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OBJECTIVE@#To investigate whether Omicron BA.1 breakthrough infection after receiving the SARS-CoV-2 vaccine could create a strong immunity barrier.@*METHODS@#Blood samples were collected at two different time points from 124 Omicron BA.1 breakthrough infected patients and 124 controls matched for age, gender, and vaccination profile. Live virus-neutralizing antibodies against five SARS-CoV-2 variants, including WT, Gamma, Beta, Delta, and Omicron BA.1, and T-lymphocyte lymphocyte counts in both groups were measured and statistically analyzed.@*RESULTS@#The neutralizing antibody titers against five different variants of SARS-CoV-2 were significantly increased in the vaccinated population infected with the Omicron BA.1 variant at 3 months after infection, but mainly increased the antibody level against the WT strain, and the antibody against the Omicron strain was the lowest. The neutralizing antibody level decreased rapidly 6 months after infection. The T-lymphocyte cell counts of patients with mild and moderate disease recovered at 3 months and completely returned to the normal state at 6 months.@*CONCLUSION@#Omicron BA.1 breakthrough infection mainly evoked humoral immune memory in the original strain after vaccination and hardly produced neutralizing antibodies specific to Omicron BA.1. Neutralizing antibodies against the different strains declined rapidly and showed features similar to those of influenza. Thus, T-lymphocytes may play an important role in recovery.
Subject(s)
Humans , Antibodies, Neutralizing , Prospective Studies , SARS-CoV-2 , Breakthrough Infections , COVID-19 Vaccines , COVID-19 , T-Lymphocytes , China/epidemiology , Antibodies, ViralABSTRACT
OBJECTIVE@#To explore the effect of transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) on toluene diisocyanate (TDI)-induced allergic airway inflammation in mice.@*METHODS@#Thirty-two mice were randomly divided into AOO group, AOO+5Z-7-Oxozeaenol group, TDI group, and TDI+5Z-7-Oxozeaenol group. Another 32 mice were randomly divided into AOO group, TDI group, TDI +5Z-7-Oxozeaenol group, and TDI +5Z-7-Oxozeaenol + Necrostatin-1 group. TAK1 inhibitor (5Z-7-Oxozeaenol, 5 mg/kg) and/or RIPK1 inhibitor (Necrostatin-1, 5 mg/kg) were used before each challenge. Airway responsiveness, airway inflammation and airway remodeling were assessed after the treatments. We also examined the effect of TDI-human serum albumin (TDI-HSA) conjugate combined with TAK1 inhibitor on the viability of mouse mononuclear macrophages (RAW264.7) using CCK8 assay. The expressions of TAK1, mitogen-activated protein kinase (MAPK) and receptor interacting serine/threonine protease 1 (RIPK1) signal pathway in the treated cells were detected with Western blotting. The effects of RIPK1 inhibitor on the viability of RAW264.7 cells and airway inflammation of the mouse models of TDI-induced asthma were evaluated.@*RESULTS@#TAK1 inhibitor aggravated TDI-induced airway inflammation, airway hyper responsiveness and airway remodeling in the mouse models (P < 0.05). Treatment with TAK1 inhibitor significantly decreased the viability of RAW264.7 cells, which was further decreased by co-treatment with TDI-HSA (P < 0.05). TAK1 inhibitor significantly decreased the level of TAK1 phosphorylation and activation of MAPK signal pathway induced by TDI-HSA (P < 0.05). Co-treatment with TAK1 inhibitor and TDI-HSA obviously increased the level of RIPK1 phosphorylation and caused persistent activation of caspase 8 (P < 0.05). RIPK1 inhibitor significantly inhibited the reduction of cell viability caused by TAK1 inhibitor and TDI-HSA (P < 0.05) and alleviated the aggravation of airway inflammation induced by TAK1 inhibitors in TDI-induced mouse models (P < 0.05).@*CONCLUSION@#Inhibition of TAK1 aggravates TDI-induced airway inflammation and hyperresponsiveness and may increase the death of macrophages by enhancing the activity of RIPK1 and causing persistent activation of caspase 8.
Subject(s)
Animals , Mice , Asthma/chemically induced , Inflammation , Macrophages , Receptor-Interacting Protein Serine-Threonine Kinases , Respiratory System , Toluene 2,4-Diisocyanate/adverse effectsABSTRACT
OBJECTIVE@#To study the correlation of IL-37 with T lymphocytes subsets and NK cells in ITP patients, and to explore its possible mechanisms involved in the pathogenesis of ITP.@*METHODS@#Forty-five patients with newly diagnosed ITP(newly diagnosed group), 32 patients of complete remission (remission group) and 22 healthy persons(control group) were selected. The serum level of IL-37 in 3 groups was determined by enzyme linked immunosorbent assay (ELISA). The mRNA expression of IL-37, IL-17 and IL-18 in peripheral blood mononuclear cells（PBMNC） in 3 groups was measured by real-time fluorescence quantitative polymerase chain reaction (PCR). The number of IL-18RαCD4 T cells and Tim-3NK cells in the peripheral blood in 3 groups was detected by flow cytometry (FCM).@*RESULTS@#The serum level of IL-37 in the peripheral blood of ITP patients in the newly diagnosed group was significantly higher than that in the control group and the remission group(P＜0.01) . The expression level of IL-37 in PBMNC of the ITP patients in newly diagnosed group was higher than that in the control group and the remission group(P＜0. 05). The expression level of IL-17 and IL-18 in PBMNC of the ITP patients in newly diagnosed group was higher than that in the control group and the remission group(P＜0. 01); the expression of IL-18Rα in CD4 T cells in newly diagnosed group was significantly higher than that in both the control and the remission group(P＜0.01).The expression of Tim-3 in NK cells in ITP patients was significantly lower than that in the control group (P＜0. 01). In ITP patients, the serum IL-37 level and IL-18RαCD4T cells ratio both negatively correlated with Plt count (r=-0.58, r=-0.48) moreo-ver the serum IL-37 level also negatively correlated with amount of CD4 T cells and NK cells (r=-0.29, r=-0.28), but positively correlated with amount of CD8 T cells (r=0.329).@*CONCLUSION@#The IL-37 and its receptors may play an immunoregulatory role in CD4 T cells and NK cells, the IL-37 may be a therapeutic target for ITP patients.
Subject(s)
Humans , CD8-Positive T-Lymphocytes , Flow Cytometry , Interleukin-1 , Allergy and Immunology , Killer Cells, Natural , Leukocytes, Mononuclear , Purpura, Thrombocytopenic, Idiopathic , T-Lymphocyte SubsetsABSTRACT
<p><b>OBJECTIVE</b>To analyze the number of myeloid-derived suppressor cells(MDSC) and the level of prostaglandin E2(PGE2) in the bone marrow of adult ITP patients, and to explore their possible mechanisms involved in the pathogenesis of this disease.</p><p><b>METHODS</b>Twenty-five patients of newly diagnosed ITP, 25 patients of complete remission group and 15 patients of control group were selected. The number of MDSC in the bone marrow between 3 groups was detect by flow cytometry (FCM). The serum level of prostaglandin E2 (PGE2) in 3 groups was determined by enzyme linked immunosorbent assay (ELISA). The relative expression of IFN-γ mRNA in bone marrow mononuclear cells was measured by real time fluorescence quantitative polymerase chain reaction (RT-qPCR) in each groups.</p><p><b>RESULTS</b>The number of MDSC in the complete remission group was significantly higher than that in the control group (P<0.05); the number of MDSC in the newly diagnosed group was higher than that in the control group; the number of MDSC in the complete remission group was higher than that in the newly diagnosed group. The serum level of PGE2 in bone marrow of ITP patients in the newly diagnosed group was higher than that of the control group（P<0.05）. The serum level of PGE2 in the bone marrow of ITP patients of the complete remission group was higher than that of the control group (P<0.05）. The level of PGE2 in bone marrow serum of ITP patients of the newly diagnosed group was lower than that in the complete remission group(P<0.05）. The relative expression level of IFN-gamma in bone marrow mononuclear cells of the ITP patients in newly diagnosed group was higher than that in the control group and the complete remission group（P<0.001）. The relative quantification (RQ) of IFN-γ in bone marrow mononuclear cells was 2.60 between the newly diagnosed group and the complete remission group.</p><p><b>CONCLUSION</b>When adult ITP disease is remitted, the number of MDSC rises and correlates with the therapeutic response and PGE2 level in the bone marrow.</p>
Subject(s)
Adult , Humans , Bone Marrow , Flow Cytometry , Myeloid-Derived Suppressor Cells , Purpura, Thrombocytopenic, Idiopathic , RNA, MessengerABSTRACT
OBJECTIVE:To investigate the toxicity effect of Jianpi shengxue granule on perinatal rats. METHODS:Based on body mass,pregnant rats were randomly divided into negative control group and Jianpi shengxue granule low-dose,medium-dose, high-dose groups(0.77,2.31,6.93 g/kg),21 in each group,and intragastrically given relevant medicines from the 15th d of preg-nancy until 21st d after delivery,once a day. Effects of Jianpi shengxue granule on general toxicity and fertility of maternal rats were observed,as well as the effects on athletic ability,learning and memory ability,fertility of F1 offspring and early viability of F2 offspring. RESULTS:In terms of toxicity effect on maternal rats,compared with control group,the days of pregnancy in Jianpi shengxue granule high-dose group was significantly prolonged(P0.05);there was no obvious effect on the indexes of offspring in Jianpi shengxue granule medium-dose,low-dose groups. CONCLUSIONS:The no toxic dose of Jianpi shengxue granule in maternal and offspring rats is 2.31 g/kg.
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Objective@#To explore characteristic and function of peripheral blood mononuclear cells (PBMNC) -induced macrophages in patients with myelodysplastic syndrome (MDS) to couple with its progression.@*Methods@#A total of 24 MDS patients (11 low-risk patients and 13 high-risk group patients) referred to Department of Hematology of Tianjin Medical University General Hospital and normal controls were enrolled from September 2014 to December 2015. PBMNC was stimulated with GM-CSF to transform to macrophages. The morphology of macrophages was observed by microscope. The quantity of macrophages, CD206 and SIRPα on surface of macrophages were detected by flow cytometry. The phagocytic function of macrophages was analyzed by fluorescence microscopy and flow cytometry.@*Results@#The morphology of macrophages from MDS patients was abnormal. The percentage of transformed macrophages was (5.17±3.47) % in patients with MDS, which was lower than that in controls significantly[ (66.18±13.43) %, t=3.529, P=0.001]. The expression of CD206 on macrophages from MDS patients was significantly lower than that of controls[ (9.73±2.59) % vs (51.15±10.82) %, t=4.551, P<0.001]. The SIRPα level of macrophages from MDS patients was significantly lower than that of controls [ (0.51±0.09) % vs (0.77±0.06) %, t=2.102, P=0.043]. The phagocytic index and the percentage of phagocytic of macrophages from MDS patients were significantly lower than those of macrophages from normal controls[0.45±0.08 vs 0.92±0.07, t=-6.253, P=0.008; (23.69±3.22) % vs (42.75±2.13) %, t=-6.982, P=0.006 respectively]by flow cytometry. The phagocytic index of MDS patients was significantly lower than that of controls (0.24±0.04 vs 0.48±0.96, t=3.464, P=0.001) by fluorescence microscopy.@*Conclusion@#The quantity, recognization receptors and phagocytosis of PBMNC-induced macrophages decreased in MDS patients.
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Objective@#To investigate the change of autophagy level of bone marrow nucleated red blood cell (RBC) in patients with myelodysplastic syndromes (MDS) .@*Methods@#Fifty-four MDS patients and thirty-three controls were enrolled in this study. The mitophagy were observed by transmission electron microscopy (TEM) . The level of autophagy-associated protein LC3B in GlycoA+ nucleated RBC was measured by flow cytometry. The expressions of ULK1 and mTOR mRNA in GlycoA+ nucleated RBC were measured by real-time PCR. The expression of the mitochondrial outer membrane protein TOM20 in GlycoA+ nucleated RBC was detected by Western blot.@*Results@#Autophagosomes or autolysosomes were scarcely observed by TEM in MDS patients. The expression of LC3B in GlycoA+ nucleated RBC in high-risk MDS patients (0.22±0.12) was significantly lower than that in normal controls (0.43±0.22, P<0.001) , and lower than that in low-risk MDS patients (0.40±0.16, P=0.001) . The expression of AMPK [0.26 (0.60) ] in GlycoA+ nucleated RBC in high-risk MDS patients was significantly lower than that in controls [1.00 (2.07) , P<0.017) . The expression of ULK1 mRNA in GlycoA+ nucleated RBC in high-risk MDS patients [0.27 (3.31) ] was significantly lower than that in controls [1.07 (4.41) , P<0.017]. The level of mTOR mRNA in GlycoA+ nucleated RBC in high-risk MDS patients [1.82 (3.74) ] was significantly higher than that in controls [1.26 (1.38) , P<0.017]. The level of LC3B in GlycoA+ nucleated RBC was negatively correlated with the HGB (r=0.529, P=0.009) in high-risk MDS patients. The expression of mitochondrial outer membrane protein TOM20 in high-risk MDS patients was 9.42±4.42.@*Conclusion@#Autophagy is impaired in nucleated RBC of MDS patients.
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Objective@#To investigate the levels of NK cells and their relevant cytokines (IL-10, TGF-β and IFN-γ) in patients with primary immune thrombocytopenia (ITP) .@*Methods@#All samples were obtained from 42 patients (22 newly diagnosed and 20 in remission) and 20 healthy volunteers. The levels of IL-10 and IFN-γ in blood serum were detected by enzyme-linked immunosorbent assay (ELISA) . The percentage of CD3- CD56+ NK cell, CD3- CD56bright CD16- NK cell, CD3- CD56dim CD16+ NK cell in peripheral blood lymphocyte were detected by flow cytometry. The NK cells were isolated by immunomagnetic microbeads. The mRNA expression levels of IL-10, TGF-β, and IFN-γ in NK cells were detected by real-time fluorescent quantitative PCR. Correlation between the above measured results was analyzed.@*Results@#① The blood serum level of IFN-γ in newly diagnosed ITP patients [ (653.0±221.6) ng/L] was higher than that in remission ITP patients [ (484.4±219.5) ng/L] and healthy control [ (390.9±253.5) ng/L] (P=0.022, P=0.001) . The blood serum level of IL-10 in newly diagnosed ITP patients was lower than that in healthy control [ (52.09±26.66) ng/L vs (79.44±38.43) ng/L, P=0.007]. ②The percentage of NK cell in newly diagnosed and remission ITP patients [ (9.53±3.93) %, (9.03±3.78) %] were significantly lower than that in healthy control [ (13.72±7.42) %] (P=0.013, P=0.007) . The ratio of CD3- CD56bright CD16- NK cell/total NK cells in newly diagnosed ITP patients was higher than that in healthy control [ (6.85±4.43) % vs (4.05±2.81) %, P=0.032]. The ratio of CD3-CD56dim CD16- NK cell/total NK cells in newly diagnosed ITP patients was lower than that in healthy control [ (93.14±4.43) % vs (95.94±2.81) %, P=0.032]. ③ There was no significant difference in the mRNA expression level of IFN-γ in NK cells of ITP patients and healthy control (all P>0.05) . The mRNA expression levels of IL-10 and TGF-β in NK cells in newly diagnosed ITP patients were significantly higher than that in healthy control (1.82±1.32 vs 1.02±1.03, P=0.023; 2.80±2.31 vs 1.46±1.37, P=0.028) . The ratio of CD3-CD56bright CD16- NK cell/total NK cells was positively correlated with the mRNA expression levels of IL-10, TGF-β in NK cells (r=0.424, P=0.001; r=0.432, P<0.001) .@*Conclusion@#NK cells may compensate for the deficiency of the number by enhancing the secretion of negative regulation cytokines, acting as "protective" roles in the disease.
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Objective To explore the effect of Balint group training on the humanistic caring ability of junior nurses. Methods About 96 junior nurses from the department of oncology were randomly assigned to an intervention group and a control group equally.Balint group training was given to the intervention group every 2 weeks in a year.The control group completed the humanistic care training according to the regular procedure in the department of the hospital.At the beginning of the study and at the end of the study,the nursing staff from the two groups and the patients under their continuous nursing care for more than 3 days were surveyed by the care efficiency scale,nursing care behavior scale and patient care perception questionnaire.Results Before the intervention,there was no significant difference between the groups in the care performance and behavior of nursing staff and the patients'care perception (P>0.05).After the intervention there was significant difference in the care performance and behavior of nursing staff and the patients' care perception (P<0.05). Conclusions Balint group training can improve the caring performance of junior nurses.Their ability to express care and establish the caring nurse-patient relationship can be improved by this group training.They become more voluntary to integrate caring behavior into the daily care and their caring behaviors can be more likely to be felt and recognize by the patients.
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Objective To investigate the synergistic regulation of KDM3B and JMJD1C in leukemia. Methods The expression level of JMJD1C and KDM3B were analyzed in multiple acute myeloid leukemia (AML) cell lines. AML cell lines NB4 and HL-60 were treated with Daminozide, followed by determination of H3K9 mono-methylation and di-methylation. AML cell lines NB4 and HL-60 were treated with Daminozide, ATRA (retinoid acid All-trans), C Vitamin and the expression of KDM3B and JMJD1C were detected by real-time quantitative PCR. Results The expression level of KDM3B and JMJD1C in the AML cell lines was negatively correlated. In NB4 and HL-60 cells treated by daminozide, H3K9 mono-methylation and di- methylation level showed a rising trend in these two cell groups. After treatment of NB4 cells with the 3 reagents, the level of mRNA of KDM3B was down-regulated while the level of mRNA of JMJD1C was up-regulated. In HL-60 cells treated by daminozide, the mRNA level of KDM3B was up-regulated and the mRNA level of JMJD1C was down-regulated. Conclusion The expression of KDM3B and JMJD1C is negatively correlated in patients with AML.
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<p><b>OBJECTIVE</b>To explore the role of the receptor for advanced glycation end products (RAGE) in regulating the expression of MUC5AC and mucus production in a mouse model of toluene diisocyanate (TDI)?induced asthma.</p><p><b>METHODS</b>BALB/c mice were randomly divided into control group, vehicle (AOO) group, TDI?induced asthma group and RAGE inhibitor (FPS?ZM1) group. PAS staining, Western blotting, and immunohistochemistry were used to analyze the changes in mucus production and MUC5AC expression in the airway of the mice, and the expression of p?ERK was detected with Western blotting. In vitro cultured human bronchial epithelial cell line 16HBE was transfected with lentiviral vector carrying short hairpin RNA targeting RAGE (shRNA?RAGE) and subsequently challenged with a TDI?human serum albumin (TDI-HSA) conjugate, and the changes in cellular MUC5AC mRNA expression as detected using RT-PCR; the protein expressions of ERK and p?ERK in the cells were examined with Western blotting. The effect of ERK inhibitor U0126 pretreatment on MUC5AC mRNA expression was also analyzed in the cells.</p><p><b>RESULTS</b>Compared with the control mice, TDI-induced asthmatic mice showed significantly higher rates of PAS positivity and increased MUC5AC and p?ERK expressions in the airway (P<0.05). Treatment with FPS?ZM1 significantly decreased PAS positivity and lowered MUC5AC and p?ERK expressions in the airway of the asthmatic mice (P<0.05). Exposure of 16HBE cells to TDI?HSA caused a significant increase in MUC5AC mRNA expression and p?ERK protein expression (P<0.05), while RAGE knockdown obviously suppressed TDI?HSA-induced upregulation of p-ERK and MUC5AC mRNA (P<0.05). Treatment with the ERK inhibitor U0126 also lowered TDI?HSA?induced up?regulation of MUC5AC mRNA in the cells (P<0.05).</p><p><b>CONCLUSION</b>RAGE signaling induces MUC5AC expression via extracellular signal-regulated kinase pathway to promote mucus overproduction in mice with TDI-induced asthma.</p>
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<p><b>OBJECTIVE</b>To explore the effect of costimulatory signaling molecules (CD80, CD86) expression on the quantity and function of B lymphocytes in peripheral blood (PB) of the patients with immune thrombocytopenia (ITP).</p><p><b>METHODS</b>A total of 55 ITP patients (30 cases were newly diagnosed and 25 cases were in remission), 25 healthy volunteers as controls were enrolled in this study. The levels of CD19(+)CD5(+), CD19(+)CD80(+), CD19(+)CD86(+), CD41a(+)IgG, CD41a(+)IgM and IgG, IgM in CD19(+)B cells were measured by flow cytometry (FCM). The correlation of CD19(+)B cells with clinical parameters of ITP patients was analyzed.</p><p><b>RESULTS</b>The level of B1 (CD19(+)CD5(+)) of newly diagnosed ITP patients was significantly higher than that of remitted ITP patients and controls (P<0.05). The level of CD19(+)CD80(+) of newly diagnosed ITP patients was significantly higher than that of remitted ITP patients and controls (P<0.05). And the expression of IgG and IgM in CD19(+)B cells of newly diagnosed ITP patients was significantly higher than that of remitted ITP patients and controls (P<0.05). The levels of IgG and IgM in remitted ITP patients after treatment were significantly lower than that before treatment (P<0.05). The level differences of IgG and IgM before and after treatment in refractory ITP patients were not statistically significant (P>0.05). The expression of CD19(+)CD80(+) in newly diagnosed ITP patients positively correlated with the level of Th1 and Th1/Th2 (r=0.502, r=0.471, P<0.05). The expression of CD19(+)CD80(+) of newly diagnosed ITP patients positively correlated with the level of IgG and IgM in CD19(+)B cells (r=0.552, r=0.467, P<0.05), and negatively correlated with PB platelet count (r= -0.424, P<0.05). The levels of IgG and IgM in CD19(+)B cells of newly diagnosed ITP patients negati- vely correlated with PB platelet count (r=-0.658, r=-0.526, P<0.05).</p><p><b>CONCLUSION</b>The enhacement of costimulatory signaling pathway of CD19(+)B cells in ITP patients results in the abnormal activation of B lymphocytes, thereby mediates the dysfunction of immune system and involves in the pathogenesis of ITP.</p>
Subject(s)
Humans , B-Lymphocytes , Flow Cytometry , Platelet Count , Purpura, Thrombocytopenic, Idiopathic , Signal Transduction , ThrombocytopeniaABSTRACT
<p><b>OBJECTIVE</b>To evaluate fractional exhaled nitric oxide (FENO) level in patients with subacute cough and its value in predicting the patients' response to inhaled corticosteroids (ICS) treatment.</p><p><b>METHODS</b>A total of 100 patients with persistent cough lasting more than 3 weeks were enrolled, including 52 patients with subacute cough and 48 with chronic cough. FENO, spirometry, and responses to ICS therapy of the patients were evaluated.</p><p><b>RESULTS</b>The recruited patients had a median (inter-quartile ranges) FENO level of 19 ppb (12-30 ppb). Patients with chronic cough had a significantly higher median FENO level than those with subacute cough (20.5 vs 16 ppb; Z=-2.245, P=0.025). A FENO level ≥25 ppb was recorded in 15 (28.8%) patients with subacute cough, as compared with 20 (41.6%) in patients with chronic cough (χ(2)=1.801, P=0.179). With a FENO ≥25 ppb as the critical value to justify ICS treatment, 15 patients with subacute cough received ICS and 14 (93.3%) of them showed obvious relief of cough after 2 weeks of therapy, a response rate similar to that of 85.0% (17/20) in patients with chronic cough receiving the treatment (χ(2)=0.588, P=0.443). In patients with subacute cough, those with cough variant asthma (CVA) or eosinophilic bronchitis (EB) had a significantly higher median FENO level than those with postinfectious cough [(16 (11-31) ppb vs 11 (8-19) ppb, P<0.01]. In the etiological analysis, CVA or EB was identified in 23 (44.2%) of the patients with subacute cough, as compared 21 (43.8%) in patients with chronic cough (χ(2)=0.002, P=0.961).</p><p><b>CONCLUSION</b>FENO may be an important indicator for etiological diagnosis of subacute cough and for predicting the response to ICS treatment.</p>
Subject(s)
Female , Humans , Male , Adrenal Cortex Hormones , Therapeutic Uses , Breath Tests , Chronic Disease , Cough , Diagnosis , Drug Therapy , Exhalation , Nitric OxideABSTRACT
<p><b>OBJECTIVE</b>To explore the expression levels of terminal complement complex (C5b-9) and CD62p on platelets and the soluble C5b-9 (sC5b-9) level in serum in patients with PNH or PNH-aplastic anemia (AA).</p><p><b>METHODS</b>Serum levels of sC5b-9, complement C3 and C4 were detected by using ELISA in 25 patients with PNH/PNH-AA. The quantities of C5b-9 and CD62p on the membrane of platelets were detected by flow cytometry.</p><p><b>RESULTS</b>①In PNH/PNH-AA group, the serum sC5b-9 level [390.27(265.73-676.87) μg/L] was lower than that in control group [540.39(344.20-1 576.78) μg/L] (P<0.01). ②The platelet PNH clone (CD59⁻CD61⁺/CD61⁺) size [50.58(23.29-81.60)%] was bigger in the PNH/PNH-AA group than that [23.57(15.58-29.02)%] in control group (P<0.01). The percentages of C5b-9 deposition (C5b-9⁺CD61⁺/CD61⁺) were higher on the PNH clone platelets (CD59⁻CD61⁺) in the PNH/PNH-AA group [(17.53 ± 6.27)%] than those on the normal platelets (CD59⁺CD61⁺) in PNH patients 11.33±5.03）%］ and control [(10.88±3.58)%] group (P<0.01). ③ The expression of CD62p (CD62p⁺CD61⁺/CD61⁺) on PNH clone platelets in PNH patients [(61.98 ± 11.71)%] was higher than that on the normal platelets in PNH patients [(43.76±11.30)%] and control group [(38.23±18.07)%] (P<0.01). In addition, the expression of CD62p on normal platelets was higher in PNH patients than control (P<0.05). ④The deposition of C5b-9 positively correlated with the expression of CD62p on the platelets (r=0.559, P=0.002).</p><p><b>CONCLUSION</b>Deficiency of CD59 antigen on platelets in PNH patients may lead to the deposition of C5b-9 on its membrane and its dysfunction, which may contribute to thrombosis events in PNH.</p>
Subject(s)
Humans , Anemia, Aplastic , Blood Platelets , Clone Cells , Complement Membrane Attack Complex , Flow Cytometry , Hemoglobinuria, Paroxysmal , P-Selectin , ThrombosisABSTRACT
<p><b>OBJECTIVE</b>To investigate the change of autophagy level of bone marrow mononuclear cells（BMMNCs）in patients with myelodysplastic syndromes（MDS）.</p><p><b>METHODS</b>Thirty- eight patients with MDS and 26 megaloblastic anemia patients were enrolled in this study. The autophagic vacuoles were observed by transmission electron microscopy （TEM） and the quantity of autophagic vacuoles was detected by monodansylcadaverine （MDC） staining. The LC3 protein positive cells were counted by immunofluorescence assays. The expression of Beclin 1, LC3A, mTOR mRNA were measured by real time PCR. The expression of Beclin 1 proteins were detected by Western blotting.</p><p><b>RESULTS</b>The autophgic vacuoles of double membrane that surrounds lysosomes appeared in MDS patients. The percentage of MDC positive cells was significantly higher in MDS patients［（9.75±2.63）%］than that of controls［（2.90± 0.89）%, P＜0.05）. The percentage of LC3 protein cells was also increased in MDS patients（6.13±1.03）% vs（1.5±0.58）%, P＜0.05）. The expression of Beclin 1 and LC3A mRNA in low-risk and intermediate-1 MDS were higher compared with controls （3.61 ± 3.02 vs 1.55 ± 1.03 and 6.56 ± 3.97 vs 1.21 ± 0.95 respectively, both P＜0.05）. The expression of mTOR mRNA was down- regulated in low- risk and intermediate-1 MDS compared with controls（0.39±0.37 vs 1.50±1.03, P＜0.05）. There were no significant difference in expression of Beclin 1, LC3 and mTOR mRNA among intermediate-2 and high-risk MDS and controls. Beclin 1 protein expression was higher in low- risk and intermediate- 1 MDS patients（1.257 ± 0.197）than that of controls（0.528±0.086）and inermediate-2 and high-risk MDS patients（0.622±0.118）.</p><p><b>CONCLUSION</b>The autophagy levels were increased in low- risk and intermediate- 1 MDS, while not enhanced in intermediate-2 MDS. Autophagy might be considered as a cell protective mechanism in MDS. The relatively defective autophagy in intermediate- 2 and high- risk MDS might contribute to disease's progression.</p>
Subject(s)
Humans , Apoptosis Regulatory Proteins , Metabolism , Autophagy , Beclin-1 , Bone Marrow Cells , Cell Biology , Membrane Proteins , Metabolism , Microscopy, Electron, Transmission , Microtubule-Associated Proteins , Metabolism , Myelodysplastic Syndromes , Pathology , TOR Serine-Threonine Kinases , Metabolism , VacuolesABSTRACT
Background Researches showed that platelet-derived growth factor (PDGF) modulate the expression of matrix metalloproteinase/tissue inhibitor of metalloproteinase (MMP/TIMP) in cells,but the association of expression of MMP/TIMP in retinal pigment epithelial (RPE) cells and the dose and active time of PDGF is unclear.Objective This study was to observe the effects of PDGF on the expressions of MMP-2,MMP-9 and TIMP-1 in cultured RPE cells in vitro.Methods RPE cell line,ARPE-19,was calculated in vitro,and the cells were divided into 5 groups when they reached 70％-80％ confluence.Different concentrations (0,0.1,1,10,50 mg/L) of PDGF was added into the medium respectively for 36 hours,and the expressing levels of mRNA and protein of MMP-2,MMP-9 and TIMP-1 were detected by reverse transcription PCR (RT-PCR) and Western blot assay.In addition,RPE cells in PDGF group were treated with 10 mg/L PDGF for 24,36,48 hours respectively to detect the expressions of mRNA and protein of MMP-2,MMP-9 and TIMP-1 in the cells and to compare with the control group without PDGF.Results PDGF stimulated proliferation of RPE cells in a dose-and time-dependent manner.As the increase of the PDGF concentrations,the expression values of MMP-2 mRNA and MMP-9 mRNA in RPE cells were gradually elevated,with a statistically significant difference among various groups (MMP-2 mRNA:F=79.304,P=0.000;MMP-9 mRNA:F =8.465,P=0.003),and the expressions of MMP-2 mRNA and MMP-9 mRNA were significantly higher in the 1,10,50 mg/L PDGF groups compared with 0 mg/L PDGF normal control group (all at P＜0.05).Also,the expression values of MMP-2 and MMP-9 proteins in RPE cells were gradually elevated with the increase of PDGF concentrations,showing statistically differences among the groups (MMP-2:F=26.550,P=0.000;MMP-9:F=80.993,P=0.000).Compared with the 0 mg/L PDGF group,MMP-2 and MMP-9 expression levels in the 1,10,50 mg/L PDGF groups were significantly up-regulated (all at P＜ 0.05).However,the expression levels of TIMP-1 mRNA and protein group in the cells were not significantly different among various groups (mRNA:F =0.143,P =0.962 ; protein:F =1.955,P =0.178).The expression levels of M MP-2 mRNA,M MP-9 mRNA in the cells were increased in the PDGF group compared with the control group at different time points (MMP-2 mRNA:Ftime =83.250,P=0.002 ; MMP-9 mRNA:Ftime =6.785,P =0.019).Also,the expression values of MMP-2 and MMP-9proteins in RPE cells were increased in the PDGF group compared with the control group at different time points (MMP-2:Ftime =1 l.185,P =0.041 ; MMP-9:Ftime =968.413,P =0.000).The expression levels of MMP-2 and MMP-9 mRNAs and proteins were significant between the two groups at different time points (all at Pgroup =0.000;all at Ptime＜0.05).While the expression changes of TIMP-1 werc not significant between the two groups and among various time points (all at P＞0.05) Conclusions PDGF up-regulates MMP-2 and MMP-9 expressions in RPE cells in a dose-and time-dependent manner.But,PDGF dose not alter the expression of TIMP-1.These results indicate that PDGF disrupt the balance of MMP/TIMP,which may damage the extracellular matrix and therefore facilitate the migration of RPE cells in the pathogenesis of proliferative vitreoretinopathy.
ABSTRACT
<p><b>OBJECTIVE</b>To explore the pathogenesis of abnormal WT1 expression in paroxysmal nocturnal hemoglobinuria (PNH).</p><p><b>METHODS</b>The expression of WT1 mRNA in CD59⁻ and CD59⁺ bone marrow mononuclear cells (BMMNC) were measured by semi-quantitative reverse transcription PCR. After WT1 gene silence by RNA interference (RNAi) technology, biological characteristics of BMMNC were investigated by flow cytometry.</p><p><b>RESULTS</b>The relative expression of WT1 mRNA in PNH CD59⁻ BMMNC (1.06 ± 0.12) was significantly higher than that in PNH CD59⁺ BMMNC (0.90 ± 0.12) and normal BMMNC (0.86 ± 0.05, P<0.05), but there was no significant difference between PNH CD59⁺ BMMNC and normal BMMNC (P>0.05). WT1 mRNA expression in PNH was positively correlated with the proportion of CD59⁻ cells (r²=0.490, P=0.016), but had no relationship with the proportion of CD59⁺ cells. After WT1 gene silence by siRNA in PNH CD59⁻ BMMNC, WT1 mRNA expression was decreased. The proportions of G0/G1 phase in PNH CD59⁻ cell blank control group and siRNA-scr transfected group were (92.73 ± 3.71)% and (93.06 ± 4.14)%, and the proportions of S phase were (6.99 ± 3.61)% and (6.73 ± 4.08)%, respectively. The proportions of G0/G1 and S phase in siRNA-WT1 transfected group was (94.46 ± 3.71)% and (5.40 ± 3.55)%, respectively. There were significant differences in the proportions of G0/G1 phase and S phase among the controls, siRNA-WT1 transfected group and siRNA-scr transfected group (P<0.05). The rate of apoptosis in siRNA-WT1 transfected group [(35.91 ± 22.36)%] was significantly higher than those in controls [(26.12 ± 17.10)%] and siRNA-scr transfected group [(27.39 ± 18.99)%] (P<0.05).</p><p><b>CONCLUSION</b>siRNA-WT1 could effectively suppress the WT1 gene expression of CD59⁻ clone in PNH patients, inhibit its proliferation, and promote its apoptosis. WT1 gene expression might contribute to PNH clone proliferation.</p>
Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Apoptosis , Bone Marrow Cells , Metabolism , Cell Cycle , Hemoglobinuria, Paroxysmal , Metabolism , Pathology , Leukocytes, Mononuclear , Metabolism , RNA Interference , RNA, Messenger , Genetics , WT1 Proteins , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the changes of relative telomere length (RTL) of peripheral blood (PB) CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺T lymphocytes, CD19⁺B lymphocytes and bone marrow (BM) CD34⁺ cells and its association with disease severity in untreated patients with immuno-related pancytopenia (IRP).</p><p><b>METHODS</b>The PB CD3⁺ , CD3⁺ CD4⁺ , CD3⁺ CD8⁺ T lymphocytes, CD19⁺ B lymphocytes, and BM CD34⁺ cells were purified by magnetic activated cell sorting (MACS), and RTL were measured with flow-fluorescence in situ hybridization (FLOW-FISH).</p><p><b>RESULTS</b>The RTL of CD3⁺, CD3⁺CD4⁺ , and CD3⁺CD8⁺T lymphocytes in untreated IRP patients were (27.754 ± 16.323)%, (7.526 ± 3.745)% and (25.854 ± 14.789)%, respectivly, which were significantly shorter than those in healthy-controls (54.555 ± 19.782)%, (12.096 ± 2.805)%, and (38.367 ± 4.626)% (P<0.05). The RTL of CD19⁺ lymphocytes in untreated IRP patients was (22.136 ± 16.142)%, which was significantly shorter than that in healthy controls (42.846 ± 16.353)% (P<0.01). There was no significant difference of BM CD34⁺ cells RTL between the untreated IRP patients (22.528 ± 21.601)% and the healthy controls (23.936 ± 19.822)% (P>0.05). There were significantly positive correlations between the RTL of B lymphocytes and the count of white blood cell (r=0.706, P=0.015). There were negative correlations between RTL of B lymphocytes and the clinical symptoms (r=-0.613, P=0.045) and positive correlations with therapeutic effect (r=0.775, P=0.005).</p><p><b>CONCLUSION</b>The shorter RTL of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺ lymphocytes, and the normal RTL of BM CD34⁺ cells in untreated IRP patients were identified, which might imply that IRP is a type of acquired autoimmune diseases.</p>
Subject(s)
Adolescent , Adult , Child , Female , Humans , Male , Middle Aged , Young Adult , B-Lymphocyte Subsets , Allergy and Immunology , Lymphocytes , Pancytopenia , Allergy and Immunology , Pathology , T-Lymphocyte Subsets , Allergy and Immunology , TelomereABSTRACT
<p><b>OBJECTIVE</b>To culture osteoblast in vitro and evaluate CCL3 receptor CCR1 expression in patients with multiple myeloma (MM).</p><p><b>METHODS</b>Bone marrow osteoblasts from MM patients were cultured in vitro with dexamethasone, β-sodium glycerophosphate and vitamin C, which were identified by alkaline phosphatase staining, Von Kossa's staining. The CCL3 receptor expression was evaluated by flow cytometry. The morphology and quantity of osteoblast were observed after exposure to CCL3.</p><p><b>RESULTS</b>Bone marrow osteoblasts from MM patients could be cultured in vitro and be identified by positive staining of alkaline phosphatase and Von Kossa's. MM-derived osteoblasts expressed higher levels of CCR1 (74.48 ± 7.31)%, compared with normal controls (48.35 ± 8.81)%. Calcium deposition of osteoblasts after exposure to CCL3 was less than that of controls.</p><p><b>CONCLUSION</b>Bone marrow osteoblasts could be cultured in vitro from MM Patients. CCL3 may contribute to the development of myeloma bone disease.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Cells, Cultured , Chemokine CCL3 , Pharmacology , Multiple Myeloma , Pathology , Osteoblasts , Cell Biology , Metabolism , Receptors, CCR1 , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To detect memory B lymphocyte (Bm) in peripheral blood (PB) of immune-related pancytopenia (IRP).</p><p><b>METHODS</b>86 patients with IRP and 11 health volunteers were enrolled in this study. Bm (CD5⁺ CD19⁺ CD27⁺) and bone marrow mononucleated cell antibodies (BMMNC-Ab) were determined via fluorescence-activated cell sorting, and clinical outcomes of these patients were analyzed.</p><p><b>RESULTS</b>(1)43 initial patients achieved obvious remission in all 52 initial cases after conventional immunosuppression therapy. 16 relapsed patients with IRP received Rituximab (RTX) and 14 cases achieved obvious remission, among which 7 cases were refractory to conventional immunosuppression therapy, 5 cases exhibited obvious remission, and 2 cases did not respond. Other 18 relapsed cases received conventional immunosuppression therapy and 13 cases achieved obvious remission. (1)The level of Bm in PB in 52 initial patients with IRP was(1.81 ± 0.97)%, and no significant difference was observed between the initial patients and health volunteers (1.75 ± 0.55)% (P>0.05). The level of Bm in PB in 34 relapsed patients with IRP was obviously higher than that in the initial IRP patients and health volunteers (P<0.05). Significant difference was observed in the level of Bm in PB in 16 relapsed IRP patients between pre-therapy and post-therapy with RTX (P<0.05). No statistical difference was found between the remission and no-response groups in relapsed patients treated with RTX. RTX regimen produced more effective outcome than conventional immunosuppression therapy, which better eliminated Bm than the latter (P<0.05). Initial patients with IRP who relapsed within a two-year follow-up period had a lower level of Bm in PB compared with un-relapsed patients (P<0.05). Majority of BMMNC- Ab antibodies in relapsed patients were IgG (82.4%) and IgM (69.2%) autoantibodies in patients with initial IRP.</p><p><b>CONCLUSION</b>The level of Bm in PB was associated with relapsed patients with IRP. Bm did not respond to conventional immunosuppression therapy,but responded to RTX.</p>