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Objective:To explored the effect of 78c in treating collagen-induced arthritis (CIA) mice and to investigate its mechanism of effects.Methods:CIA mice model and CD38 +NK cells were treated with 78c. Cytokine concentrations and lymphocyte subtypes were measured in the mice peripheral blood and culture medium using flow cytometry. Mikenyi cell isolation kit was used to isolate CD4 + T cells and NK cells from peripheral blood mononuclear cells of healthy volunteers. CD38 + NK cells were enriched using the Miltenyi CD38 microbeads from the extracted NK cells. CD38 + NK cells with 78c pretreatment or not were cocultured with CD4 +T cells in transwells. The least significant difference (LSD) method was used for comparison between the two groups, and one-way analysis of variance was used for multi-group significance. Pearson correlation analysis was used for correlation analysis. Results:78c treatment significantly suppressed joint inflammation, inhibited the toe thickness of CIA mice, and reduced the number of while cell, neutrophils, platelets, and concentrations of IFN-γ, IL-6 and TNF-α ( t=6.10, P<0.001; t=4.00, P=0.002; t=3.09, P=0.012; t=2.31, P=0.043; t=3.58, P=0.005; t=2.68, P=0.002) in the CIA mice. The proportion of CD38 +NK cells decreased from (3.9±0.9)% to (2.4±0.3)% ( t=2.49, P=0.032), the proportion of regulatory T cell (Treg) increased from (0.81±0.33)% to (1.41±0.26)% ( t=2.74, P=0.021), and the concentration of IL-10 also increased from (99±37) pg/ml to (199±9) pg/ml( t=2.76 , P=0.020). The proportion of Treg in CD4 +T cells cocultured with 78c-pretreated CD38 +NK cells increased from (0.52±0.04)% to (0.69±0.08)% ( t=3.33, P=0.029) , the T helper cells (Th)17/Treg ratio decreased from (4.44±0.26) to (2.59±0.64) ( t=4.76 , P=0.009), and the Th1/Th2 ratio decreased from (14.8±1.6) to (8.1±1.3)( t=5.70 , P=0.005). Conclusion:78c can reduce the proportion of CD38 +NK cells, thereby reducing the inhibition of CD38 +NK cells on CD4 +T cell differentiation into Treg cells, leading to the restoration of immune balance. The results of this study suggest that 78c is a potential therapeutic agent for rheumatoid arthritis.
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Abstract Objective The present study was conducted to estimate histologically the proportion of avascularity of fracture ends in case of nonunion of long bones. Methods A total of 15 cases of established quiescent nonunion were operated according to the standard protocol and the fracture ends were evaluated histologically. The biopsied tissue was briefly fixed with formalin, embedded with paraffin (FFPE), and 5-micron sections were stained with hematoxylin and eosin according to standard protocols. Immunohistochemistry with anti-CD31 antibody (JC70A clone, DBS) was performed manually using standard protocols. Results All cases of quiescent nonunion were included; radiologically, 2 cases were oligotrophic, and 13 cases were of atrophic nonunion. A total of 20% of the patients were females, 40% were in the age group between 31and 40 years old, and, radiologically, all cases were of atrophic nonunion. All cases showed positivity for CD-31 on immunohistochemistry. The blood vessel density was category I in 13.33% of the cases and category II in 86.67% of the cases. Four cases presented with mild inflammation and two presented with moderate inflammation. The average vessel count was 10 per high power field in the age groups between 20 and 30, 31 and 40, and 41and 50 years old. The age group between 61 and 70 years old showed an average vessel count of 4 per high power field. The difference in the vessel counts of oligotrophic and atrophic nonunion was not significant. No correlation was observed in the density of vessel count and duration of nonunion Conclusion The nomenclature for the classification of nonunion into atrophic, oligotrophic, and hypertrophic needs revision. Our findings do not support that atrophic and oligotrophic nonunion are histologically different.
Resumo Objetivo O presente estudo estimou a proporção de avascularidade histológica das extremidades das fraturas em caso de pseudoartrose de ossos longos. Métodos No total, 15 casos de pseudoartrose quiescente estabelecida foram operados de acordo com o protocolo padrão e as extremidades da fratura foram avaliadas histologicamente. Em resumo, o tecido biopsiado foi fixado em formalina e embebido em parafina (FFPE); secções de 5 mícrons foram coradas com hematoxilina e eosina de acordo com os protocolos padrões. A imunohistoquímica com anticorpo anti-CD31 (clone JC70A, DBS) foi realizada manualmente segundo protocolos padrões. Resultados Todos os casos de pseudoartrose quiescente foram incluídos; 2 eram de pseudoartrose oligotrófica e 13 eram de pseudoartrose atrófica à radiologia. Destes, 20% eram de pacientes do sexo feminino, 40% de indivíduos entre 31 e 40 anos de idade e todos os casos eram de pseudoartrose atrófica à radiologia. Todos os casos eram positivos para CD-31 à imunohistoquímica. A densidade dos vasos sanguíneos era de categoria I em 13,33% dos casos e de categoria II em 86,67%. Quatro casos apresentavam inflamação branda e dois apresentavam inflamação moderada. O número médio de vasos era de 10 por campo de alta potência na faixa etária de 20 a 30, de 31 a 40 e de 41 a 50 anos. A faixa etária de 61 a 70 anos apresentava, em média, 4 vasos por campo de alta potência. A diferença nos números de vasos em pseudoarthroses oligotróficas e atróficas não foi significativa. Não houve correlação entre a densidade de vasos e a duração da pseudoartrose. Conclusão A nomenclatura de classificação da pseudoartrose em atrófica, oligotrófica e hipertrófica precisa ser revista. Nossos achados não indicam que a pseudoartrose atrófica e oligotrófica sejam histologicamente diferentes.
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Humans , Male , Female , Adult , Middle Aged , Aged , Pseudarthrosis , Cross-Sectional Studies , Platelet Endothelial Cell Adhesion Molecule-1 , Fractures, Bone/surgery , Fractures, UnunitedABSTRACT
Objective:To explore the markers predicting the efficacy of anti-programmed death receptor-1 (PD-1)/PD-1 ligand (PD-L1) immunotherapy for non-small cell lung cancer (NSCLC) and analyze their relationships with the tumor immune microenvironment.Methods:(1) Gene expression profile data and relevant clinical data of 55 NSCLC patients receiving anti-PD-1/PD-L1 monotherapy from two independent clinical cohorts were downloaded from the GEO database. Genes associated with progression-free survival (PFS) were screened using univariate Cox regression analysis. (2) Twenty-six pathological tissue specimens of NSCLC patients who received anti-PD-1/PD-L1 monotherapy in the Cancer Hospital of Chinese Academy of Medical Sciences (CICAMS) from April 2016 to August 2019 prior to the screening of genes were selected to verify the predictive value of the screened genes using immunohistochemistry. (3) The relationship between efficacy predictive markers and PD-L1 and infiltrating immune cells in the tumor immune microenvironment was analyzed using NSCLC gene expression profile downloaded from the TCGA database.Results:Univariate Cox regression analysis revealed that only T-Cell Surface Glycoprotein CD3 Gamma Chain (CD3G) was a risk predictive gene for PFS in NSCLC patients in both clinical cohorts of the GEO database (GSE93157: P = 0.049; GSE136961: P = 0.034). Further Kaplan-Meier survival curves showed that PFS was significantly prolonged in the high CD3G expression group ( P = 0.012). The results of survival analysis in the CICAMS clinical cohort also demonstrated that NSCLC patients with high CD3G expression had a better prognosis after receiving anti-PD-1/PD-L1 monotherapy ( P = 0.001) compared with those with low CD3G expression. Univariate and multivariate Cox regression analyses also showed that CD3G was an independent predictor of PFS (univariate: P = 0.002; multivariate: P = 0.001). The tumor immune microenvironment analysis showed that CD3G was positively correlated with PD-L1 expression (lung adenocarcinoma: r = 0.44, P < 0.001; lung squamous cell carcinoma: r = 0.37, P < 0.001) and the infiltration level of CD8 + T cells (lung adenocarcinoma: r = 0.13, P = 0.002; lung squamous cell carcinoma: r = 0.70, P < 0.001). CD3G was also positively correlated with the expression of CD8 + T cell chemokines CCL5, CXCL9, CXCL10 and CXCL11. Conclusion:Patients with NSCLC with high CD3G expression have greater clinical benefits after anti-PD-1/PD-L1 monotherapy compared with those with low CD3G expression. CD3G can be used as a potential marker to predict the efficacy of immunotherapy for NSCLC.
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Objective@#To investigate the characteristic and prognostic significance of leukemia stem cells associated antigens expressions including CD34, CD38, CD123, CD96 and TIM-3 in t (8;21) AML.@*Methods@#Bone marrow samples of 47 t (8;21) AML patients were collected at diagnosis from October 2015 to April 2018 in Peking University Peoples’ Hospital, then flow cytometry method was performed to detect the expression frequencies of CD34, CD38, CD123, CD96 and TIM-3 to analyze the relationship between leukemia stem cells associated antigens expressions and relapse.@*Results@#Of 47 t (8;21) AML patients tested, the median percentages of CD34+CD38-, CD34+ CD38-CD123+, CD34+CD38- CD96+ and CD34+ CD38- TIM-3+ cells among nucleated cells were 2.37%, 0.24%, 0.27% and 0.06%, respectively. All the frequencies of CD34+CD38-, CD34+CD38-CD123+, CD34+CD38-CD96+ and CD34+ CD38-TIM-3+ cells had no impact on the achievement of CR after the first course of induction. All higher frequencies of CD34+CD38-, CD34+CD38-CD123+, CD34+CD38-CD96+ cells were related to higher 2-year CIR rate. Whereas, the frequency of CD34+ CD38- TIM-3+ cells had no impact on CIR rate. Both high frequency of CD34+ CD38- cells and the high level of minimal residual diseases (patients with <3-log reduction in the RUNX1-RUNX1T1 transcript level after the second consolidation therapy) were independent poor prognostic factors of CIR[P=0.025, HR=6.9 (95%CI 1.3-37.4) ; P=0.031, HR=11.1 (95%CI 1.2-99.2) ].@*Conclusion@#Different leukemia stem cells associated antigens had distinct prognostic significance in t (8;21) AML. High frequencies of CD34+ CD38-, CD34+ CD38- CD123+ and CD34+CD38-CD96+ cells at diagnosis predicted relapse in patients with t (8;21) AML.
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Objective: To investigate the characteristic and prognostic significance of leukemia stem cells associated antigens expressions including CD34, CD38, CD123, CD96 and TIM-3 in t (8;21) AML. Methods: Bone marrow samples of 47 t (8;21) AML patients were collected at diagnosis from October 2015 to April 2018 in Peking University Peoples' Hospital, then flow cytometry method was performed to detect the expression frequencies of CD34, CD38, CD123, CD96 and TIM-3 to analyze the relationship between leukemia stem cells associated antigens expressions and relapse. Results: Of 47 t (8;21) AML patients tested, the median percentages of CD34(+)CD38(-), CD34(+) CD38(-)CD123(+), CD34(+)CD38(-) CD96(+) and CD34(+) CD38(-) TIM-3(+) cells among nucleated cells were 2.37%, 0.24%, 0.27% and 0.06%, respectively. All the frequencies of CD34(+)CD38(-), CD34(+)CD38(-)CD123(+), CD34(+)CD38(-)CD96(+) and CD34(+) CD38(-)TIM-3(+) cells had no impact on the achievement of CR after the first course of induction. All higher frequencies of CD34(+)CD38(-), CD34(+)CD38(-)CD123(+), CD34(+)CD38(-)CD96(+) cells were related to higher 2-year CIR rate. Whereas, the frequency of CD34(+) CD38(-) TIM-3(+) cells had no impact on CIR rate. Both high frequency of CD34(+) CD38(-) cells and the high level of minimal residual diseases (patients with <3-log reduction in the RUNX1-RUNX1T1 transcript level after the second consolidation therapy) were independent poor prognostic factors of CIR[P=0.025, HR=6.9 (95%CI 1.3-37.4) ; P=0.031, HR=11.1 (95%CI 1.2-99.2) ]. Conclusion: Different leukemia stem cells associated antigens had distinct prognostic significance in t (8;21) AML. High frequencies of CD34(+) CD38(-), CD34(+) CD38(-) CD123(+) and CD34(+)CD38(-)CD96(+) cells at diagnosis predicted relapse in patients with t (8;21) AML.
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Humans , ADP-ribosyl Cyclase 1 , Antigens, CD , Flow Cytometry , Interleukin-3 Receptor alpha Subunit , Leukemia, Myeloid, Acute , Neoplastic Stem Cells , Prognosis , Stem CellsABSTRACT
Objective@#To study the specific killing effect of CD4 membrane protein targeted chimeric antigen receptor modified T (CAR-T) cell.@*Methods@#The second generation CD4 targeted chimeric antigen receptor containing 4-1BB costimulation domain was insert into lentiviral vector through recombinant DNA technology. Lentivirus was prepared and packaged by 293T cells with four plasmids. Beads activated T cells were transduced with lentivirus and the transduction efficiency was checked with Protein L and flow cytometry. T cell subsets and IFN-γ concentrations were detected with probe-tagged antibody and cytometric bead assay.@*Results@#①The transduction efficiency of activated T cells with prepared lentivirus were 50.0%-70.0%. A subset of CD8+ T cell acquired dim expression of CD4 membrane protein after activation. CD4+T cell and CD8+CD4dim T cell were gradually killed by CD4 targeted CAR-T post lentivirus transduction. ②The kill efficacy of CD4 targeted CAR-T cell and control T cell toward KARPAS 299 T cell at an E∶T ratio of 8∶1 for 24 h was (96.9±2.1)% and (11.2±3.1)%, CAR-T cell has a higher killing efficacy than control T cell (t=7.137, P=0.028). The IFN-γ concentrations in culture supernatant of CAR-T cell with K562-CD4 cell, CAR-T cell with K562 cell and CAR-T cell alone were (15 648±2 168), (1 978±354) and (1 785±268) pg/ml, CAR-T cell cocultured with K562-CD4 cell produced more IFN-γ than the other two controls (P<0.01).@*Conclusions@#CD4 targeted CAR-T has an immunophenotype of CD8+CD4-T cell. CD4 targeted CAR-T cell has killing efficacy toward normal CD4+T cell and CD4+T lymphoma cell. CD4 targeted CAR-T cell also has a killing efficacy toward CD4dim target cell.
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Objective: To study the recovery effect of Siwutang on the anemia induced by chemotherapy in the mice, and to clarify its mechanism. Methods: A total of 24 mice were randomly divided into control group (n=8, not given any drugs), model group (n= 8, given 5-fluorouracil), Siwutang group (n= 8, given 5-fluorouracil + Siwutang). After administration for 15 d, the peripheral blood of mice was taken, and the morphology and the number of red cells were observed under microscope. The number of peripheral blood red cells and the levels of hemoglobin of the mice in various groups were calculated by full-automatic hemocytometer; Double staining was used to mark the surface antigens of red cells, then antibody binding and staining were performed. Flow cytometry was used to detect the number of peripheral blood-labelled and spleen-labelled red cells. Results: Compared with control group, the number of red cells in peripheral blood of the mice in model group was significantly decreased (P<0. 05), and the cells shrinkaged. Compared with model group, the number of red cells in peripheral blood of the mice in Siwutang group was significantly increased (P<0. 05) and the cells showed rounding. Compared with control group, the level of hemoglobin in peripheral blood of the mice in model group was significantly decreased (P<0. 05); compared with model group, the level of hemoglobin in peripheral blood of the mice in Siwutang group was significantly increased (P<0. 01). The flow cytometry results showed that compared with control group, the expression level of CD71/Terll9 in peripheral blood of the mice in model group was significantly decreased (P<0. 01), and the number of red cells in various quadrants was decreased (P<0. 01); compared with model group, the expression level of CD71/Terll9 in peripheral blood of the mice in Siwutang group was significantly increased (P<0. 01), and the number of red cells in various quadrants was increased (P<0. 01); compared with control group, the expression level of CD71/Terll9 in spleen blood of the mice in model group was significantly decreased (P< 0. 01), and the number of cells in various quadrants was decreased (P<0. 01); compared with model group, the expression level of CD71/Terll9 in spleen blood of the mice in Siwutang group was significantly increased (P<0.01), the number of red cells in UL quadrant was decreased (P<0. 01), and the number of red cells in UR and LR quadrants was increased (P<0.01). Conclusion: Siwutang can promote the recovery of anemia induced by chemotherapy in the mice, increase the number of red cells, improve the morphology of red blood cells, increase the level of hemoglobin in peripheral blood, and increase the expression levels of marker antigens on the surface of red cells in the peripheral blood and spleen.
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Objective: To study the specific killing effect of CD4 membrane protein targeted chimeric antigen receptor modified T (CAR-T) cell. Methods: The second generation CD4 targeted chimeric antigen receptor containing 4-1BB costimulation domain was insert into lentiviral vector through recombinant DNA technology. Lentivirus was prepared and packaged by 293T cells with four plasmids. Beads activated T cells were transduced with lentivirus and the transduction efficiency was checked with Protein L and flow cytometry. T cell subsets and IFN-γ concentrations were detected with probe-tagged antibody and cytometric bead assay. Results: ①The transduction efficiency of activated T cells with prepared lentivirus were 50.0%-70.0%. A subset of CD8+ T cell acquired dim expression of CD4 membrane protein after activation. CD4+T cell and CD8+CD4dim T cell were gradually killed by CD4 targeted CAR-T post lentivirus transduction. ②The kill efficacy of CD4 targeted CAR-T cell and control T cell toward KARPAS 299 T cell at an E∶T ratio of 8∶1 for 24 h was (96.9±2.1)% and (11.2±3.1)%, CAR-T cell has a higher killing efficacy than control T cell (t=7.137, P=0.028). The IFN-γ concentrations in culture supernatant of CAR-T cell with K562-CD4 cell, CAR-T cell with K562 cell and CAR-T cell alone were (15 648±2 168), (1 978±354) and (1 785±268) pg/ml, CAR-T cell cocultured with K562-CD4 cell produced more IFN-γ than the other two controls (P<0.01). Conclusions: CD4 targeted CAR-T has an immunophenotype of CD8+CD4-T cell. CD4 targeted CAR-T cell has killing efficacy toward normal CD4+T cell and CD4+T lymphoma cell. CD4 targeted CAR-T cell also has a killing efficacy toward CD4dim target cell.
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Humans , CD4 Antigens , CD8-Positive T-Lymphocytes , Cell Line, Tumor , Lymphoma , Receptors, Antigen, T-Cell , Receptors, Chimeric AntigenABSTRACT
Objective To investigate the expression of CD55 and its implication on prognosis for patients with gastric stromal tumors.Methods Expression of CD55 was detected by immunohistochemistry staining and the correlation between CD55 and clinicopathological features and prognosis were analyzed on 60 cases of primary gastric stromal tumors from January 2010 to October 2012.Results Of the 60 patients with gastric stromal tumors there were 33 males and 27 females.CD55 was mainly located in the cytoplasm of gastric stromal tumors.There was no statistical difference between CD55 expression and patients' gender and age (P ＞ 0.05).Moreover,the expression of CD55 was closely related to tumor size,mitotic counts,2008 NIH classifications,and distant metastasis at the first visit to the hospital (P ＜ 0.05).During a median follow-up of 58.5 (range 23-78) months,14 patients had tumor progression.Log-rank univariate survival analysis showed tumor size,mitotic counts,2008 NIH classifications,radical resection,distant metastasis at the first clinical visit and CD55 expression were related to progression-free survival (P ＜ 0.001).COX multivariate survival analysis showed that tumor size (HR 11.504,95％ CI:1.085-122.011,P =0.043) and CD55 expression (HR 11.819,95％ CI:1.827-76.477,P =0.01) were independent prognostic factors.Conclusions Up-regulated CD55 might play an important role in the development and metastasis of gastric stromal tumors.
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Objective: To evaluate the clinical indications of CD151 in patients with lung adenocarcinoma with positive expressions of vascular endothelial growth factor A (VEGFA) or vascular endothelial growth factor receptor (VEGFR). Methods: The expressions of VEGFA, VEGFR1, VEGFR2 and CD151 in 116 specimens of patients with lung adenocarcinoma after surgery were detected by immunohistochemistry. The association of CD151 expression with the clinical features of patients with lung adenocarcinoma with positive expression of VEGFA or VEGFR was analyzed. The effect of CD151 expression on the disease-free survival (DFS) of patients with lung adenocarcinoma with positive expression of VEGFA or VEGFR was evaluated. Results: The positive rates of VEGFA, VEGFR1, VEGFR2 and CD151 in lung adenocarcinoma tissues were 69.83% (81/116), 69.83% (81/116), 44.83% (52/116), and 42.24% (49/116), respectively. For patients with lung adenocarcinoma with positive expression of VEGFA, positive expression of CD151 was positively correlated with clinical TNM stage (r = 0.24, P = 0.04) as well as lymph node metastasis (r = 0.26, P = 0.02). CD151 was an independent factor predicting DFS of patients with lung adenocarcinoma with positive expression of VEGFA [hazard ratio (HR) = 1.80, 95% confidence interval (CI): 1.00 - 3.19, P = 0.048]. The median DFS of CD151-positive patients with positive expression of VEGFA was significantly shorter than that of CD151-negative patients (20 vs 34 months, P < 0.05). For patients with lung adenocarcinoma with positive expression of VEGFR1, positive expression of CD151 was positively correlated with TNM stage (r = 0.28, P = 0.01) and lymph node metastasis (r = 0.31, P < 0.01). Moreover, CD151 was an independent factor predicting DFS of patients with lung adenocarcinoma with positive expression of VEGFR2 (HR = 2.10 C95% CI: 1.02 - 4.33, P = 0.044), and the median DFS of CD151-positive patients with positive expression of VEGFR2 was significantly shorter than that of CD151-negative patients (21 vs 42 months, P < 0.05). Conclusion: CD151 is an independent factor predicting DFS of patients with lung adenocarcinoma with positive expression of VEGFA or VEGFR2.
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BACKGROUND: Immunohistochemical demonstration of CD20 in diffuse large B-cell lymphoma (DLBCL) is prerequisite not only for the diagnosis but also for assigning patients to rituximab-containing chemotherapy. However, little is known about the impact of abundance of CD20 expression assessed by immunohistochemistry on the clinical outcome of DLBCL. We performed a semi-quantitative immunohistochemical analysis of CD20 expression in DLBCL to examine the prognostic implication of the level of CD20 expression. METHODS: Pre-treatment diagnostic tissue samples from 48 DLBCL patients who were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) regimen were represented in a tissue microarray and immunostained for CD20. The relative abundance of CD20 expression was semi-quantitatively scored using a web-based ImmunoMembrane plug-in. Receiver operating characteristic curve analysis was used to determine a prognostically relevant cut-off score in order to dichotomize the patients into CD20-high versus CD20-low groups. RESULTS: The levels of CD20 expression were heterogeneous among the patients, with a wide and linear distribution of scores. Patients in CD20-low group showed significantly poor clinical outcome. CONCLUSIONS: The levels of CD20 expression in DLBCL are heterogeneous among the patients with DLBCL. A subgroup of the patients with CD20 expression levels below the cut-off score showed poor clinical outcome.
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Humans , Antigens, CD20 , B-Lymphocytes , Cyclophosphamide , Diagnosis , Doxorubicin , Drug Therapy , Immunohistochemistry , Lymphoma, B-Cell , Prednisone , ROC Curve , Tissue Array Analysis , Vincristine , RituximabABSTRACT
Objective To determine the expression of CD24 in colorectal carcinoma, and to explore the relationship between CD24 and the clinicopathological features of colorectal carcinoma.Methods The expression of CD24 in 62 cases of colorectal carcinoma, 47 cases of adenomas, 15 cases of colorectal polyps and 30 cases of the adjacent non-cancerous tissues were observed by immunohistochemical assay.The relationship between CD24 and the clinicopathological features was analyzed.Results The positive rates of CD24 in colorectal carcinoma 72.6％ and adenomas 63.8％ were significantly higher than those in colorectal hyperplastic polyps 13.3％ (x2 =17.83, P =0.00;x2 =11.61, P =0.00) and adjacent non-cancerous tissues 6.7％ (x2 =35.15, P =0.00;x2 =24.64, P =0.00).The expression of CD24 in colorectal carcinoma had a significant correlation with the tumor diameter (x2 =5.48, P =0.02), tumor differentiation (x2 =8.86, P =0.00), Duke staging (x2 =11.47, P =0.00) and lymph node metastasis (x2 =8.92, P =0.00).Conclusion The expression of CD24 is high in colorectal carcinoma, having a significant correlation with the size of tumor, degree of differentiation, Duke stage and lymph node metastasis.
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BACKGROUND:Progressive fracture of the cartilage is considered the characteristic lesion in later osteoarthritis, the expression of osteoarthritis-related factors such as hyaluronic acid, osteopontin and CD44 in osteoarthritic cartilage is increased. OBJECTIVE:To investigate the effect of hyaluronic acid on the expression of osteopontin mRNA and CD44 mRNA of chondrocytes in the in vitro cultured chondrocytes of patients with knee osteoarthritis. METHODThe cartilage samples obtained from osteoarthritic patients were cultured and purified into acquire chondrocytes in vitro, and the cells were divided into three groupblank control group, hyaluronic acid (100 mg/mL) group and hyaluronidase (200 mg/mL) group. After 48 hours of cellculture, real-time quantitative polymerase chain reaction assay was used to detect the expression of CD44 mRNA and osteopontin mRNA. The difference of the expression levels before and after the intervention of hyaluronic acid was compared and analyzed using SPSS 17.0 software. RESULTS AND CONCLUSION:Compared with the blank control group, hyaluronic acid (100 mg/mL) upregulated osteopontin mRNA expression in the chondrocytes, hyaluronidase (200 mg/mL) also reduced osteopontin mRNA expression in the chondrocytes. The CD44 mRNA expression in the chondrocytes of hyaluronic acid (100 mg/mL) group and hyaluronidase (200 mg/mL) group was lower than that in the blank control group. Hyaluronic acid can upregulate the expression of the osteopontin mRNA expression in the osteoarthritic chondrocytes;the biphasic effects of hyaluronic acid on CD44 mRNA expression in osteoarthritic chondrocytes might be associated with the molecule weight of hyaluronic acid.
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Objective To investigate the mechanism of protective effects of resveratrol on tissueengineered cartilage.Methods The chondrogenesis of alginate-encapsulated bone marrow mesenchymal stem cells (BMSCs) were evaluated by toluidine blue staining and immunostain.The morphology of BMSCs-derived chondrocytes cultured on chitosan-gelatin scaffolds (CGS) was evaluated by scanning electron microscope and laser confocal microscope.When these cells on CGS were pre-stimulated with interleukin-1β (IL-1β) or cotreated with IL-1β and resveratrol in the absence and presence of specific β1-integrin blocking antibody,collagen type Ⅱ,aggrecan,matrix metalloproteinase-13 (MMP-13) expression,and the translocation of nuclear factor kappaB (NF-κB) were analyzed by Western blotting.ANOVA was used for statistical analysis.Results Alginate bead culture plus conditional medium together could induce the cartilage-specific collagen type Ⅱ,aggrecan expression and extracellular matrix accumulation in differentiated chondrocytes.CGS supported differentiated cell attachment,proliferation,and migration.When those cells cultured on CGS were stimulated with IL-1β alone,collagen type Ⅱ and aggrecan expression was inhibited.However,MMP-13 expression increased.By Western blotting semi-quantitative analysis,the expression level of cartilage-specific collagen type Ⅱ of the control group was 0.484±0.006; the expression level of resveratrol intervention group was 0.474±0.014.The difference between these two groups was not statistically significant (P＞0.05).The expression level of the IL-1β intervention group reduced to 0.155±0.009,which was statistically significant different from the above two groups(P＜0.05).Resveratrol could antagonist the negative effect of IL-1β,and increase collagen type Ⅱ to 0.468±0.014,the difference between these two was statistically significant (P＜0.05),and no significant difference when compared to the control group (P＞0.05).Specific β1-integrin blocking antibody could abrogate these effects of resveratrol,decrease collagen Ⅱ expression to 0.169±0.011,the difference was significant (P＜0.05),but there was no difference when compared to the IL-1β group (P＞0.05).Aggrecan semi-quantitative expression has the same trend in the expression of type Ⅱ collagen while the expression of MMP-13,NF-κB had the reversal trend.These indicated that the resveratrol reversed the catabolic effects by reducing the nuclear translocation of NF-κB.Specific β1-integrin blocking antibody abrogated these effects of resveratrol.Conclusion Resveratrol,by regulating β1-integrin,acts as a NF-κB nuclear trans-location inhibitor to protect tissue-engineered cartilage.
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INTRODUÇÃO: O fato do vírus da Hepatite C (HCV) estabelecer uma infecção crônica persistente, na maioria dos casos, mesmo sendo reconhecido e alvejado pelos sistemas imune inato e adaptativo sugere que o mesmo tenha desenvolvido estratégias eficazes para driblar a ação desses sistemas. O HCV interfere na fase inicial de ativação da resposta imune adaptativa alterando a função das células dendríticas (DCs), o que provavelmente leva a uma ativação deficiente das células natural killer (NKs) e de linfócitos T. Portanto, a realização de estudos sobre DCs e NKs na infecção pelo HCV se torna de fundamental importância para a compreensão da patogênese e persistência desta infecção. MÉTODOS: Foram selecionados indivíduos com resolução espontânea da infecção pelo HCV, indivíduos com infecção crônica e indivíduos saudáveis. A técnica de citometria de fluxo foi utilizada para a determinação da frequência e do fenótipo de células dendríticas e NKs nesses indivíduos. Além disso, foi avaliada a atividade citotóxica das células NKs sob estímulo de IL-12 e IL-18, e também da linhagem K-562. RESULTADOS: A frequência de DC mielóides (mDC) expressando CD86, nos indivíduos crônicos, foi elevada e uma correlação positiva com a carga viral foi observada. Na análise do ensaio funcional foi observado que as populações de células NKs CD7+ CD57+ apresentaram maior expressão da molécula CD107a e baixa produção de IFNy nos indivíduos com infecção crônica. A constante exposição das células imunes ao IFN-alfa, induzido durante a infecção pelo HCV, resulta na polarização do fenótipo citotóxico, caracterizado por células NK ativadas com elevado poder de degranulação, mas com deficiente produção de IFN-y. CONCLUSÕES: As frequências das células DCs e NKs eram semelhantes em todos os indivíduos. A expressão da molécula CD86 na superfície das mDCs pode ter sido induzida pela presença do HCV, uma vez que foi observada correlação positiva com a carga viral...
INTRODUCTION: Hepatitis C virus (HCV) develops a chronic persistent infection in most of the cases, even being recognized and targeted by the innate and adaptive immune systems, suggests that the virus have developed effective strategies to circumvent the action of these systems. HCV interferes in the initial activation of the adaptive immune response by altering the function of dendritic cells (DCs), which probably leads to a deficient activation of natural killer cells (NK) and T lymphocytes. Therefore, studies of DCs and NK in HCV infection are very important for understanding the pathogenesis and the persistence of this infection. METHODS: We selected subjects with spontaneous resolution of HCV infection, with chronic infection and healthy subjects. Flow Cytometry was used to determine the frequency and phenotype of dendritic cells and NK cells of these individuals. In addition, we evaluated the NK cell cytotoxic activity in response to stimulation of IL-12 and IL-18 and in co-cultivation with the cell line K-562. RESULTS: In individuals with chronic infection, the frequency of myeloid (m) DC cells expressing CD86 was elevated and a positive correlation between these cells and viral load was observed. It was observed in chronic infected individuals that NK cells co-expressing CD7 and CD57 showed higher expression of CD107a and low production of IFN gamma. The constant exposure of immune cells to IFN-alfa induced during HCV infection results in the polarization of cytotoxic phenotype characterized by activated NK cells with high power degranulation, but with impaired production of IFN-y. CONCLUSIONS: The frequency of DCs and NK cells were similar in all individuals. The expression of CD86 molecule on the surface of mDCs may have been induced by the presence of HCV, since a positive correlation was observed with viral load. Cytotoxic NK cells, highly differentiated and unable to produce IFN-y, were the most frequent in chronic HCV infection...
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Humans , Dendritic Cells , Flow Cytometry , Hepatitis C, Chronic , Immunity, Innate , Interferon-gamma Release Tests , Killer Cells, NaturalABSTRACT
Objective To investigate the expression of liver cancer stem cells(LCSCs) biomarkers and tumour angiogenesis in patients with hepatocellular carcinoma (HCC) and its clinical significance.Methods Immunohistochemistry was used to detect the expression of CD90,CD133 and CD34 in tumor tissues from 61 patients with HCC undergoing curative hepatectomy.Results The positive rates of CD90and CD133 in HCC tissues were 29.5％ (18/61)and 31.1％ (19/61),the positive rates of CD90 and/or CD133 in HCC were 45.9％ (28/61).The positive rate of LCSCs biomarkers in patients with vascular invasion,tumor differentiation Ⅲ/Ⅳ,non-complete encapsulation,and TNM stage Ⅲ/Ⅳ was significantly higher than those with non-vascular invasion,tumor differentiation Ⅰ / Ⅱ,with complete encapsulation,and TNM stage Ⅰ/Ⅱ.There were a significant positive correlation between LCSCs biomarkers expression and MVD(r =0.5,P ＜ 0.05).As compared with negative LCSCs expression and MVD low group,patients with a positive LCSCs biomarkers expression and MVD high in tumours had significantly lower overall survival (OS) and poor relapse-free survival (RFS).Conclusions High expression levels of LCSCs biomarkers are related to markers of angiogenesis microvessel density (MVD).By combining two of those we can classify paitients and anticipate the prognosis after surgery.
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BACKGROUND: Paroxysmal nocturnal hemoglobinuria is a hematological disease with complex physiopathology. It is genetically characterized by a somatic mutation in the PIG-A gene (phosphatidylinositol glycan anchor biosynthesis, class A), in which the best known antigens are DAF (decay accelerating factor or CD55) and MIRL (membrane inhibitor of reactive lysis or CD59). OBJECTIVE: To determine the frequency of paroxysmal nocturnal hemoglobinuria in patients attended at the HEMOPA foundation from November 2008 to July 2009. METHOD: Thirty patients, with ages ranging from two to 79 years old and suspected of having paroxysmal nocturnal hemoglobinuria were examined. All patients were immunophenotyped by flow cytometry for the CD5, CD59, CD16 and CD45 antigens. RESULTS: Paroxysmal nocturnal hemoglobinuria was identified in nine of the thirty patients investigated. Another 3 cases had inconclusive results with CD59-negative labeling only for neutrophils. The highest frequency of paroxysmal nocturnal hemoglobinuria patients (7/9) and inconclusive cases (2/3) were between 19 years old and 48 years old, with a median of 28 years. CONCLUSION: These results show the importance of flow cytometry to identify cases in which patients are deficient in only one antigen (CD59).
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Humans , Male , Female , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Aged , Immunophenotyping , CD59 Antigens , CD55 Antigens , Flow Cytometry , Hemoglobinuria, Paroxysmal/diagnosisABSTRACT
BACKGROUND: The leukocyte common antigen (CD45) is a transmembrane-type protein tyrosine phosphatase that has five isoforms. METHODS: We generated seven murine mAbs against human CD45 by injecting cells from different origins, such as human thymocytes, PBMCs, and leukemic cell lines. By using various immunological methods including flow cytometry, immunohistochemistry, and immunoprecipitation, we evaluated the reactivity of those mAbs to CD45 of thymus as well as tonsil lysates. Furthermore, we transiently transfected COS-7 cells with each of gene constructs that express five human CD45 isoforms respectively, and examined the specificities of the mAbs against the transfected isoforms. RESULTS: In case of thymocytes, lymphocytes, and monocytes, all the seven mAbs demonstrated positive reactivities whereas none was reactive to erythrocytes and platelets. The majority of immune cells in formalin-fixed paraffin-embedded thymus and tonsil tissues displayed strong membranous immunoreactivity, and the main antigen was detected near 220 kDa in all cases. Among the mAbs, four mAbs (AP4, DN11, SHL-1, and P6) recognized a region commonly present in all the five isoforms. One mAb, YG27, recognized four isoforms (ABC, AB, BC, and O). Two mAbs, P1 and P14, recognized the isoforms that contain exon A encoded regions (ABC and AB). CONCLUSION: In this study, we confirmed that AP4, DN11, SHL-1, YG27 and P6, are mAbs reactive with the CD45 antigen whereas P1 and P14 are reactive with the CD45RA antigen.
Subject(s)
Animals , Humans , Antibodies, Monoclonal , Leukocyte Common Antigens , Blood Platelets , Cell Line , COS Cells , Erythrocytes , Exons , Flow Cytometry , Immunohistochemistry , Immunoprecipitation , Leukocytes , Lymphocytes , Monocytes , Palatine Tonsil , Protein Isoforms , Protein Tyrosine Phosphatases , Thymocytes , Thymus GlandABSTRACT
Objective To detect T-lymphocyte subgroup in patients with malignant lymphoma, and investigate the relationship between the number of the cells and the oncogenesis and development of malignant lymphoma. Methods The counts of T-lymphocyte subgroup in peripheral blood of 106 patients with lymphoma were detected by flow-cytometry, and analyzed in comparison with that of 106 normal people as control. Results The counts of CD+3 cell and the ratio of CD+4/CD+8 in patients with malignant lymphoma were significantly lower than that of control group (P＜0.05). The number of CD+3 were significantly lower and CD+8 were higher than that of control group in patients with NHL at early ( Ⅰ, Ⅱ ) stage (P＜0.05). The counts of CD+3 CD+4 cell and the rate of CD+4/CD+8 in patients with NHL at advanced (Ⅲ ,Ⅳ) stage were significantly ldwer than that of control group (P＜0.05). Conclusion The function of immune is decreased in patients with malignant lymphoma, and these changes had correlation with clinic stage. It is of active significance by detecting T-lymphocyte subgroup in peripheral blood in patients with malignant lymphoma to evaluate the function of immune and find out the relationship between the number of the immune cells and the oneogenesis and development of malignant lymphoma.
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Objective To study the effect of berberine on the lipid metabolism and the expression of ox-LDL,CD36 and PPAR? in the peritoneal macrophage(PM) and alveolar macrophage(AM) from the type 2 diabetic rats. Methods Sprague-Dauley rats were randomly divided into groups of normal control,high-fat diet,type 2 diabetes and berberine treatment.The levels of blood glucose,insulin and lipid were measured biochemically,the content of ox-LDL in macrophage and the protein expression of CD36 and PPAR? in macrophage were assayed by ELISA,the mRNA expression of CD36 and PPAR? in macrophage was disclosed by RT-PCR. Results Compared with the control group, the levels of glucose,insulin and total cholesterol (TC),triglycerides(TG),low density lipoprotein-cholesterol (LDL-C) in blood were increased significantly(P0.05). The content of ox-LDL,the protein and mRNA expressions of CD36 and PPAR? in PM and AM increased in type 2 diabetic group(P