[Effects of applying human umbilical cord mesenchymal stem cell exosomes through different pathways to treat full-thickness skin defect wounds in mice].

Objective: To investigate the effects of human umbilical cord mesenchymal stem cell (hUCMSC) exosomes in the treatment of full-thickness skin defect wounds in mice through local wound application, subcutaneous injection at the wound margin, and tail vein injection, and to explore the optimal administration route of hUCMSC exosomes for wound treatment. Methods: This study was an experimental study. hUCMSC exosomes were extracted from the discarded umbilical cord tissue of three normal delivery women aged 25-35 years in the Department of Obstetrics and Gynecology of Baogang Hospital of Inner Mongolia and successfully identified. Totally 120 male BALB/c mice aged 6-8 weeks were selected, and full-thickness skin defect wounds were prepared on the back of them. According to the random number table, the injured mice were divided into control group (without drug administration), local wound application group, wound margin subcutaneous injection group, and tail vein injection group (with 30 mice in each group). Mice in the latter three groups were given 0.2 mL phosphate buffer solution containing 200 µg hUCMSC exosomes by local wound application, subcutaneous injection at the wound margin, and tail vein injection, respectively. On post injury day (PID) 7, 14, and 21, the general condition of the wound was observed, and the wound healing rate was calculated; the wound tissue was collected, the pathological changes and collagen fibers were observed respectively by hematoxylin-eosin staining and Masson staining, the number of new microvessels was observed by CD31 immunohistochemical staining, and the content of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) was detected by enzyme-linked immunosorbent assay. The sample number was 10 in each group at each time point. Results: On PID 7, 14, and 21, the wounds of mice in the 4 groups all healed gradually, and the wound healing of the mice in wound margin subcutaneous injection group was the best; the wound healing rates of mice in the three administration groups were significantly higher than those in control group (P<0.05), the wound healing rates of mice in wound margin subcutaneous injection group and tail vein injection group were significantly higher than those in local wound application group (P<0.05), and the wound healing rates of mice in wound margin subcutaneous injection group were significantly higher than those in tail vein injection group (P<0.05). On PID 7, 14, and 21, the growth and epithelialization speed of the wound tissue of mice in the three administration groups were significantly accelerated, and the collagen fibers in the wounds of mice in the three administration groups were larger in number and more neatly arranged in comparison with the control group. On PID 7, 14, and 21, under every 200-fold visual field, the number of new microvessels in the wound tissue of mice in local wound application group was 24.1±2.5, 50.7±4.1, and 44.2±2.3, respectively, the number of new microvessels in the wound tissue of mice in wound margin subcutaneous injection group was 32.2±2.9, 67.5±4.9, and 53.6±3.7, respectively, and the number of new microvessels in the wound tissue of mice in tail vein injection group was 27.8±2.4, 59.1±3.7, and 49.6±2.6, respectively, which was significantly more than 20.6±1.7, 46.7±3.4, and 40.9±2.8 in control group (P<0.05); the number of new microvessels in the wound tissue of mice in wound margin subcutaneous injection group and tail vein injection group was significantly more than that in local wound application group (P<0.05); the number of new microvessels in the wound tissue of mice in wound margin subcutaneous injection group was significantly more than that in tail vein injection group (P<0.05). On PID 7, 14, and 21, the content of TNF-α and IL-6 in the wound tissue of mice in the three administration groups was significantly less than that in control group (P<0.05), the content of TNF-α and IL-6 in the wound tissue of mice in wound margin subcutaneous injection group and tail vein injection group was significantly less than that in local wound application group (P<0.05), and the content of TNF-α and IL-6 in the wound tissue of mice in wound margin subcutaneous injection group was significantly less than that in tail vein injection group (P<0.05). Conclusions: Local wound application, subcutaneous injection at the wound margin, and tail vein injection of hUCMSC exosomes can all promote the wound healing of full-thickness skin defects in mice through alleviating excessive inflammatory response and promoting angiogenesis. Among them, subcutaneous injection at the wound margin has a better therapeutic effect, indicating subcutaneous injection at the wound margin is the optimal administration route for hUCMSC exosomes in wound treatment.

[The regulatory function of tumor-infiltrating Th9 cells to anti-tumor activity of CD8(+) T cells in patients with gastric cancer].

Objective: To investigate the levels of Th9 cells and interleukin-9 (IL-9), and to assess the regulatory activity of Th9/IL-9 to anti-tumor immune response in patients with gastric cancer. Methods: Thirty-four patients with gastric cancer who received operation in the First Affiliated Hospital of Xinxiang Medical University between October 2018 and August 2019 were included. Twenty individuals who received physical examination in the same period were also enrolled. Peripheral blood was collected, and then plasma and peripheral blood mononuclear cells (PBMCs) were isolated. Tumor-infiltrating lymphocytes (TILs) and autologous gastric cancer cells were isolated from resected gastric cancer tissues. CD4(+) T cells, CD8(+) T cells, and CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells were purified from PBMCs and TILs. Plasma IL-9 level was measured by enzyme linked immunosorbent assay (ELISA). The percentage of CD3(+) CD4(+) IL-9(+) Th9 cells in PBMCs and TILSs was assessed by flow cytometry. The mRNA levels of IL-9 and transcriptional factors purine-rich nucleic acid binding protein 1 (PU.1) were semi-quantified by real-time quantitative polymerase chain reaction (RT-qPCR). PBMCs and TILs from gastric cancer patients were stimulated with recombinant human IL-9. Cellular proliferation was measured by cell counting kit-8. The phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and STAT6 were investigated by western blot. Cytokine production was measured by ELISA. Purified CD8(+) T cells from TILs of gastric cancer patients were stimulated with recombinant human IL-9. CD8(+) T cells and autologous gastric cancer cells were cocultured in direct contact and indirect contact manner. The percentage of target cell death was calculated by measuring the lactate dehydrogenase (LDH) level. These cretion of γ-Interferon (γ-IFN) and tumor necrosis factor-α (TNF-α) was measured by ELISA. CD4(+) CCR4(-)CCR6(-)CXCR3(-)cells, CD8(+) T cells, and autologous gastric cancer cells were directly cocultured, and anti-IL-9 neutralizing antibody was added. The target cell death was measured. Results: The percentages of CD3(+) CD4(+) IL-9(+) Th9 cells in PBMCs of control group and PBMCs of gastric cancer group were (1.21±0.25)% and (1.14±0.19)%, respectively. The difference was not statistically significant (P=0.280). The percentage of CD3(+) CD4(+) IL-9(+) Th9 cells in TILs of gastric cancer group was (2.30±0.55)%, which was higher than those in PBMCs of control group and PBMCs of gastric cancer group (P<0.001). The plasma IL-9 level in control group and gastric cancer group were (5.04±1.51) and (4.93±1.25) ng/ml. The difference was not statistically significant (P=0.787). The relative levels of IL-9 mRNA in PBMCs of control group and PBMCs of gastric cancer group were 1.33±0.39 and 1.36±0.27. The difference was not statistically significant (P=0.691). The relative level of IL-9 mRNA in TILs of gastric cancer group was 2.90±0.75, which was higher than those in PBMCs of control group (P<0.001) and PBMCs of gastric cancer group (P<0.001). The relative levels of PU.1 mRNA in PBMCs of control group and PBMCs of gastric cancer group were 1.21±0.12 and 1.20±0.11. The difference was not statistically significant (t=0.21, P=0.833). PU.1 mRNA relative level in TILs of gastric cancer group was 2.81±0.65, which was higher than those in PBMCs of control group (P<0.001) and PBMCs of gastric cancer group (P<0.001). Recombinant human IL-9 stimulation did not affect the proliferation of PBMCs and TILs of gastric cancer patients (P>0.05), but elevated the phosphorylation level of STAT6 and induced the secretions of γ-IFN, IL-17, and IL-22 by TILs (P<0.05). In direct contact culture system, IL-9 stimulation promoted tumor-infiltrating CD8(+) T cells-induced autologous gastric cancer cell death [(20.62±2.27)% vs. (16.08±2.61)%, P<0.01)]. In indirect contact culture system, IL-9 stimulation did not increase CD8(+) T cell-induced autologous gastric cancer cell death [(5.21±0.70)% vs. (5.31±1.22)%, P=0.998)]. However, the secretion levels of γ-IFN were elevated in response to IL-9 stimulation in both culture systems [direct contact culture system: (100.40±12.05) pg/ml vs. (76.45±8.56) pg/ml; indirect contact culture system: (78.00±9.98) pg/ml vs. (42.09±10.71) pg/ml; P<0.01]. The TNF-α secretion level did not significantly changed (P>0.05). In direct contact culture system, the percentage of target cells was (22.01±3.05) % and γ-IFN secretion level was (104.5±12.84) pg/ml in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells group, which was higher than (16.08±2.61)% and (76.45±8.56) pg/ml in CD8(+) T cells+ gastric cancer cells group (P<0.01). However, the percentage of target cells was (14.47±3.14)% and γ-IFN secretion level was (70.45±19.43) pg/ml in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells+ anti-IL-9 neutralizing antibody group, which were lower than those in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells group (P<0.01). Conclusion: Tumor-infiltrating Th9 cells and the secreting IL-9 promote the activity of CD8(+) T cells in gastric cancer patients, and enhance anti-tumor immune response.

[Transplantation of compound tissue flap of toe to reconstruct the thumb with necrosis caused by electric burns in four patients].

From January 2010 to December 2017, 4 patients of thumb with necrosis caused by electric burns (all male, aged from 31 to 58 years) were admitted to our hospital, with 1 patient of second degree injury of right thumb, 2 patients of third degree injury of right thumb, and 1 patient of third degree injury of left thumb. Routine debridement under general anesthesia was performed within 7 days after injury. The compound tissue flap of contralateral second toe was transplanted to reconstruct the thumb with third degree defect, and compound tissue flap of ipsilateral distal hallex was transplanted to reconstruct the thumb with second degree defect. Dorsalis pedics artery was anastomosed with radial artery, saphenous vein or dorsalis pedics vein was anastomosed with cephalic vein. The donor site was transplanted with split-thickness skin graft from autologous thigh. All the tissue flaps and skin grafts survived in 2 weeks after surgery. Within 1 year of follow-up, the reconstructed thumbs can achieve radial abduction and palmar abduction with good function. Reconstruction of thumb with free transplantation of compound tissue flap of toe is a good method to repair thumb with necrosis caused by electric burn.

[Replication of 3D laparoscopic membrane anatomic radical gastrectomy for gastric cancer].

According to the current evidence-based medicine researches, the eastern and western countries have reached a consensus that D2 operation is a standardized procedure for advanced gastric cancer.However, the postoperative five-year survival rate is still not satisfactory. Professor Gong Jianping of Tongji Hospital, Tongji Medical Gollege of Huazhong University of Science and Technology proposed a theory of membrane anatomy (the third element of surgical anatomy) and the concept of cancer leakage-an epoch-making concept in surgical anatomy. The Department of Gastrointestinal Surgery, The Affiliated Hospital of Guizhou Medical University was honored to be selected as one of the first domestic replication units of 3D laparoscopic radical gastrectomy under membrane anatomy. Professor Gong Jianping has visited our hospital several times for surgical demonstration, explanation of membrane anatomy theory and replication training. Through the understanding of membrane anatomy theory, we found that 3D laparoscopic radical gastrectomy guided by membrane anatomy can achieve good results, e.g less bleeding, complete resection, complete lymph node dissection and avoidance of side damage, meanwhile the operation is simple and safe. At the same time, it can avoid the shedding of cancer cells, so as to reduce the iatrogenic leakage of cancer and improve the efficacy of radical gastrectomy. In addition, the standardized procedure of laparoscopic radical gastrectomy makes it scientific, reproducible and easy to be popularized.

Apolipoprotein B100 danger-associated signal 1 (ApoBDS-1) triggers platelet activation and boosts platelet-leukocyte proinflammatory responses.

Low-density lipoproteins (LDL), occurring in vivo in both their native and oxidative form, modulate platelet function and thereby contribute to atherothrombosis. We recently identified and demonstrated that 'ApoB100 danger-associated signal 1' (ApoBDS-1), a native peptide derived from Apolipoprotein B-100 (ApoB100) of LDL, induces inflammatory responses in innate immune cells. Platelets are critically involved in the development as well as in the lethal consequences of atherothrombotic diseases, but whether ApoBDS-1 has also an impact on platelet function is unknown. In this study we examined the effect of ApoBDS-1 on human platelet function and platelet-leukocyte interactions in vitro. Stimulation with ApoBDS-1 induced platelet activation, degranulation, adhesion and release of proinflammatory cytokines. ApoBDS-1-stimulated platelets triggered innate immune responses by augmenting leukocyte activation, adhesion and transmigration to/through activated HUVEC monolayers, under flow conditions. These platelet-activating effects were sequence-specific, and stimulation of platelets with ApoBDS-1 activated intracellular signalling pathways, including Ca2+, PI3K/Akt, PLC, and p38- and ERK-MAPK. Moreover, our data indicates that ApoBDS-1-induced platelet activation is partially dependent of positive feedback from ADP on P2Y1 and P2Y12, and TxA2. In conclusion, we demonstrate that ApoBDS-1 is an effective platelet agonist, boosting platelet-leukocyte's proinflammatory responses, and potentially contributing to the multifaceted inflammatory-promoting effects of LDL in the pathogenesis of atherothrombosis.

CIAPIN1 gene silencing enhances chemosensitivity in a drug-resistant animal model in vivo

Overexpression of cytokine-induced apoptosis inhibitor 1 (CIAPIN1) contributes to multidrug resistance (MDR) in breast cancer. This study aimed to evaluate the potential of CIAPIN1 gene silencing by RNA interference (RNAi) as a treatment for drug-resistant breast cancer and to investigate the effect of CIAPIN1 on the drug resistance of breast cancer in vivo. We used lentivirus-vector-based RNAi to knock down CIAPIN1 in nude mice bearing MDR breast cancer tumors and found that lentivirus-vector-mediated silencing of CIAPIN1 could efficiently and significantly inhibit tumor growth when combined with chemotherapy in vivo. Furthermore, Western blot analysis showed that both CIAPIN1 and P-glycoprotein expression were efficiently downregulated, and P53 was upregulated, after RNAi. Therefore, we concluded that lentivirus-vector-mediated RNAi targeting of CIAPIN1 is a potential approach to reverse MDR of breast cancer. In addition, CIAPIN1 may participate in MDR of breast cancer by regulating P-glycoprotein and P53 expression.

CIAPIN1 gene silencing enhances chemosensitivity in a drug-resistant animal model in vivo.

Overexpression of cytokine-induced apoptosis inhibitor 1 (CIAPIN1) contributes to multidrug resistance (MDR) in breast cancer. This study aimed to evaluate the potential of CIAPIN1 gene silencing by RNA interference (RNAi) as a treatment for drug-resistant breast cancer and to investigate the effect of CIAPIN1 on the drug resistance of breast cancer in vivo. We used lentivirus-vector-based RNAi to knock down CIAPIN1 in nude mice bearing MDR breast cancer tumors and found that lentivirus-vector-mediated silencing of CIAPIN1 could efficiently and significantly inhibit tumor growth when combined with chemotherapy in vivo. Furthermore, Western blot analysis showed that both CIAPIN1 and P-glycoprotein expression were efficiently downregulated, and P53 was upregulated, after RNAi. Therefore, we concluded that lentivirus-vector-mediated RNAi targeting of CIAPIN1 is a potential approach to reverse MDR of breast cancer. In addition, CIAPIN1 may participate in MDR of breast cancer by regulating P-glycoprotein and P53 expression.

Increased rate of apoptosis in intimal arterial smooth muscle cells through endogenous activation of TNF receptors.

Intimal proliferation of smooth muscle cells (SMCs) is a key event in the vascular response to injury, including the early stages of atherosclerosis and restenosis after angioplasty. Tumor necrosis factor-alpha (TNF-alpha) has been reported to stimulate growth of cultured human SMCs, but activation of TNF receptors is also known to induce cell death by apoptosis. We report here that SMCs isolated from the neointima of injured rat aortas are characterized by increased expression of TNF-alpha in response to interleukin-1beta and gamma-interferon compared with medial SMCs. Basal and serum-stimulated DNA synthesis was higher in intimal than in medial SMCs. In contrast to previous findings on human SMCs, exposure to interleukin-1beta/gamma-interferon or TNF-alpha did not affect the growth of rat medial SMCs, inhibited DNA synthesis, and decreased cell numbers in cultures of intimal SMCs. Incubation of intimal SMCs with these cytokines also resulted in induction of terminal dUTP nick end-labeling positivity and caspase-3 expression, suggesting cell death by apoptosis, whereas medial cells were markedly less sensitive in this respect. Cytokine-induced apoptosis in intimal cells was effectively inhibited by treatment with antibodies against TNF receptors. These findings suggest that endogenous activation of TNF receptors may represent a way to limit accumulation of SMCs in injured arteries. This mechanism may also be important in SMC death in advanced atherosclerotic plaques.

Activation of nuclear factor kappaB and induction of inducible nitric oxide synthase by Ureaplasma urealyticum in macrophages.

Chronic lung disease (CLD) of prematurity is an inflammatory disease with a multifactorial etiology. The importance of Ureaplasma urealyticum in the development of CLD is debated, and steroids produce some improvement in neonates with this disease. In the present study, the capability of U. urealyticum to stimulate rat alveolar macrophages to produce nitric oxide (NO), express inducible nitric oxide synthase (iNOS), and activate nuclear factor kappaB (NF-kappaB) in vitro was characterized. The effect of NO on the growth of U. urealyticum was also investigated. In addition, the impact of dexamethasone and budesonide on these processes was examined. We found that U. urealyticum antigen (> or =4 x 10(7) color-changing units/ml) stimulated alveolar macrophages to produce NO in a dose- and time-dependent manner (P<0.05). This effect was further enhanced by gamma interferon (100 IU/ml; P<0.05) but was attenuated by budesonide and dexamethasone (10(-4) to 10(-6) M) (P<0.05). The mRNA and protein levels of iNOS were also induced in response to U. urealyticum and inhibited by steroids. U. urealyticum antigen triggered NF-kappaB activation, a possible mechanism for the induced iNOS expression, which also was inhibited by steroids. NO induced by U. urealyticum caused a sixfold reduction of its own growth after infection for 10 h. Our findings imply that U. urealyticum may be an important factor in the development of CLD. The host defense response against U. urealyticum infection may also be influenced by NO. The down-regulatory effect of steroids on NF-kappaB activation, iNOS expression, and NO production might partly explain the beneficial effect of steroids in neonates with CLD.

Augmented expression of inducible NO synthase in vascular smooth muscle cells during aging is associated with enhanced NF-kappaB activation.

Vascular smooth muscle cells (SMCs) are important targets for endothelium-derived nitric oxide (NO), but this production is attenuated in injured and diseased arteries and during aging. However, SMCs can produce NO themselves by expressing an inducible form of NO synthase (iNOS) under inflammatory conditions and in the repair process after arterial injury. We examined iNOS expression in SMCs derived from the aortic media of newborn, young adult, and old rats. Our results show that SMCs from newborn rats cannot produce significant amounts of NO on stimulation with interferon-gamma plus lipopolysaccharide or interleukin-1beta. In contrast, SMCs from old rats exhibit markedly enhanced iNOS activity. The difference in iNOS activity between the newborn and the old SMCs was closely correlated with levels of iNOS protein, mRNA, and gene promoter activity. Similarly, intercellular adhesion molecule-1 (ICAM-1) was also expressed more abundantly in the old than in the newborn SMCs in response to cytokines. Both iNOS and ICAM-1 are transcriptionally regulated by nuclear factor kappaB (NF-kappaB). Our data demonstrate an intense transactivation of NF-kappaB in old SMCs on tumor necrosis factor-alpha stimulation but only a weak one in newborn SMCs. The difference in the NF-kappaB activation could be explained by a much faster and more extensive IkappaBalpha degradation in old than in newborn SMCs. These data indicate that the capability to respond to proinflammatory stimuli by activating NF-kappaB differs between SMCs at different stages of development. This results in differential capability to express NF-kappaB-dependent genes such as iNOS and ICAM-1, which could have implications for host defense and the pathogenesis of vascular diseases.

The contribution of inducible nitric oxide and cytomegalovirus to the stability of complex carotid plaque.

BACKGROUND: Although the association between inflammation and atherosclerosis is well established, the biologic events that trigger the local inflammatory response within plaque are not fully understood. Cytotoxic free radicals and infectious agents, both of which are associated with an inflammatory response, have previously been implicated in the initiation and progression of atherosclerosis. In this study, we analyzed carotid plaque for evidence of oxidative vascular injury by determining the presence and distribution of inducible nitric oxide synthase (iNOS) expression and nitrotyrosine formation and for evidence of infection with cytomegalovirus. METHODS: Carotid plaque from 51 patients who underwent endarterectomy for either primary (n = 37) or recurrent (n = 14) stenosis were examined histologically (hematoxylin-eosin staining and Masson's trichrome staining) and with immunohistochemistry with specific antibodies to alpha-smooth muscle actin, macrophages (CD68), T-lymphocytes (CD3), and T-cell activation (human leukocyte antigen-DR). Twenty-eight specimens from patients with primary (n = 15) and recurrent (n = 13) stenosis were examined for the presence of iNOS and nitrotyrosine with immunohistochemistry and in situ hybridization (iNOS). Twenty-three additional specimens (22 primary, and 1 recurrent) were analyzed with antibodies to p53, cytomegalovirus, and the polymerase chain reaction (cytomegalovirus, n = 8). RESULTS: Primary atherosclerotic lesions were either complex heterogenous cellular plaques (n = 29) or relatively acellular fibrous plaques (n = 8). Ten of 14 recurrent plaques were either complex or fibrous lesions, and the remaining four were typical of myointimal thickening. CD68-positive staining cells were detected in all specimens regardless of their structural morphology. CD3-positive cells were interspersed between macrophages in all heterogeneous cellular plaques and only infrequently noted in fibrous plaques. iNOS and nitrotyrosine immunoreactivity were detected in macrophages and smooth muscle cells in all complex and fibrous plaques and in two of four myointimal plaques. The presence of iNOS and nitrotyrosine in plaque correlated with the existence of symptoms in 80% of primary and 62% of recurrent lesions. Cytomegalovirus was detected in only two of 23 carotid specimens (9%). CONCLUSION: The association between ischemic cerebrovascular symptoms and iNOS and nitrotyrosine immunoreactivity in complex primary and recurrent carotid plaque and the infrequent occurrence of cytomegalovirus in primary carotid lesions suggests that ongoing free radical oxidative damage rather than viral infection may contribute to plaque instability in patients with complex and fibrous carotid plaques.

Isolation, characterization, and immunological detection of neutrophil-activating peptide 2: a proteolytic degradation product of platelet basic protein.

Neutrophil-activating peptide 2 (NAP-2), corresponding to platelet basic protein fragment 25-94, was prepared by chymotryptic digestion of its precursors, low affinity platelet factor 4 or beta-thromboglobulin, followed by purification by high performance liquid chromatography. NAP-2 (0.1-1.5 microns) caused the release of human granulocyte elastase from cytochalasin B-treated neutrophils in a dose-dependent manner. In the same system, beta-thromboglobulin, human platelet factor 4, S-pyridylethyl NAP-2, and platelet basic protein C-terminal fragment (77-94) were inactive, whereas platelet basic protein fragment 22-89 had low, but significant, activity. Sensitive immunological identification of NAP-2 based on nonequilibrium isoelectric focusing and immunoblotting is described.