng/L): 15.33+/-3.20, 14.66+/-3.24, 14.20+/-3.33, IL-10 (microg/L): 71.54+/-4.73, 70.71+/-4.34, 69.35+/-4.60, all P<0.01]. Compared with control group, HVHF treatment group could down-regulate mRNA expression of TLR4 in myocardium (t=3.58, P<0.01). Correlation analysis revealed significant positive-correlation between tissue TLR4 mRNA expression and contents of TNF-alpha, IL-6, IL-10 in circulation (r(1)=0.785, r(2)=0.569, r(3)=0.653, all P<0.05). Injury to the myocardium was significantly ameliorated in therapy group compared with control group as shown by electron microscopic observation. CONCLUSION: HVHF can down-regulate mRNA expression of TLR4 in myocardium in LPS induced shock in dogs, and myocardial inflammatory response was alleviated resulting in amelioration of myocardial injury.

[Anti-prostate cancer activity of Survivin-T34A mutant in vitro and in vivo].

OBJECTIVE: To investigate the effect of Survivin-T34A mutant on murine prostate cancer and its apoptosis-inducing efficacy in vivo and in vitro. METHODS: In vitro, prostate cancer cells TRAMP-C1 were transfected with Survivin-T34A plasmid encapsulated by cationic liposome. The apoptosis of TRAMP-C1 was evaluated with flow cytometry. C57BL/6 mice model with TRAMP-C1 prostate cancer was established. Twenty four male mice with TRAMP-C1 prostate cancers were divided randomly into three groups, which were intravenously injected with normal saline, empty vector PORF-9-null encapsulated by cationic liposome and Survivin-T34A plasmid encapsulated by cationic liposome respectively twice a week for eight doses. The size of tumors was measured and the tumor sections of each group were stained with TUNEL reagent for apoptosis detection. RESULTS: An apoptotic index of 46% of TRAMP-C1 transfected with Survivin-T34A plasmid encapsulated by cationic liposome was observed. The tumor volume of Survivin-T34A group of C57BL/6 mice with TRAMP-C1 prostate cancer was far smaller than those in the control groups (P < 0.05) and the tumors treated with Survivin-T34A showed significant increase of apoptosis compared with those of control groups (P < 0.05). CONCLUSION: Survivin-T34A mutant efficiently inhibits the growth of prostate cancer, which is based on the mechanism of Survivin-T34A mutant inducing apoptosis of tumor cells.

CCDC7 Activates Interleukin-6 and Vascular Endothelial Growth Factor to Promote Proliferation via the JAK-STAT3 Pathway in Cervical Cancer Cells.

OBJECTIVE: Tumor growth is one of the most lethal attributes of human malignancy. The expression of CCDC7, a novel gene which has multiple functions, has been shown to be associated with tumor growth and poor prognosis in patients with cancer. However, the specific functions of CCDC7 remain unclear. Here, we investigated the molecular mechanisms underlying the effects of CCDC7 on proliferation in cervical cancer. MATERIALS AND METHODS: The MTT and EdU assays were performed to evaluate the function of CCDC7. The immunohistochemical, quantitative real-time PCR (qRT-PCR), ELISA and Western blot assay were used to detect the gene and protein expression in tissues and cells. A xenograft test was conducted to detect the impact of CCDC7 on tumor development in vivo . RESULTS: In immunohistochemical analysis of 193 cases, normal cervical tissue and cervical cancer tissue show that CCDC7 expression is closely correlated with the development of cervical cancer and was positively correlated with the clinical stage and histological grade. Overexpression or knockdown of CCDC7 affected cell proliferation in cervical cancer cells in vitro. In a nude mouse xenograft model in vivo, knockdown of CCDC7 inhibited cell proliferation and tumor growth. Furthermore, CCDC7 overexpression upregulated interleukin (IL)-6 and vascular endothelial growth factor (VEGF) at mRNA and protein levels, and treatment with recombinant IL-6 or VEGF proteins also increased CCDC7 expression. In a case set of 80 patients with cervical cancer, we found that CCDC7, IL-6, and VEGF affected patient prognosis. Finally, inhibition of various signaling pathways using specific inhibitors indicated that CCDC7 blocked the decrease in cell proliferation observed following suppression of the JAK-STAT3 pathway, suggesting that CCDC7 functioned via this critical signaling network. CONCLUSION: Those findings indicated that CCDC7 may be a novel target for the treatment of cervical cancer and may have applications as a predictive marker for tumor growth in cervical carcinoma.

The Acceleration of Diabetic Wound Healing by Low-Intensity Extracorporeal Shockwave Involves in the GSK-3ß Pathway.

Previous studies have demonstrated that extracorporeal shock wave therapy (ESWT) could accelerate diabetic wound healing and that the inhibition of glycogen synthase kinase-3ß (GSK-3ß) is involved in epithelial differentiation during wound healing. This study investigated whether the enhancement of diabetic wound healing by ESWT is associated with the GSK-3ß-mediated Wnt/ß-catenin signaling pathway. A dorsal skin wounding defect model using streptozotocin-induced diabetic rodents was established. Rats were divided into 4 groups: group 1, normal controls without diabetes; group 2, diabetic controls without treatment; group 3, diabetic rats receiving ESWT; and group 4, rats receiving 6-bromoindirubin-3'oxime (BIO), a GSK-3ß inhibitor, to trigger Wnt/ß-catenin signaling. Tissue samples were collected and analyzed by immunohistochemical (IHC) staining and quantitative RT-PCR. The ESWT and BIO-treated groups both exhibited significant promotion of wound healing compared to the healing in controls without treatment. RT-PCR analysis of Wnt-1, -3a, -4, -5a, and -10 and ß-catenin expression showed significantly increased expression in the ESWT group. The IHC staining showed that Wnt-3a and -5a and ß-catenin levels were significantly increased in the ESWT and BIO treatment groups compared to the control groups. ESWT enhancement of diabetic wound healing is associated with modulation of the GSK-3ß-mediated Wnt/ß-catenin signaling pathway.

Alloantigen-pulsed host dendritic cells induce T-cell regulation and prolong allograft survival in a rat model of hindlimb allotransplantation.

BACKGROUND: Composite tissue allotransplantation is restricted due to the risks presented by long-term therapeutic immunosuppression. This study is conducted to investigate whether treatment with recipient immature dendritic cells (DCs) pulsed with donor alloantigens can prolong allograft survival and induce T-cell regulation in a rodent model. MATERIALS AND METHODS: Orthotopic hindlimb transplants from Brown-Norway (RT1(n)) to Lewis (RT1(1)) rats were performed (day 0). DCs were propagated from the recipient bone marrow and pulsed with the donor alloantigen lysate. Group 1 (control group) did not receive any treatment. Groups 2 and 3 received cyclosporine A (CsA) at a concentration of 10 and 16 mg.kg(-1).day(-1), respectively, on days 0-20 following composite tissue allotransplantation. Group 4 received antilymphocyte serum (i.p. administered 4 d before and 1 d after transplantation) therapy. Group 5 received combined treatment with CsA (10 mg.kg(-1).day(-1), days 0-20) and donor alloantigen-pulsed recipient DCs (i.v. administered on days 7, 14, and 21). Group 6 received combined treatment with CsA (10 mg.kg(-1).day(-1) on days 0-20), antilymphocyte serum (administered i.p. 4 d before and 1 d after transplantation), and DCs (administered i.v. on days 7, 14, and 21). Graft rejection was defined as epidermolysis/desquamation of the donor skin. The mixed lymphocyte reaction was performed to determine the donor T-cell reactivity. Tissue samples were biopsied to analyze the histological changes, and flow cytometry was performed to quantify the donor T-cells. RESULTS: Allograft survival was significantly prolonged (>200 d) in Group 6 when compared with the other groups (P < 0.001). The mixed lymphocyte reaction performed for Group 6 revealed hyporesponsiveness of the T-cells to donor alloantigens. Flow cytometric analysis in Group 6 revealed a significant increase in the percentage of CD4(+)/CD25(+) and CD4(+)/foxP3(+) T-cells expression, and significant increase in the percentage of donor cells (RT1(n)) in the recipient peripheral blood. Immunohistochemical staining of allo-skin revealed a significant increase in the proportion of CD25(+) cells in the subcutaneous and dermis layers in Group 6, as compared to other groups. CONCLUSION: Treatment with donor alloantigen-pulsed recipient immature DCs in combination with transient immunosuppression prolongs allograft survival and induced tolerance by inducing T-cell hyporesponsiveness to donor alloantigens and increasing the CD4(+)/CD25(+) T-cell population.