After escaping from the hyperacute rejection (HAR), the xenograft has to be faced the challenge of acute vascular, acute cellular and even chronic rejection. Endothelial cells have been confirmed as a kind of antigen processing cell (APC) in allo-rejection. The porcine aortic endothelial cell (PAEC) expressed SLA-II and B7 which are the characteristics of professional APC. PAEC also has plenty of alpha-Gal residues, whether the antigen play any role in the post-HAR is still unknown. Human and porcine peripheral blood lymphocyte (PBLC) were isolated and divided into two parts, one for the effectors and the another were incubated with mitomycin C (MMC) as stimulators. The two kinds of PBLC were mixed-cultured within five days. Cultured PAEC from NJZ Pig was incubated with MMC and divided into two: One digested with alpha-galactosidase. The two kinds of PAEC were taken as stimulators to mixed-culture with human PBLC for five days. All the proliferation was detected with 3H-TdR intermingled in the system. The results showed that allo-MLR was ber than xeno-MLR in the cases. The proliferation was much ber when PAEC was used as the stimulator than that of porcine PBLC. However, the response was remarkably decreased after the digestion of alpha-Gal with alpha-galactosidase. The conclusion was that the low response of porcine-to-human MLR in vitro might be related to the predominant indirect pathway of antigen recognition in this system. While PAEC was used as the stimulator the proliferation in MLR was ber which might be concerned that PAEC itself was an APC as well as xeno-antigen sources, thus the direct pathway was predominant and worked more efficiently. The alpha-Gal might induce T cell proliferation through the linkage with the biological big molecules working as a complete antigen. The other post-HAR antigen might also exist in PAEC such as SLA-II, etc.
ObjectiveTo investigate the inhibitory effect of T lymphocyte transplantation of EphrinAl-Caspase-3 on the growth of breast cancer.MethodsSix-week-old BALB/c nude mice were used to inoculate breast cancer cells to construct a nude mouse model of breast cancer. They were randomly divided into 3 groups according to random number table: PBS group received intratumoral injection of 10 μL PBS, and negative control group received intratumoral injection of 1×106 T lymphocytes uninfected with adenovirus, 1×106 EphrinAl-Caspase3-T lymphocytes were injected intratumorally into the infected group, and the tumors size (0, 3, 6, 9, 12 and 15 d) were measured with vernier calipers every 3 days until end of experiment. The content of EphrinAl-Caspase-3 in the tissues of the nude mice was measured. The presence of T lymphocytes expressing green fluorescent protein and the ratio of Caspase-3-positive and Ki-67-positive cell were observed by pathological examination.ResultsOn the day 0 and day 3, there were no significant difference in tumor volume between the 3 groups (P>0.05). On the 6th day and later, the difference between the infected group and the PBS group/negative control group were statistically significant (P<0.05), but there were no significant difference in tumor volume between the PBS group and negative control group at each time point (P>0.05). The presence of scattered green fluorescent protein-labeled EphrinAl-Caspase-3-T lymphocytes was observed in the tumor tissues of the infected group, while the presence of green fluorescent protein were not detected in the PBS group and the negative control group. In the infected cells, ratio of Caspase-3-positive cell was up-regulated and ratio of Ki-67-positive cell was down-regulated. The expression of EphrinAl-Caspase-3 could be detected on the 3rd day in the infected group, and at the peak on the 6-day, then the amount of secretion gradually decreased. The expression of EphrinAl-Caspase-3 were not detected in the PBS group and the negative control group at each time point.ConclusionEphrinAl-Caspase-3 can significantly inhibit the growth of breast cancer cells and promote apoptosis.
Objective To investigate the correlation between monocyte-lymphocyte ratio (MLR) and intensive care unit (ICU) results in ICU hospitalized patients. Methods Clinical data were extracted from Medical Information Mart for Intensive Care Ⅲ database, which contained health data of more than 50000 patients. The main result was 30-day mortality, and the secondary result was 90-day mortality. The Cox proportional hazards model was used to reveal the association between MLR and ICU results. Multivariable analyses were used to control for confounders. Results A total of 7295 ICU patients were included. For the 30-day mortality, the hazard ratio (HR) and 95% confidence interval (CI) of the second (0.23≤MLR<0.47) and the third (MLR≥0.47) groups were 1.28 (1.01, 1.61) and 2.70 (2.20, 3.31), respectively, compared to the first group (MLR<0.23). The HR and 95%CI of the third group were still significant after being adjusted by the two different models [2.26 (1.84, 2.77), adjusted by model 1; 2.05 (1.67, 2.52), adjusted by model 2]. A similar trend was observed in the 90-day mortality. Patients with a history of coronary and stroke of the third group had a significant higher 30-day mortality risk [HR and 95%CI were 3.28 (1.99, 5.40) and 3.20 (1.56, 6.56), respectively]. Conclusion MLR is a promising clinical biomarker, which has certain predictive value for the 30-day and 90-day mortality of patients in ICU.
Human lymphocyte function-associated antigen 3 (hLFA3) has been identified as an important T cell accessory molecule. Rhesus monkeys (Macaca mulatta) have been widely used as animal models for human immune disorders. Due to the species-specificity of immune system, it is necessary to study M. mulatta LFA3 (mmLFA3). In this study, the gene encoding mmLFA3 CD2-binding domain (mmLFA3Sh) was amplified by polymerase chain reaction (PCR) and genetically fused to human IgG1 Fc fragment in pPIC9K to construct the expression plasmid pPIC9K-mmLFA3Sh-Ig. Approximately 3-4 mg mmLFA3Sh-Ig protein was recovered from 1 L of inductive media, and mmLFA3Sh-Ig produced by the P. pastoris can bind to the CD2 positive cells, and suppress the monkey and human lymphocytes proliferation induced by Con A and alloantigen in a dose-dependent manner. These results suggested that mmLFA3Sh-Ig might be used as a novel tool for pathogenesis and experimental immunotherapy of Rhesus monkey immune disorders.
ObjectiveTo investigate the method for generating anchor chemric T lymphocytes that can target tumor associated glycoprotein-72 (TAG72) antigen and analyze their repressive effects on proliferation of TAG72 positive hepatocarcinoma cells. MethodsFirstly, peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated. And then, CD8+ T cells were isolated from PBMCs via magnetic activated cell sorting (MACS). These lymphocytes were transfected with recombinant vector, anti-TAG72-scFv-CD28-pcDNA3, through Lipofectamine2000 to gernerate anchor chimeric TAG72-specific CD8+ T cells. SMMC7721 (TAG72 positive) hepatocarcinoma cells were co-cultured with chimeric T lymphocytes and their cell cycles were analyzed by flow cytometry (FCM). ResultsAnchor chmeric T lymphcytes targetting TAG72 recognized TAG72 positive SMM7721 cells and repressive effects on their proliferation were observed by flow cytometry. ConclusionAnchor chmeric T lymphcytes targetting TAG72 on tumor surface can specifically recognize TAG72 positive hepatocarcinoma cells and may exert repressive effect on their proliferation.
Objective To study the immunological tolerance induced by blocking the second signal of T cell with extrinsic cytotoxic T lymphocyteassociated antigen 4 immuno globlin(CTLA4-Ig). Methods Fifty-four BALB/C mice, inbred strains, were employed as recipients of bone allografts, using a model of heterotopic muscle pouch. The 54 mice were divided into 3 groups and18 for each group. The first group, in which the donor was C57BL/6 with intraperitoneal injection ofL6(as a control), was named AL group. The second group,also C57BL/6 with injection CTLA4-Ig, was named AC group. The third group,homologous BALB/C with injection of PBS buffer solution, was named AB group.The serum antibody, lymphocyte proliferation of the second stimulation by splenic cell and bone supernatant of donor, the analysis of lymphocyte subsets, a regraft experiment and histology were determined 2, 4 and 6 weeks after transplantation. The second transplantation was to regraft C57BL/6(BC group) and C3H(BHgroup) mice respectively after first 12 mice being transplantated with C57BL/6 and injected with CTLA4-Ig as to detect donor-specificity of immunological tolerance. Results Compared with AB group, AL group created more intensive immune rejection: CD4 T cell subsets(Plt;0.05), the serum antibody(Plt;0.05) and lymphocyteproliferation of the second stimulation by splenic cell and bone supernatant ofdonor (Plt;0.01 and 0.05) were significantly increased. However, the results of AC group showed that CTLA4-Ig significantly inhibited the immune rejection: CD4T cell subsets(Pgt;0.05), the serum antibody (Pgt;0.05), and lymphocyte proliferation of the second stimulation(Pgt;0.05) were similar to those of AB group. Histological observation of AC group showed that lymphocyte infiltration disappeared,cartilage and new bone formed, and bone marrow cavities emerged. A regraft experiment showed that CD4 T cell subsets (Plt;0.05) and lymphocyte proliferation of the second stimulation by splenic cell and bone supernatant of donor(Plt;0.05), BC group was significantly lower than those of BH group. So theimmunological tolerance induced by CTLA4-Ig was of donor-specificity. Conclusion The immunological tolerance induced by CTLA4-Ig was prolonged for 6 weeks. This study provides a brand-new path for bone transplantation, which can be helpful to other organ transplantation.
ObjectiveTo evaluate prognostic value of change of immune status in locally advanced gastric cancer (LAGC) patients. Methods We retrospective collected 210 LAGC patients who underwent treatment in our department from January 2013 to December 2018, then we collected lymphocyte-to-monocyte ratio (LMR) and cLMR (change of lymphocyte-to-monocyte ratio, cLMR) before operation and after three cycles of adjuvant chemotherapy. We had developed a new immune state change score (ICS) based on preoperative LMR (pLMR) and cLMR, and explored its prognostic value. The definition of ICS in this study was: ICS=1, pLMR≤4.53 and cLMR≤1; ICS=2, pLMR≤4.53 and cLMR>1, or pLMR>4.53 and cLMR≤1; ICS=3, pLMR>4.53 and cLMR>1. Results The results of multivariate Cox proportional hazard regression model showed that ICS was an influencing factor for overall survival [ICS=2, RR=0.397, 95%CI (0.260, 0.608), P<0.001; ICS=3, RR=0.080, 95%CI (0.040, 0.162), P<0.001), patients with ICS scores of 2 and 3 had better overall survival. In addition, the prognostic accuracy of ICS was superior to pLMR and Clmr, and the C-index of ICS [0.806, 95%CI (0.746, 0.865)] was higher than that of pLMR [0.717, 95%CI (0.635, 0.799), P=0.003)] and cLMR [0.723, 95%CI (0.641, 0.806), P=0.005)]. Based on this, a Nomogram model included ICS, CEA, and pTNM staging was constructed to predict the 3-year and 5-year survival rates of patients. The calibration curve and C-index [0.821, 95%CI (0.783, 0.859)] showed high discrimination and accuracy of Nomogram, and decision curve analysis confirmed that the model had good clinical application value. Conclusions The dynamic changes in the patient’s immune status before and after adjuvant therapy are related to the overall survival of LAGC patients. As an evaluating system which combined the cLMR and pLMR, ICS can better predict the prognosis of LAGC patients.
Objective To explore the effect of CD3AK cells on T lymphocyte subsets and its functions of patients with primary liver carcinoma (PLC). MethodsFiftyeight patients with PLC were divided into two groups, CD3AK group (n=37), control group (n=21). Eleven blood donors were taken as normal control. All patients from the treatment group were given 2×109 CD3AK cells once a day for 5 days on the 1st day after operation, having received IL2 at a dose of 100 units per 24 hour by intravenous injection starting 30 minutes before administration of CD3AK cells. T lymphocyte subsets, IL2R, NK activity, LAK activity and proliferative activity were determined. ResultsThe CD4/CD8 ratio, expression of IL2R, proliferative activity, NK activity and LAK activity of T lymphocytes from the treatment group were significantly higher than those of control respectively.Conclusion The adoptive transfer of CD3AK cells could improve the disorder of T lymphocyte subsets and its functions and provide the patients with PLC with fresh abundant effectors against tumor.
Objective To investigate the prediction of baseline neutrophil-lymphocyte ratio (NLR) on the prognosis of unresectable hepatocellular carcinoma (uHCC) treated with transarterial chemoembolization (TACE) + lenvatinib + camrelizumab. Method The clinical data of 58 patients treated with TACE + lenvatinib + camrelizumab in the Department of Liver Surgery of West China Hospital of Sichuan University from June 2020 to May 2021 were analyzed retrospectively. Results Among the 58 cases included, 7 cases were complete response (CR), 37 cases were partial response (PR), 11 cases were stable disease (SD), and 3 cases were progressive disease (PD). All cases had different degrees of adverse events, including 58 cases of grade 1, 36 cases of grade 2, 35 cases of grade 3, and 1 case of grade 4. The overall response rate (ORR) and disease control rate (DCR) based on modified Response Evaluation Criteria in Solid Tumors (mRECIST) were 75.9% (44/58) and 94.8% (55/58), respectively. The hepatectomy rate was 31.0% (18/58) and the conversion success rate was 37.9% (22/58). Multivariate logistic regression analysis showed that NLR was an independent risk factor for ORR (OR=0.093, P=0.008). All cases were followed up for 16–60 weeks, with a median follow-up of 34 weeks. Overall survival situation (χ2=4.163, P=0.041) and progression free survival situation (χ2=10.626, P=0.001) in the low NLR group were better than those of the high NLR group. Conclusion NLR has clinical significance in predicting the prognosis of uHCC cases underwent TACE + lenvatinib + camrelizumab, which is worthy of further study.
Objective To investigate the effects of down-regulating of Rfng gene ( 1, 3-Nacetylglucosaminyltransferases) in lung CD4 + T cells of asthmatic rat model by small interfering RNA ( siRNA) and explore the role of Rfng in pathogenesis of asthma. Methods An asthmatic rat model was established by OVA sensitization and challenge. Total T cells were isolated from lung tissue of asthmatic rats, and CD4 + T lymphocytes were purified using magnetic beads. CD4 + T lymphocytes were transfected by siRNA targeting Rfng gene. The mRNA and protein expressions of Rfng were detected by quantitative PCR and Western blot. Quantitative PCR was performed to determine the mRNA levels of Th1 /Th2 cytokines and related genes including IL-12, IFN-γ, IL-4, IL-5, T-bet, and GATA3. ELISA was performed to determine the concentrations of IL-12, IFN-γ, IL-4, and IL-5 in supernatant. Results The mRNA and protein expression of Rfng in RNAi group decreased significantly. IL-12, IFN-γ, T-bet increased and while IL-4, IL-5, and GATA3 decreased significantly. The concentrations of IL-12 and IFN-γ in the supernatant increased significantly, while IL-4 and IL-5 decreased significantly. Conclusions Down regulation of Rfng affects T cell differentiation. It is presumed that Fringe contribute to the pathogenesis of asthma.