BACKGROUND: Peripheral helper T (T PH ) cells are uniquely positioned within pathologically inflamed non-lymphoid tissues to stimulate B-cell responses and antibody production. However, the phenotype, function, and clinical relevance of T PH cells in hepatocellular carcinoma (HCC) are currently unknown. METHODS: Blood, tumor, and peritumoral liver tissue samples from 39 HCC patients (Sep 2016-Aug 2017) and 101 HCC patients (Sep 2011-Dec 2012) at the Third Affiliated Hospital of Sun Yat-sen University were used. Flow cytometry was used to quantify the expression, phenotype, and function of T PH cells. Log-rank tests were performed to evaluate disease-free survival and overall survival in samples from 39 patients and 101 patients with HCC. T PH cells, CD19 + B cells, and T follicular helper (T FH ) cells were cultured separately in vitro or isolated from C57/B6L mice in vivo for functional assays. RESULTS: T PH cells highly infiltrated tumor tissues, which was correlated with tumor size, early recurrence, and shorter survival time. The tumor-infiltrated T PH cells showed a unique ICOS hi CXCL13 + IL-21 - MAF + BCL-6 - phenotype and triggered naïve B-cell differentiation into regulatory B cells. Triggering programmed cell death protein 1 (PD-1) induced the production of C-X-C motif chemokine ligand 13 (CXCL13) by T PH cells, which then suppressed tumor-specific immunity and promoted disease progression. CONCLUSION Our study reveals a novel regulatory mechanism of T PH cell-regulatory B-cell-mediated immunosuppression and provides an important perspective for determining the balance between the differentiation of protumorigenic T PH cells and that of antitumorigenic T FH cells in the HCC microenvironment.
Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/metabolism* ; Humans ; T-Lymphocytes, Helper-Inducer/metabolism* ; Animals ; Mice ; Male ; Female ; Mice, Inbred C57BL ; Middle Aged ; B-Lymphocytes, Regulatory/metabolism* ; Flow Cytometry ; Interleukin-21 ; Aged ; Chemokine CXCL13/metabolism*

Objective To explore the effect of estrogen deficiency on bone remodeling characteristics in an ovariectomized mouse model.Methods Ten 12-week-old female C57BL/6J mice were randomly divided into the sham operation group and the ovariectomy(OVX)group,with 5 mice in each group.The uteri of mice were isolated at 16 weeks postoperatively.The morphology was observed,and uterine weights were counted.CT scans were performed to analyze the distal femur of the mice,and anti-tartrate acid phosphatase(TRAP)staining was performed to assess the number of osteoclasts in paraffin sections of femoral tissue.Bone marrow-derived mesenchymal stem cells(BM-MSCs)and bone marrow-derived macrophages(BMMs)were sorted from femoral bone marrow of mice using CD11b magnetic beads.Alkaline phosphatase(ALP)staining was performed to assess the osteogenic differentiation potential of BM-MSCs.Reverse transcription-quantitative PCR(RT-qPCR)was performed to detect the expression levels of genes related to osteogenic differentiation of BM-MSCs(Runx2,Sp7,and Spp1)and osteoclast differentiation of BMMs(Ctsk,Acp5,and Cd40).Results Compared with the sham group,the uterus of the OVX group showed significant atrophy and weight loss,and bone mineral density(BMD),bone volume fraction(BV/TV),bone surface area tissue volume ratio(BS/TV),number of trabeculae(Tb.N),trabecular thickness(Tb.Th),cortical bone mineral density(Ct.BMD)and cortical bone thickness(Ct.Th)were significantly reduced,and bone trabecular separation(Tb.Sp)was significantly increased(P＜0.01).The mRNA expression levels of Runx2,Sp7 and Spp1 were reduced in BM-MSCs of the OVX group compared with the sham group(P＜0.01).The osteogenic differentiation was also diminished after induction.The mRNA expression levels of Ctsk,Acp5 and Cd40 were elevated in the BMMs of the OVX group,and the number of osteoclasts in bone marrow of the distal femur was greater in the OVX group than that in the sham group(P＜0.01).Conclusion OVX-induced estrogen deficiency can enhance osteoclast differentiation and attenuate osteogenic differentiation capacity,ultimately causing dysregulation of bone homeostasis and resulting in osteoporosis.

Objective To explore the effect of estrogen deficiency on bone remodeling characteristics in an ovariectomized mouse model.Methods Ten 12-week-old female C57BL/6J mice were randomly divided into the sham operation group and the ovariectomy(OVX)group,with 5 mice in each group.The uteri of mice were isolated at 16 weeks postoperatively.The morphology was observed,and uterine weights were counted.CT scans were performed to analyze the distal femur of the mice,and anti-tartrate acid phosphatase(TRAP)staining was performed to assess the number of osteoclasts in paraffin sections of femoral tissue.Bone marrow-derived mesenchymal stem cells(BM-MSCs)and bone marrow-derived macrophages(BMMs)were sorted from femoral bone marrow of mice using CD11b magnetic beads.Alkaline phosphatase(ALP)staining was performed to assess the osteogenic differentiation potential of BM-MSCs.Reverse transcription-quantitative PCR(RT-qPCR)was performed to detect the expression levels of genes related to osteogenic differentiation of BM-MSCs(Runx2,Sp7,and Spp1)and osteoclast differentiation of BMMs(Ctsk,Acp5,and Cd40).Results Compared with the sham group,the uterus of the OVX group showed significant atrophy and weight loss,and bone mineral density(BMD),bone volume fraction(BV/TV),bone surface area tissue volume ratio(BS/TV),number of trabeculae(Tb.N),trabecular thickness(Tb.Th),cortical bone mineral density(Ct.BMD)and cortical bone thickness(Ct.Th)were significantly reduced,and bone trabecular separation(Tb.Sp)was significantly increased(P＜0.01).The mRNA expression levels of Runx2,Sp7 and Spp1 were reduced in BM-MSCs of the OVX group compared with the sham group(P＜0.01).The osteogenic differentiation was also diminished after induction.The mRNA expression levels of Ctsk,Acp5 and Cd40 were elevated in the BMMs of the OVX group,and the number of osteoclasts in bone marrow of the distal femur was greater in the OVX group than that in the sham group(P＜0.01).Conclusion OVX-induced estrogen deficiency can enhance osteoclast differentiation and attenuate osteogenic differentiation capacity,ultimately causing dysregulation of bone homeostasis and resulting in osteoporosis.

The treatment of advanced hepatocellular carcinoma (HCC) is limited and the prognosis is poor, which seriously endangers the public health. Results of clinical trials have confirmed the validity of atelizumab plus bevacizumab in patients with advanced HCC. The authors introduce the clinical experience of a patient with stage Ⅲa HCC undergoing local therapy of hepatic artery chemoembolization, and combined with atelizumab plus bevacizumab. The results show that patient with successfully transformational therapy, and receiving surgical resection with a good clinical effect.

Autophagy is essential for the maintenance of cellular homeostasis and its dysfunction has been linked to various diseases. Autophagy is a membrane driven process and tightly regulated by membrane-associated proteins. Here, we summarized membrane lipid composition, and membrane-associated proteins relevant to autophagy from a spatiotemporal perspective. In particular, we focused on three important membrane remodeling processes in autophagy, lipid transfer for phagophore elongation, membrane scission for phagophore closure, and autophagosome-lysosome membrane fusion. We discussed the significance of the discoveries in this field and possible avenues to follow for future studies. Finally, we summarized the membrane-associated biochemical techniques and assays used to study membrane properties, with a discussion of their applications in autophagy.

Objective To establish a non-venous bypass orthotopic liver transplantation model in Bama miniature pigs with high repeatability and stability. Methods Twelve Bama miniature pigs were randomly divided into the donor group (n=6) and recipient group (n=6). Pigs underwent non-venous bypass orthotopic liver transplantation. The time of anhepatic phase during operation was shortened, blood pressure during anhepatic phase was stably maintained, and management of anesthesia and body fluid during operation were strengthened. The operation time, anhepatic phase and survival status of the recipients were observed and recorded. The intraoperative heart rate, mean arterial pressure (MAP) and changes in arterial blood gas analysis were monitored. The perioperative liver function was evaluated. Results Among 6 Bama miniature pigs, 1 died from transplantation failure intraoperatively. The operation time of the remaining 5 pigs was (247±27) min and the time of anhepatic phase was (46±4) min. Three animals survived for more than 2 weeks. Compared with the preanhepatic phase, the heart rate of the animals was significantly faster, MAP was considerably reduced to (46±6) mmHg, blood pH value, base excess (BE) and HCO₃^- level were all significantly decreased and serum level of K⁺ was significantly elevated during the anhepatic phase (all P < 0.05). In the neohepatic phase, MAP of Bama miniature pigs was significantly increased, heart rate was dramatically slower.Blood pH value, BE, HCO₃^- level were significantly increased and serum level of K⁺ was significantly declined (all P < 0.05). During abdominal closure, MAP, blood gas indexes and serum level of K⁺ were almost recovered to those in the preanhepatic phase. Compared with preoperative levels, the levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), lactate dehydrogenase(LDH)and alkaline phosphatase(ALP)were significantly increased after operation (all P < 0.05), the change in AST was the most obvious, and it gradually decreased at postoperative 2 d. The level of γ-gutamyl transferase(GGT) did not significantly elevated. The level of total bilirubin (TB) was evidently elevated at postoperative 5 d. Compared with the preoperative levels, the levels of total protein (TP) and albumin (ALB) were significantly decreased after operation (both P < 0.05), and began to gradually increase at postoperative 1 d. Conclusions The non-venous bypass orthotopic liver transplantation model of Bama miniature pig is convenient, with highly reproducible and survival rate, which can be utilized as a standardized liver transplantation model.

Objective: To explore the influence of three-level collaboration network of pediatric burns in Anhui province on treatment effects of burn children. Methods: The data of medical records of pediatric burn children transferred from Lu′an People′s Hospital and Fuyang People′s Hospital to the First Affiliated Hospital of Anhui Medical University from January 2014 to December 2015 and January 2016 to September 2017 (before and after establishing three-level collaboration network of pediatric burns treatment) were analyzed: percentage of transferred burn children to hospitalized burn children in corresponding period, gender, age, burn degree, treatment method, treatment result, occurrence and treatment result of shock, and operative and non-operative treatment time and cost. Rehabilitation result of burn children transferred back to local hospitals in 2016 and 2017. Data were processed with t test, chi-square test, Mann-Whitney U test, and Fisher′s exact test. Results: (1) Percentage of burn children transferred from January 2014 to December 2015 was 34.3% (291/848) of the total number of hospitalized burn children in the same period of time, which was close to 30.4% (210/691) of burn children transferred from January 2016 to September 2017 (χ²=2.672, P>0.05). (2) Gender, age, burn degree, and treatment method of burn children transferred from the two periods of time were close (χ²=3.382, Z=-1.917, -1.911, χ²=3.133, P>0.05). (3) Cure rates of children with mild, moderate, and severe burns transferred from January 2016 to September 2017 were significantly higher than those of burn children transferred from January 2014 to December 2015 (χ²=11.777, 6.948, 4.310, P<0.05). Cure rates of children with extremely severe burns transferred from the two periods of time were close (χ²=1.181, P>0.05). (4) Children with mild and moderate burns transferred from the two periods of time were with no shock. The incidence of shock of children with severe burns transferred from January 2014 to December 2015 was 6.0% (4/67), and 3 children among them were cured. The incidence of shock of children with severe burns transferred from January 2016 to September 2017 was 3.9% (2/51), and both children were cured. The incidences and cures of shock of children with severe burns transferred from the two periods of time were close (χ²=0.006, P>0.05). Incidence of shock of children with extremely severe burns transferred from January 2014 to December 2015 was 57.1% (32/56), significantly higher than that of burn children transferred from January 2016 to September 2017 [34.5% (10/29), χ²=3.925, P<0.05]. Shock of 25 children with extremely severe burns transferred from January 2014 to December 2015 were cured, and shock of 9 children with extremely severe burns transferred from January 2016 to September 2017 were cured. The cures of shock of children with extremely severe burns transferred from the two periods of time were close ( χ²=0.139, P>0.05). (5) Time of operative treatment of children with moderate, severe, and extremely severe burns transferred from January 2014 to December 2015 was obviously longer than that of burn children transferred from January 2016 to September 2017 (t=2.335, 2.065, 2.310, P<0.05). Time of operative treatment of children with mild burns transferred from the two periods of time was close (Z=-0.417, P>0.05). Costs of operative treatment of children with moderate and severe burns transferred from January 2014 to December 2015 were significantly more than those of burn children transferred from January 2016 to September 2017 (Z=-3.324, t=2.167, P<0.05). Costs of operative treatment of children with mild and extremely severe burns transferred from the two periods of time were close (t=0.627, 0.808, P>0.05). (6)Time of non-operative treatment of children with mild, moderate, and severe burns transferred from January 2014 to December 2015 was obviously longer than that of burn children transferred from January 2016 to September 2017 (t=2.335, Z=-2.095, t=2.152, P<0.05). Time of non-operative treatment of children with extremely severe burns transferred from the two periods of time was close (t=0.450, P>0.05). Costs of non-operative treatment of children with moderate and severe burns transferred from January 2014 to December 2015 were obviously higher than those of burn children transferred from January 2016 to September 2017 (Z=-2.164, t=2.040, P<0.05). Costs of non-operative treatment of children with mild and extremely severe burns transferred from the two periods of time were close (t=0.146, 1.235, P>0.05). (7) Sixty-seven burn children transferred from January 2016 to September 2017 were transferred back to local hospitals for rehabilitation under the guidance of experts of the First Affiliated Hospital of Anhui Medical University, with 25 patients in 2016 and 42 patients in 2017. Effective rehabilitation rates of burn children transferred back to local hospitals for rehabilitation in 2016 and 2017 were both 100%. Conclusions The three-level collaboration network of pediatric burns treatment in Anhui province can effectively increase cure rate of children with mild, moderate, and severe burns, reduce incidence of shock of children with extremely severe burns, shorten time of operative treatment of burn children with moderate, severe, and extremely severe burns, and time of non-operative treatment of children with mild, moderate, and severe burns, reduce treatment costs of children with moderate and severe burns, and improve rehabilitation effectiveness of children transferred from Lu′an People′s Hospital and Fuyang People′s Hospital to the the First Affiliated Hospital of Anhui Medical University.

Objective:To investigate the effect of early enteral nutrition combined with Bacillus in the treatment of patients with digestive tract perforation.Methods:59 patients with emergency digestive tract perforation were randomly divided into experimental group (EN plus Bacillus,n =31) and control group (TPN,n =28).The patients in the experimental group were given enteral nutrition plus Bacillus after first days (＜ 24 h),and the control group was treated with parenteral nutrition support on the first day after surgery.The average hospitalization time,length of ICU stay,incidence of complications,and serum C-reactive protein in the two groups were analyzed.Results:The average length of hospitalization time,ICU stay,recovery of bowel function and eating time were significantly shorter in the experimental group than in the control group(P ＜ 0.05).There was no significant difference in nutrition index between the two groups during the perioperative period (P ＞ 0.05).The C-reactive protein in the experimental group was significantly lower than that in the control group fourth days after operation (P ＜ 0.05).The incidence of postoperative wound infection in the experimental group was significantly lower than that of the control group (P ＜ 0.05).There was no significant difference in the incidence of other complications.Conclusion:Early administration of enteral nutrition combined with Bacillus can significantly shorten the intestinal function recovery time,decreasethe average hospitalization days,reduce the postoperative wound infection rate and thus improve the outcome of patients with digestive tract perforation.

Objective To evaluate the value of intermittent splenic artery occlusion in the treatment of iatrogenic splenic injury and the cause of ischemia reperfusion injury.Methods Experimental animals using dogs were divided into experimental group and control group according to whether the splenic artery was blocked.The animal model of iatrogenic splenic injury was established by general anesthesia.Experimental group was treated with intermittent splenic artery blockage combined with gelatin sponge oppression of the wound to stop bleeding.The first time of splenic artery occlusion was 15 min and continued for 10 min and for another 5 rmin if still bleeding.Then hemostatic effect after removing blocking and oppression was observed.Control group was only treated with gelatin sponge compression to stop bleeding.Blood samples and tissue samples were collected before blocking and after reperfusion for 2 hours in experimental group,and before compression and relieving the compression for 2 hours in control group.IL-1 (interleukin),SOD (superoxide disproportionation enzyme),MPO (myeloperoxidase),MDA (malondialdehyde) and caspase-3 (aspartame acid cysteine protease) were detected.Results The control group still had bleeding,while the experimental group had successfully stopped bleeding.Serum markers before and after occlusion in the experinental group were IL-1 (124.4 ± 106.8 vs.121.2 ± 105.1),SOD (4.7 ± 2.7 vs.5.2 ± 1.8) and MDA (8.8 ±6.5 vs10.8 ±7.5).There were no significant differences on serum markers in two groups after occlusion/oppression including IL-1 (121.2 ± 105.1 vs.162.8 ± 73.8),SOD (5.2 ± 1.8 vs.4.7 ± 2.8) and MDA (10.8 ±7.5 vs.9.6 ±6.6) (P＞0.05).Histological indicators before and after occlusion in the experimental group included MPO (0.62 ±0.23 vs.0.68 ±0.21) and Caspase-3 (0.90 ±0.29 vs.0.86 ± 0.26),and there was no statistical difference on MPO (0.68 ±0.21 vs.0.86 ±0.23 after two sets of experiments) and Caspase-3 level (0.86 ± 0.26 vs.1.21 ± 0.18) (P ＞ 0.05) in two groups after occlusion/ oppression.Conclusion The therapeutic effect of intermittent splenic artery occlusion combined with gelatin sponge compression hemostasis is safe and effective and without obvious ischemia reperfusion injury.

Objective To explore the features of peripheral blood immune cells in long-term survival recipients after liver transplantation.Methods The expression of T subsets (Th1,Th2,Th17,Th22,Tregs),NK cells,NKt cells,Bregs,MDSC in long-term survival recipients (postoperative follow-up time ≥5 years,30 cases),short-term survival recipients(postoperative follow-up time ≤1 year,15 cases) and healthy control (15 cases) were determined by flowcytometry.Results Th17 cells were significantly higher in the long-term group compared with short-term group and healthy control group(P ＜0.01).Tregs in long-term group compared with short-term group were significantly higher (P ＜ 0.01),but the difference was not statistically significant compared with healthy control group (P ＞ 0.05).NK cells were significantly higher in long-term group compared with short-term group and healthy control group (P ＜ 0.01).MDSC were significantly higher in long-term group compared with short-term group and healthy control group (P ＜0.01).Conclusions Th17,Tregs,NK cells and MDSC were significantly higher in long-term survival of liver recipients,which may be related to immune tolerance.