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Objective:To explore the expression of type 2 complement receptor (CR2) in mesangial cells of the renal tissue in IgA nephropathy (IgAN) and its possible mechanism involved in complement C3 deposition.Methods:The demographic data, samples of plasma and renal tissues of primary IgAN patients diagnosed by renal biopsy in the Guangdong Provincial People's Hospital from August 2021 to May 2022 were collected. According to the fluorescent intensity of mesangial complement C3 deposition, the patients were divided into complement C3 deposition ≥2+ group and complement C3 deposition <2+ group. The circulating IgA and complement C3 levels were detected by enzyme linked immunosorbent assay (ELISA). The influencing factors of kidney prognosis, plasma IgA and complement C3 levels were compared between the two groups. Immunofluorescence was used to detect the expression of IgA, complement C3 and CR2 in the renal mesangial cells of IgAN patients and normal renal tissues around renal carcinoma. Human mesangial cells were cultured in vitro and randomly divided into control group and experimental group. The experimental group was incubated with IgA protein (2 g/L) for 8 hours. The expressions of CR2 protein and mRNA were measured by Western blotting and real-time fluorescence quantitative PCR. The biological function of differential genes was analyzed by gene ontology (GO) and Kyto encyclopedia of genes and genomes (KEGG) enrichment analysis. Results:A total of 75 patients with IgAN were included in this study, including 50 patients in the complement C3 deposition ≥2+ group and 25 patients in the complement C3 deposition <2+ group. The proportions of patients with urine red blood cell count negative, 1+, 2+ and 3+-4+ in the complement C3 deposition ≥2+ group were 2.0%, 8.0%, 18.0% 72.0%, respectively, which were more serious than those in the complement C3 deposition <2+ group (4.0%, 4.0%, 52.0%, 40.0%) ( Z=-2.320, P=0.020). Meanwhile, the proportion of S1 in Oxford pathological classification in the complement C3 deposition ≥2+ group was higher than that in the complement C3 deposition <2+ group (68.0% vs. 40.0%, χ2=5.389, P=0.020), and there were no statistically significant differences in gender, age, 24-hour urinary protein, serum creatinine, other indicators of Oxford pathological classification between the two groups. ELISA results showed that plasma IgA concentration in the complement C3 deposition ≥2+ group was higher than that in the complement C3 deposition <2+ group [3.62 (2.95, 5.53) g/L vs. 2.72 (2.15, 4.24) g/L, Z=2.405, P=0.016], and the plasma complement C3 concentration was lower than that in the complement C3 deposition <2+ group [199.6 (116.0, 328.0) mg/L vs. 319.2 (158.3, 454.5) mg/L, Z=-2.383, P=0.017]. Spearman correlation analysis showed that the complement C3 deposition intensity was positively correlated with IgA deposition intensity in mesangial area ( rs=0.441, P<0.001). Immunofluorescence results showed that there was colocalization of IgA and complement C3 in the glomeruli of IgAN patients. The expression of CR2 in the kidney was consistent with complement C3 deposition, and CR2 was colocalization with complement C3. In vitro experiments, the expression of CR2 in IgA protein group was higher than that in the control group ( P<0.05). GO and KEGG enrichment analysis found that IgA protein induced active changes in various pathways of mesangial cells. Conclusion:IgA protein induces mesangial cells to express CR2 and participates in complement C3 deposition, which may be an important mechanism of complement C3 activation in IgAN.
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Objective To investigate the change of CD35 expression on neutrophils in the peripheral blood and the relationship between the change and disease activity in patient with myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis (MPO-AAV).Methods Forty untreated patients with active MPO-AAV(patient group)and forty healthy volunteers (control group) were enrolled into this study,and Bermingham vasculitis activity score (BVAS) for every patient was recorded.Flow cytometry (FCM) was employed to detect the CD35 and MPO expression on the neurtrophil,and enzyme linked immunosorbent assay (ELISA) was taken to test the levels of autoantibody against MPO-Antineutrophil cytoplasmic antibody (MPO-ANCA),fragment a from the activated complement factor B (Ba) and MPO in peripheral blood from both group.All test results were compared between the 2 groups by t test,Non-parametric test,Spearman correlation analysis.In addition,the relations among the laboratory results and the relationship between BVAS and the laboratory results were analyzed respectively.Results Compared with the control group,the expression level,which was represented as mean flourscence indensity (MFI),of CD35 and neutrophil membrane MPO on peripheral blood neutrophils was significantly increased [(2 014±968) vs (1 454±511),t=3.024,P=0.002 and (709±244) vs (580±158),t=2.806,P＜0.01,respectively],and the MPO expression level in neutrophils was significantly lower [(1 525±1 033) vs (3 196±2 126),t=-4.468,P＜0.01].Ba and MPO levels in serum of the patient group was significantly higher than that in the control group [37.89(26.17,63.14) μg/L vs 27.99(18.64,46.52) μg/L,Z=-2.521,P=0.012 and 546.16(450.55,729.96) U/L vs 327.93(279.02,365.10) U/L,Z=7.121,P＜0.01,respectively].In patient group,the expression level of CD35 had a significant positive relationship with peripheral blood neutrophil count (r=0.573,P＜0.01),serum Ba (r=0.433,P=0.005) and BVAS (r=0.368,P=0.020),respectively,whereas,there was a negative correlation between the MPO expressed on the neutrophils and that in the neutrophils (r=-0.458,P=0.003),and a positive relationship between MPO-ANCA and BVAS (r=0.351,P=0.026).Conclusion There is significant increased expression of CD35 on the neutrophil of patient with MPO-AAV,which might protect the neutrophil from destruction by the activated complement alternative pathway,and more neutrophils consequently contribute to the MPO-AAV pathogenesis.Inhibition of CD35 expression might become one of the potential new pathways for the treatment of MPO-AAV.
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ObjectiveTo investigate the changes in serum soluble complement receptor type 1 (sCR1) at 3 and 6 months after antiviral therapy with telbivudine in patients with chronic hepatitis B (CHB) or liver cirrhosis and the influence of telbivudine on serum sCR1 level. MethodsA total of 57 patients with HBeAg-positive CHB or liver cirrhosis were enrolled and given the antiviral therapy with telbivudine. Venous blood was collected before treatment and at 3 and 6 months after treatment, and enzyme-linked immunosorbent assay was used to measure serum sCR1 level. The paired t-test was used for comparison within one group. ResultsAt 3 and 6 months after the antiviral treatment with telbivudine, the serum sCR1 level changed significantly compared with corresponding baseline values (t=4.864 and 6.238, both P＜0.05). The patients with CHB or liver cirrhosis showed significant changes in the serum sCR1 level at 3 and 6 months after treatment compared with corresponding baseline values (t=3425,5468,4047,7378 all P＜0.05). The patients with CHB had a lower serum sCR1 level at baseline and at 3 and 6 months after treatment than those with liver cirrhosis, but the serum sCR1 level at each time point showed no significant differences between the two groups (all P＞005). ConclusionIn patients with HBeAg-positive CHB or liver cirrhosis, serum sCR1 level is reduced significantly after antiviral therapy with telbivudine.
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Objective To evaluate the role of complement receptor 3 (CR3) on murine macrophages in the recognition of Penicillium marneffei.Methods RAW264.7 murine macrophage cells were cultured in vitro,and divided into four groups to be cocultured with inactivated and live Penicillium mameffei yeast cells as well as inactivated and live Penicillium marneffei conidia respectively at 37 ℃ in 5％ CO2 for one hour.The RAW264.7 cells incubated with phosphate-buffered saline (PBS) served as the blank control group.Then,reverse transcription-PCR was conducted to detect CR3 mRNA expression,Western blot to measure CR3 protein expression,flow cytometry to determine phagocytosis rate,enzyme-linked immunosorbent assay (ELISA) to quantify cytokine levels in culture supernatant.Some RAW264.7 macrophages were transfected with a specific siRNA targeting CR3 gene and cocultured with inactivated Penicillium marneffei conidia,subsequently,phagocytosis rate and supematant cytokine levels were determined.Data were processed by the SPSS 16.0 software,and one-way analysis of variance (ANOVA) was conducted for inter-group comparisons of these parameters.Results No significant differences were observed in the mRNA or protein expressions of CR3 among the four groups of RAW264.7 cells cocuhured with different forms of Penicillium marneffei (both P ＞ 0.05).The phagocytosis rate was 95.14％,89.56％,91.03％ and 90.78％ in RAW264.7 cells cocultured with inactivated conidia and yeast cells,as well as live conidia and yeast cells of Penicillium marneffei,respectively (P ＞ 0.05).The levels of interleukin (IL)-2,interferon (IFN)-γ,IL-4 and IL-10 in culture supernatant were increased at different degrees after one-hour coculture in the four coculture groups compared with the blank control group,but no statistical difference was noted among the four coculture groups in the supernatant levels of these cytokines (all P ＞ 0.05).After coculture with inactivated Penicillium marneffei conidia,the siRNA-transfected RAW264.7 cells showed a statistical decrease in phagocytosis rate (10.89％ vs.92.78％,P ＜ 0.05) and supernatant levels of IL-2,IFN-γ IL-4 and IL-10 compared with untransfected RAW264.7 cells.Conclusions In early stage of innate immunity,CR3 on macrophages may be one of the pattern recognition receptors participating in the recognition and mediation of phagocytosis of Penicillium marneffei.It's possible that both Thl-and Th2-type cytokines,such as IL-2,IFN-γ,IL-4 and IL-10,are involved in the immune response of macrophages against Penicillium marneffei.
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Objective To investigate the role of erythrocyte complement receptor type 1 (CR1) in the pathogenesis of chronic urticaria.Methods Venous blood samples were collected from 59 patients with chronic urticaria (including 14 cases of dermatographism and 45 chronic idiopathic urticaria) and 29 healthy human controls.Flow cytometry was carried out to quantify the expression level of CR1,and double-antibody sandwich enzyme-linked immunosorbent assay to determine the serum level of immunoglobulin E (IgE),complement C3,C4 and 50％ complement hemolytic activity (CH50).Differences in these parameters were analyzed by one-way ANOVA and independent samples t-test,and correlation between these paramenters by Pearson correlation analysis.Results The expression level (expressed as mean fluorescence intensity per 10 000 erythrocytes) of CR1 was significantly higher in patients with dermatographism and chronic idiopathic urticaria than in the healthy controls (35.06 ± 2.06 and 29.17 ± 1.53 vs.20.46 ± 2.57,t =4.20 and 3.33,both P ＜ 0.05),while no statistical difference was observed between the patients with dermatographism and chronic idiopathic urticaria (P ＞ 0.05).Increased total serum IgE levels were observed in patients with dermatographism and chronic idiopathic urticaria compared with the healthy controls ((769.89 ± 123.0) μg/L and (340.09 ± 29.74) μg/L vs.(107.63 ± 88.79) μg/L,t =5.58,5.85,both P ＜ 0.05),and in patients with dermatographism compared with those with chronic idiopathic urticaria (t =3.49,P ＜ 0.05).For patients with chronic urticaria,there was a statistical difference in the expression level of CR1 between individuals (n=22) with total serum IgE levels ranging from 0 to 240 μg/L and those (n =17) higher than 500 μg/L (24.45 ± 10.83 vs.33.09 ± 11.86,t =3.33,P＜ 0.05).The total serum IgE levels were positively correlated with the level of CR1 (r =0.27,P ＜ 0.05),but uncorrelated with that of complement C3 (r =0.16,P ＞ 0.05) or C4 (r =-0.08,P＞ 0.05).The level of complement C3 was positively correlated with that of C4 (r =0.54,P ＜ 0.01).One-way ANOVA revealed no significant difference in the serum levels of complement C3,C4,or CH50 between the patients with dermatographism,patients with chronic idiopathic urticaria and healthy controls (all P ＞ 0.05).Conclusion CR1 is abnormally expressed in patients with chronic urticaria.
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Objective To explore the hereditary susceptibility of children with asthma through studying the relationship between erythroeyte CR1 genomic density polymorphism and erythrocyte immune function. Methods The rates of RBC-C3_(3b)RR and RBC-ICRR were detected to the asthma group consisted of 65 children with asthma and the control group consisted of 28 normal children. The CR1 activity and genomic density polymorphism of erythrocyte from the two groups were detected by Hind Ⅲ restriction enzyme digestion, polymerase chain reaction restriction fragment length polymorphism. Results Frequencies of high expression gene (HH), mid expression gene (HL) and low expression gene (LL) genotypes were 43.08% (28/65), 36.92% (24/65) and 20.00% (13/65) in asthma group, and 78.57% (22/28), 17.86% (5/28) and 3.57%(1/28) in control group respectively. A significant difference was found in the distribution frequency of CR1 genotype between the two groups(P< 0.01).The rates of RBC-C_(3b)RR were significant lower and the rates of RBC-ICRR were significant higher in asthma group than those in control group (P < 0.01). The rates of RBC-C_(3b) RR in HH, HL and LL were decreased in order (P < 0.01),while the rates of RBC-ICRR in HH,HL and LL were increased in order (P < 0.01). Conclusion It suggests that CR1 gene polymorphism may play an important role in determining susceptibility to asthma.
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Objective To study complement receptor typeⅠ(CR1)activity of erythrocytes in pa-tients with SLE.Methods Using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP),CR1rosette(RBC-CR1R)and immuno-complex rosette(RBC-ICR)on red cell,the ery-throcyte complement receptor I type(ECR1)genomic density polymorphism(HH type,HL type,LL type)and erythrocyte CR1immune activity were determined in32patients with SLE and in48normal individuals.Results It was found that HH type rate of ECR1density polymorphism in patients with active SLE was significantly lower(10/16,62.5%)than that(13/16,81.3%)in patients with stable SLE.The level of CR1immune activity in HH type was significantly higher than that in HL,LL type of SLE,and significantly dif-ferent from that in48normal individuals(P
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Objective Through studing the genotype and quantitatively analysing expression of type Ⅰ complement receptor(CR1) on erythrocytes from the patients with systemic lupus erythematosus(SLE). To investigate the relationship between CR1 and SLE. Methods Polymerase chain reaction(PCR) and Hind Ⅲ restriction enzyme digestion were used to analyse the genotype of CR1, and the quantitative expression of CR1 was demonstrated by FACscan flow cytometry. Results Compared with normal controls, high expression genotype of CR1 was significantly decreased, but low expression genotype was significantly increased in SLE patients. The number of CR1 on erythrocytes in SLE patients was significantly lower than that in normal controls. There was a negative relationship between the expression of CR1 and disease activity index of SLE (SLEDAI). Conclusion The expression of CR1 on erythrocytes might be related to the development of SLE, and it is helpful to detect the CR1 on erythrocytes for the determination of SLE patient′s condition.
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Objective To study CD35on erythrocytes and erythrocyte chemokine recepter(ECKR)in patients with psoriasis in order to explore red blood cell(RBC)innate immune function and its possible role in the pathogenesis of psoriasis.Methods The rapid natural immune reaction on cancer cells was measured with RBCs isolated from fresh blood.Expression of CD35on erythrocytes was detected by flow cy-tometry.The level of IL-8,which was bound to isolated erythrocytes,was measured by enzyme linked im-munosorbent assay(ELISA)in the supernatant after centrifugation,which represented ECKR binding activity.Results Rosette formation rates of RBC adhering to cancer cells and expression of CD35on RBCs were significantly higher in psoriatic patients than those in control group(P
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The traditional Chinese medicine Antiinflammation No6, composed of Flos Lonicerae, Herba Taraxaci, Folium Isatidis and Herba Houttuyniae and proved to be highly effective clinically in the treatment of certain acute bacterial and viral infections, has been found in the present study to inhibit the prostaglandin E productin by zymosan-stimulated rat peritoneal macrophages (RPM?). In contrast, the production of lysozyme by non-stimulated RPM?, as well as the expression of C_3b receptors on the macrophage plasma membrane, was markedly enhanced by the drug. All these results seem to be in accordance with the authors' speculation that the basic mechanisms underlying the clinical effect of the drug may well be due to, at least in part, its potentiating action on the immune system.
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Objective To evaluate of red blood cell as giving instruction in whole white blood cell immunological activity by new nature experimental system of hemaimmune reaction rood map. Methods Plasma 0.3ml were added to whole blood cells (including: red blood cell and white blood cells) or white blood cells 0.2ml, and incubated for 1h at 37℃. The content of IL-8 and IL-12 was determined by enzyme linked immunadsorbent assay (ELISA) method. The expression level of CD4, CD8, CD35 and CXCR4 on white blood cells was determined by method of Flow Cytometry. Results The content of IL-8 (5.96?4.26) and IL-12 (9.84?2.23) in whole blood and plasma nature group was significantly lower than that (13.59?3.69?B?pg~ -1 ?ml~ -1 ) and (15.09?9.86?B?pg~ -1 ?ml~ -1 ) in white blood cell and plasma isolation group (P
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Objective To study the changes in the expressions of CXCR4 of leucocytes as modulated by erythrocytes from cord blood. Methods 0.2ml suspension of whole blood cells (or 0.2ml suspension of leucocytes) was added to 0.3ml of auto plasma, and 0.2ml suspension of S180 (5?10~6/ml) was added as stimuli. The suspension was incubated at 37 ℃ for 1 hour. Normal saline instead of S180 was used as control The expression changes in CD35 on erythrocytes and CXCR4 on leucocytes were determined with flow cytometry assay. The data could be grouped under four heads: (1) cancer cells 0.2ml were added to whole blood cells 0.2ml and plasma 0.3ml; (2) NS 0.2ml were added to whole blood cells 0.2ml and plasma 0.3ml; (3) cancer cells 0.2ml were added to white blood cells 0.2ml and plasma 0.3ml; (4) NS 0.2ml were added to white blood cells and plasma 0.3ml. Results When activated by S180, the expression of CD35 on cord erythrocytes was decline not so low as that of adult′; while the expression of CXCR4 on cord leucocytes was increased not so high as that of adults′. When activated by S180, the expression of CD35 on adult erythrocytes was significantly decreased than that of group of not activating (40.21%?11.52% and 28.65%?8.08% respectively), while the expression of CXCR4 on adult erythrocytes was significantly increased (40.62%?17.89% and 64.18%?11.69% respectively, P
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Objective To investigate the role of CD35 on erythrocytes played in antigen activating immunological reaction of lymphocytes. Methods Suspensions of lymphocytes(1?10~6/ml)and erythrocytes (1?10~8/ml) were respectively separated from anticoaguted whole blood of healthy adults with the lymphocyte separation medium. Using inactivated ascites carcinoma cell S180(1?10~6/ml) as activation antigen and autologous plasma as reactive medium, the role of erythrocytes in regulating the anti-neoplasm immunological reaction of lymphocytes was appraised. The expression of CD25 on lymphocytes was detected and compared using flow cytometry assay with CD35 on erythrocytes blocked by monoclonal antibody or complements in plasma were destroyed by EDTA. Results The expression of CD25 on lymphocytes (18.22?4.27%) was significantly decreased when CD35 on erythrocytes was blocked (P