Optimization of the dose-effect relationship of N-ethyl-N-nitrocarbamide and cyclophosphamide in Pig-a gene mutation detection in rats / 中国比较医学杂志

Objective The effect of different doses of ethylnitrosourea(ENU)and cyclophosphamide(CP)on the loss rate of CD59 on peripheral blood erythrocytes was explored to optimize the detection method of Pig-a gene mutation. Methods According to the weight and loss rate of CD59 on peripheral blood erythrocytes,rats were divided into 4 groups:the control group,CP 40 mg/kg group,ENU 10 mg/kg group and ENU 40 mg/kg group(n=6). The control group was injected i.p. with PBS,other groups were injected i.p. with corresponding solutions. The body weight of rats on days 0,7,14,21, 28, 42 and 56 were recorded. At the same time, blood samples were collected and incubated with antibodies,and the loss rate of RBCCD59-was detected by flow cytometry. Results Compared with the control group, at different time points, the body weight and weight gain of ENU 10 mg/kg group and ENU 40 mg/kg group had no statistically significant difference(P > 0.05),while those in the CP 40 mg/kg group were significantly decreased(P <0.05). The loss rate of RBCCD59-was significantly increased in the CP 40 mg/kg group at 28,42 and 56 days, ENU 10 mg/kg group at 42 and 56 days,and ENU 40 mg/kg group at 7,14,21,28,42 and 56 days,(P < 0.05). The results showed a dose-response relationship. Conclusions Under the conditions of this Pig-a mutation detection method,ENU is superior to CP on raising loss rate of RBCCD59-,ENU 40 mg/kg is better than 10 mg/kg,and 28 days is suitable as the test period.

Role of PPARγ in exogenous protectin D1-induced reduction of endotoxin-induced acute lung injury in mice / 中华麻醉学杂志

Objective To evaluate the role of peroxisome proliferator-activated receptor gamma (PPARγ) in exogenous protectin D1 ( PD1)-induced reduction of endotoxin-induced acute lung injury (ALI) in mice. Methods Thirty-two clean-grade healthy male BABL∕C mice, weighing 20-25 g, aged 6-8 weeks, were divided into 4 groups (n＝8 each) using a random number table method: sham operation group (group S), ALI group, PD1 group and PPARγ antagonist GW9662 group. Mice underwent oral tra-cheal intubation, normal saline was instilled, and 1 h later normal saline was injected via the tail vein in group S. Mice underwent oral tracheal intubation, lipopolysaccharide (LPS) 3 mg∕kg was instilled, and 1 h later normal saline was injected via the tail vein in group ALI. Mice underwent oral tracheal intubation, LPS 3 mg∕kg was instilled, and 1 h later PD1 200 ng was injected via the tail vein in group PD1. Mice un-derwent oral tracheal intubation, LPS 3 mg∕kg was instilled, and 1 h later GW9662 1 mg∕kg and PD1 200 ng were injected via the tail vein in group GW9662. Mice were sacrificed at 24 h after intratracheal instilla-tion of LPS, the left lung was lavaged with phosphate buffer solution, and the broncho-alveolar lavage fluid (BALF) was collected for determination of neutrophil count and concentrations of interleukin-1beta ( IL-1β), tumor necrosis factor-alpha ( TNF-α) and IL-6 ( by enzyme-linked immunosorbent assay). Right lung tissues were obtained and cut into sections which were stained with haematoxylin and eosin and exam-ined with a light microscope for microscopic examination of the pathological changes which were scored (lung injury score) and for determination of the expression of PPARγ in lung tissues. Results Compared with group S, the neutrophil counts in BALF, concentrations of IL-1β, TNF-α and IL-6 and lung injury score were significantly increased, and the expression of PPARγ was down-regulated in group ALI ( P＜0. 01). Compared with group ALI, the neutrophil counts in BALF, concentrations of IL-1β, TNF-α and IL-6 and lung injury score were significantly decreased, and the expression of PPARγ was up-regulated in group PD1 (P＜0. 01). Compared with group PD1, the neutrophil counts in BALF, concentrations of IL-1β, TNF-α and IL-6 and lung injury score were significantly increased, and the expression of PPARγ was down-regulated in group GW9662 ( P＜0. 01). Conclusion PPARγ activation is involved in exogenous protectin D1-induced reduction of LPS-induced ALI in mice.

Diagnostic Significance of Detecting Peripheral Blood Granulocyte CD55/CD59 and FLAER in Anemia and PNH / 现代检验医学杂志

Objective To investigate the clinical significance of CD55,CD59 and Aeromonas hydrophila toxin variant (FLAER) in the diagnosis of anemia and paroxysmal nocturnal hemoglobinuria (PNH).Methods Collected 30 healthy controls,22 cases of PNH,33 cases of aplastic anemia (AA),37 cases of iron deficiency anemia (IDA),45 cases of megaloblastic anemia (MA),30 cases of hemolytic anemia (HA) and 31 cases of myelodysplastic syndrome (MDS) from January 2009 to March 2017,CD55,CD59 and FLAER negative cell ratio of peripheral blood neutrophil of them were detected by multipa rameter flow cytometry.Results The detection rates of FLAER in PNH,AA and MDS groups were higher than those of CD55 and CD59,but there was significant difference in AA (x2 =7.759,5.518,P=0.005,0.019＜0.05).The average CD55,CD59 and FLAER deletion rate in PNH and AA group were significantly higher than those in normal control group and other groups (t=2.163～17.890,P=0.000～0.038＜0.05).The number of FLAER in PNH group was higher than CD59 and CD59 was higher than CD55 with the statistically significant difference (t=2.503 ～ 6.308,P=0.000 ～0.016＜ 0.05).Conclusion CD55,CD59 and FLAER have important value in the diagnosis of PNH and differential diagnosis with other anemia diseases,and can also be used to detect the presence of MDS and AA in patients with PNH.FLAER outperforms CD59,CD59 outperforms CD55.

Effect of down-regulation of CD59 gene on apoptosis of acute T lymphocyte Jurkat cell lines / 中国免疫学杂志

Objective:To study the change of related molecular about apoptosis we reduce the expression of CD59 on acute T lymphocyte Jurkat cell lines .Methods: RNA interference (RNAi) was used to reduce the expression of CD59 gene by lentivirus,confocal was applyed to observe the transfection efficiency and the location of CD59 molecular then Real-time-PCR and Western blot were used to select the most effective group to do the rest experiment;Western blot was used to detect the change of expression about Bcl-2,Bax,Caspase-3 and Survivin;ELISA was used to investigate the expression of IL-3 and TNF-β.Results: Confocal observed each group′s transfection efficiency over 90%,CD59 molecules were mainly located in cell membrane;Real-time-PCR and Western blot showed group A had the best down-regulation efficiency;we defined RNAi-CD59-A as experimental group for subsequent experiments named RNAi-CD59;the experimental group can enhance the expression of Bax,caspase-3 (P<0.05),inhibit the expression of Bcl-2 and Survivin(P<0.05);ELISA showed that the expression of IL-3 in the down-regulation group increase(P<0.05),the expression of TNF-β decrease (P<0.05).Conclusion: Down-regulation CD59 can promote the expression of apoptosis molecular in acute leukemia Jurkat cell lines restrain the expression of proliferation molecular.

Changes of erythrocytes surface molecule CD35,CD58 and CD59 expression in recipients infected with cytomegalovirus after renal transplantation / 器官移植

Objective To investigate the change rules and its significance of erythrocytes surface molecule CD35 , CD58 and CD59 expression in recipients infected with cytomegalovirus (CMV)after renal transplantation. Methods Eighty-two recipients undergoing allogeneic renal transplantation were selected and divided into the negative (n=21 )and positive CMV groups (n=61 )based on the qualitative detection of CMV-pp65 antigen in peripheral blood. According to the results of CMV-pp65 (+)leucocyte count,all 61 patients in positive CMV group were further divided into low (n=55)and high active infection subgroups (n =6 ). Healthy adults were recruited into the normal control group (n =30 ). The expression levels of CMV-pp65 antigen,erythrocytes surface molecule CD35,CD58 and CD59 were measured by flow cytometry. Results Compared with normal control group,the expression levels of erythrocytes surface molecule CD35 , CD58 and CD59 in the positive CMV group were significantly down-regulated,and the CD35 and CD59 expression in the negative CMV group were considerably down-regulated (all P<0. 05 ). Compared with negative CMV group,the expression levels of CD58 and CD59 in the positive CMV group were significantly down-regulated (both P<0. 05 ). The expression levels of CD35 and CD59 in the high active infection subgroup were significantly lower than those in the low active infection subgroup (both P<0. 05 ). Conclusions The more severe active CMV infection after renal transplantation,the lower expression of erythrocytes surface molecule CD35,CD58 and CD59,hinting that red cell immune dysfunction is probably involved with active CMV infection.

Expression of CD46, CD55, and CD59 in colon cancer Expression of CD46, CD55, and CD59 in colon cancer / 中国肿瘤临床

Objective:To determine whether CD46, CD55, and CD59 are differentially expressed in neoplastic and adjacent nor-mal colon tissues and to investigate their influence on clinicopathologic variables. Methods:Immunohistochemistry (a modified two-step method) was used to detect the expression of CD46, CD55, and CD59 in a tissue microarray of 121 cases of colon cancer and corre-sponding adjacent non-tumor tissues with detailed clinical information, including gender, age, differentiation, TNM classification, tu-mor location, and tumor histotype. The colon carcinoma microarray was constructed from patients' samples obtained from the Depart-ment of Gastrointestinal Surgery of Xijing Hospital of the Fourth Military Medical University between October 2004 and June 2006. The correlation between expression and clinicopathologic features was analyzed. Results:The expression levels of CD46, CD55, and CD59 were significantly higher in colon cancer tissues compared with those in normal adjacent colon tissues (P0.05). The expression levels of CD55 and CD59 were correlated with the grade of colon cancer differentiation. Low levels of CD55 and CD59 were detected in cancer cells of highly differentiated cancer, whereas stronger staining for CD55 and CD59 was mainly observed in cancer cells of moderately and poorly differentiated colon cancer (P<0.05). In addition, the expression levels of CD55 and CD59 were higher in stages III and IV colon cancer than those in stages I and II according to TNM classification (P<0.05). Conclusion:CD46, CD55, and CD59 are up-regulated in colon cancer. Specifically, CD55 and CD59 are of clinical relevance to differentiation and TNM staging of colon cancer, and their expression might be closely related to clinical biological behaviors.

Roles of CD5+, CD19+, CD41a+, CD55+ and CD59+ in Chronic Immune Thrombocytopenic Purpura (ITP).

Aims: The aim of this study was to investigate of the roles of CD5+ and CD19+ on lymphocytes, CD5+ on B lymphocytes, CD41a+ on platelets and CD55+ and CD59+ on erythrocytes in platelet destruction; and evaluate them according to the patient response status to steroid therapy and platelet counts in chronic immune thrombocytopenic purpura (ITP). Study Design: This study included 20 chronic ITP patients and 20 healthy controls. We investigated the roles of CD5+ and CD19+ expression on lymphocytes, CD5+ expression on B lymphocytes, CD41a+ expression on platelets, and CD55+ and CD59+ expression on erythrocytes, as well as the platelet counts in healthy and chronic ITP patients. Additionally, these markers were evaluated according to the patient response status to steroid therapy and platelet counts. Place and Duration of Study: This study took place at the Department of Internal Medicine and Haematology, Meram Medical Faculty at Selçuk University in Turkey, between November, 2008 and July, 2009. Methodology: A total of 40 patients (26 women, 14 men, age range: 19-79 years) were studied. The study group included 20 chronic ITP patients (12 women and 8 men, age range: 19-78 years) and the control group included 20 healthy volunteers (14 women and 6 men, age range: 22-79 years). The platelet counts and expressions of CD5+ and CD19+ on lymphocytes, CD5+ on B lymphocytes, CD41a+ on platelets, and CD55+ and CD59+ on erythrocytes were analysed in the patients and control subjects. The chronic ITP patients were evaluated according to their requirements of treatment. Five patients whose platelet counts were above 50,000 mm–3 were observed without treatment. The other 15 patients whose platelet counts were under 50.000 mm–3 and had bleeding, or whose platelet counts were under 20,000 mm–3, were given methylprednisolone treatments (1 mg/kg/day orally). Three of the 15 patients discontinued treatment for various reasons. The twelve patients who continued the methylprednisolone treatment were divided into two subgroups according to their responder status of steroid treatment. The patients whose platelet counts slowly increased above 30,000 mm–3 within three months included the steroid treatment responder subgroups. The chronic ITP patients were also divided into two subgroups according to the severity of their thrombocytopenia. The limit of the platelet count was 30,000 mm–3 for severe thrombocytopenia. These parameters were analysed according to the response status of the steroid treatment and platelet counts. The platelet counts, and the expressions of these markers, were compared between the subgroups. Results: The level of CD5+ on B lymphocyte expression (2.19 ± 1.65) in peripheral blood lymphocytes was significantly higher in the immune thrombocytopenic purpura patients than in the controls (P = .05). The CD55+ + CD59+ expression on erythrocytes (98.03 ± 1.77) was significantly higher in the ITP patients than in the controls (P = .05). There was no significant relationship between the expression of CD5+, CD19+ or CD5+ on B lymphocytes, CD41a+ expression on platelets or CD55+ and CD59+ expression on erythrocytes, according to the response status to steroid therapy in the patient group (P > 0.05). Additionally, the patients were evaluated according to platelet counts, and there was a significantly positive correlation between the level of CD41a+ expression on the platelets and the platelet count (P = .05). Conclusion: The level of CD5+ on B lymphocytes was significantly higher in the ITP patients than in the controls. A relationship between CD55+ plus CD59+ expression on erythrocytes and immune destruction of platelets was not observed in the chronic ITP patients.

Estudo do SNP C1QA-A27GG e das proteínas reguladoras do complemento CD55 e CD59 como biomarcadores de resposta clínica do rituximabe no linfoma de grandes células B / [Study of SNP C1QA-A27GG and complement regulatory proteins CD55 and CD59 as biomarkers of clinical response to rituximab in lymphoma of large B cells]

A combinação do anti-CD20 rituximabe (R) e quimioterapia (CHOP-R) melhorou o desfecho dos pacientes com linfoma difuso de grandes células B (LDGCB), entretanto nem todos os pacientes respondem satisfatoriamente. Embora o mecanismo antitumoral do rituximabe in vivo não tenha sido claramente definido, acredita-se que uma das principais vias seja a citotoxicidade dependente de complemento (CDC). Um polimorfismo de nucleotídeo simples (SNP) no gene do componente do complemento Cq1 (C1QA-A276G) e a superexpressão das proteínas reguladoras do complemento (PRC) foram associados à resposta clínica de pacientes com linfomas B, entretanto estes biomarcadores não foram explorados em uma grande série de pacientes com LDGCB. Linhagens celulares derivadas de LNH-B foram empregadas para provar que a expressão de PRC contribui à inibição da CDC-R. Observamos também, que a resistência à CDC-R pode ser revertida por bloqueio funcional de CD55 e CD59 utilizando anticorpos neutralizantes. Uma coorte retrospectiva de 202 pacientes com LDGCB que receberam R-CHOP como primeira linha de tratamento, foi avaliada quanto: a expressão dos marcadores CD55 e CD59 por imunohistoquímica (IHQ) em material tumoral fixado e incluído em parafina, a distribuição dos genótipos C1QA-A276G determinados por PCR em tempo real e as características clínicas, resposta terapêutica e sobrevida. A taxa de sobrevida global (SG) e de sobrevida livre de progressão (SLP) da nossa coorte foi 69,8% e 66,3%, respectivamente...

The introduction of the anti-CD20 rituximab to the chemotherapy (CHOP) has significantly improved the treatment outcome of diffuse large B-Cell lymphoma (DLBCL) patients, however, not all patients respond satisfactorily. Even though the antitumor mechanism of rituximab in vivo is not clearly understood, it is thought that complement-dependent cytotoxicity (CDC-R) is one of the major pathways. A single-nucleotide polymorphism (SNP) in the component of complement C1q (C1QA-A276G) and overexpression of complement regulatory proteins (CRP) were associate to the clinical response of B-lymphomas patients, nevertheless, these markers have not been explored in a large series of patients with DLBCL. Cell lines from NHL-B were employed to prove that the expression of CRP contributes to inhibition of CDC-R. We also observed that resistance to CDC-R could be reversed by functional blockade of CD55 and CD59 using neutralizing antibodies. A retrospective cohort of 202 patients with DLBCL receiving R-CHOP as first-line treatment was evaluated for: the expression of CD55 and CD59 markers by immunohistochemistry (IHC) in fixed paraffin embedded tumor material, the distribution of genotypes C1QA-A276G determined by real time PCR and the clinical characteristics, response to therapy and survival. The rate of overall survival (OS) and progression-free survival (PFS) of our cohort was 69.8% and 66.3%, respectively. The application of IPI and IPI-R coefficients, successfully distinguish groups of patients with PFS (P <0.001) and OS (P <0.001)...

CD59 has a positive effect on LAT induced T lymphocytes activation / 中国免疫学杂志

Objective:To investigate the function of CD 59 in LAT induced T lymphocytes'proliferation and activation.Methods:Transfected LAT-GFP recombinant lentiviral vectors into Jurkat cells and established a fusion-protein stable express cell line ( Jurkat-GFP ).Junket-GFP cells were transfected with pSUPER-siCD59 plasmids by electroporetion or stimulated by anti-CD59 antibody.The cellular locations of CD 59 and LAT were observed under fluorescence microscope with the immunofluorescence cytochem -istry.The cells proliferation were measured by MTT assay.Furthermore,Western blot was used to detect the total and phosphorylation levels of several down-stream proteins after T cell activated .Results: Jurkat-GFP cells successfully transfected with pSUPER-siCD59 plasmids showed lower fluorescence staining.CD59 and LAT distributed uniformly on the cell surface before stimulated with anti-CD59 antibody and formed clusters once upon stimulation.Jurkat-GFP cells stimulated with anti-CD59 antibody showed a higher level of pro-liferation and protein phosphorylation ,compared with the others.Conclusion:CD59 contributed to LAT induced signaling transduction of T lymphocytes ,and stimulated CD59 molecule partly promoted T cell activation.

Generation of 1E8 Single Chain Fv-Fc Construct Against Human CD59

BACKGROUND: Therapeutic approaches using monoclonal antibodies (mAbs) against complement regulatory proteins (CRPs:i.e.,CD46,CD55 and CD59) have been reported for adjuvant cancer therapy. In this study, we generated a recombinant 1E8 single-chain anti-CD59 antibody (scFv-Fc) and tested anti-cancer effect.by using complement dependent cytotoxicity (CDC). METHODS: We isolated mRNA from 1E8 hybridoma cells and amplified the variable regions of the heavy chain (VH) and light chain (VL) genes using reverse-transcriptase polymerase chain reaction (RT-PCR). Using a linker, the amplified sequences for the heavy and light chains were each connected to the sequence for a single polypeptide chain that was designed to be expressed. The VL and VH fragments were cloned into the pOptiVEC-TOPO vector that contained the human CH2-CH3 fragment. Then, 293T cells were transfected with the 1E8 single-chain Fv-Fc (scFv-Fc) constructs. CD59 expression was evaluated in the prostate cancer cell lines using flow cytometry. The enhancement of CDC effect by mouse 1E8 and 1E8 scFv-Fc were evaluated using a cytotoxicity assay. RESULTS: The scFv-Fc constructs were expressed by the transfected 293T cells and secreted into the culture medium. The immunoreactivity of the secreted scFv-Fc construct was similar to that of the mouse 1E8 for CCRF-CEM cells. The molecular masses of 1E8 scFv-Fc were about 120 kDa and 55 kDa under reducing and non-reducing conditions, respectively. The DNA sequence of 1E8 scFv-Fc was obtained and presented. CD59 was highly expressed by the prostate cancer cell line. The recombinant 1E8 scFv-Fc mAb revealed significantly enhanced CDC effect similar with mouse 1E8 for prostate cancer cells. CONCLUSION: A 1E8 scFv-Fc construct for adjuvant cancer therapy was developed.

Anaemia & expression levels of CD35, CD55 & CD59 on red blood cells in Plasmodium falciparum malaria patients from India.

Background & objectives: Severe anaemia in Plasmodium falciparum (Pf) associated malaria is a leading cause of death despite low levels of parasitaemia. In an effort to understand the pathogenesis of anaemia we studied expression level of RBC complement regulatory proteins, CR1 (CD35), CD55 and CD59 with haemoglobin status in a group of malaria cases from Assam, Goa and Chennai, and in healthy controls. Methods: Flowcytometry was used to study expression of CR1, CD55 and CD59 in 50 Pf cases and 30 normal healthy volunteers. Giemsa stained thick and thin blood films were used for microscopic detection and identification of malarial parasites and parasite count. Results: No correlation was found between degree of expression of RBC surface receptors CR1, CD55 and CD59 with haemoglobin level. However, expression of CD55 was less in malaria cases than in healthy controls. Interpretation & conclusions: The present findings indicate that malaria infection changes the expression profile of complement regulatory protein CD55 irrespective of severity status of anaemia. Further studies are needed to explore the pathophysiology of anaemia in malaria cases in Assam where expression of RBC complement receptors appears to be low even in normal healthy population.

Frequency of paroxysmal nocturnal hemoglobinuria in patients attended in Belém, Pará, Brazil

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).

Study of the expression of complement regulatory protein CD59 on CD4+ T cells and its relationship with apoptosis in HIV infected patients / 中国基层医药

Objective To explore CD59 expression on CD4+T cells in HIV infected patients and its relationship with apoptosis.Methods 12 HIV infected patients and 10 healthy donors were performed in this study.The PBMC(peripheral blood monocyte)were collected and cell surface cytokine were stained,and then were evaluated with the BD FACSCanto flow cytometry.The expression of CD59 on T lymphocyte subsets were analyzed by FACSDiva software,and the apoptosis rate of CD59+CD4+T cells and CD59-CD4+T cells in every group was analyzed respectively,then the results were compared between groups.Results Compared with healthy donor,the expression of CD59 on T cells in HIV infected patients was significantly hisher(t=5.198,P＜0.01),and the apoptosis rate of CD59+CD4+T cells had significantly higher(t=5.968,P＜0.01).The apoptosis rate of CD59-CD4+T cells was no difference between two groups (t=0.1353,P=0.8577).Condnsion HIV infection increase CD59 expression on CD4+T cells,and CD59+CD4+T cells were prone to apoptosis.

Effect of infection with Toxoplasma gondii on the complement regulatory proteins in the mouse placenta / 中华微生物学和免疫学杂志

Objective To investigate the expression of complement regulatory proteins on placentas of pregnant C57BL/6 mice infected with Toxoplasma gondii in order to explore the molecular immunological mechanism for abnormal pregnancy induced by T. gondii infection. Methods Twenty-four pregnant C57BL/6 mice were randomly divided into two groups equally. The infection group was intraperitoneally injected with 200 of living T, gondii RH strain tachyzoites on the 8th day of gestation, and the normal group of mice was injected with physiological saline. All mice were killed on day 14 after gestation and placentas were collected. The expression levels of Crry, GPI-DAF and CD59a mRNA were analyzed by real-time quantitative PCR, and the positive rates of Crry and GPI-DAF were measured with flow cytometry. Results The died fetus rates of infected group and control were 80. 95% and 4. 41% , respectively. The infected group was significantly higher than of that the control group (P＜0.01). The expression levels of Crry, GPT-DAF and CD59a mRNA in the infected and control group were 0.786 ±0. 199, 0.594 ±0.096, 0.880 ±0. 179 and 0.550 ±0.077, 0.221 ±0.074, 0.591 ± 0.075 , respectively, and the difference of three kind of complement regulation proteins between two groups was all significant (P＜0.01). The positive percentages of Crry and GPI-DAF cells of infected and control group were (10. 03 ± 2. 11) % , (2.95 ±1.04)% and (3. 15 ± 1. 32) % , (0. 66 ±0. 26) % , respectively, and the difference of the two kind complement regulation proteins between two groups was also significant ( P ＜ 0. 01). Conclusion The expression level of mouse placental complement regulatory proteins was increased after infection with T. gondii, and then immunological microenvironment at the fetomaternal interface was destroyed. It may be one of important immunological mechanism for abnormal pregnancy induced by T. gondii infection.

Flow cytometric analysis of erythrocytes in paroxysmal nocturnal hemoglobinuria reveals superiority of CD59 as a diagnostic marker compared to CD55.

Context: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by complement-mediated hemolysis due to reduced expression of glycosyl phosphatidylinositol-anchored complement deactivating proteins such as CD55 and CD59 on RBC. Flow cytometric analysis of CD55 and CD59 expression by RBC is a reliable tool for the diagnosis of PNH. Aims: Detection and quantification of PNH clone and comparison of the relative role of CD55 and CD59 expression by RBC in the diagnosis of PNH. Materials and Methods: Flow cytometric analysis of RBC was performed in blood samples of 239 patients by direct immunofluorescence using monoclonal anti-CD55 and anti-CD59 antibodies. CD55 and CD59 expressions by RBC were compared in 54 cases in which PNH clones were detected. Results: Out of 54 cases, 85% and 72% revealed CD59 and CD55 negative populations, respectively. Various combinations of type II and III erythrocytes could be identified in all cases having CD59 deficient RBC. In contrast, distinct populations of CD55-deficient RBC were seen in only 33% cases. In the remaining (67%) cases, CD55 negative RBC caused sloping of the ascending limb of the histogram resulting in difficulties in interpretation. Fifteen percent cases had false CD55-deficient RBC and in 23% cases anti-CD55 antibody failed to identify PNH clones which were detected by CD59. Conclusion: CD59 is a better marker for the diagnosis of PNH. Although CD55 negativity supported the diagnosis of PNH in cases with CD59-deficient RBC, its role as an independent diagnostic marker for PNH is questionable due to its lower sensitivity and specificity.

The role of CD55/CD59 complement regulatory proteins on peripheral blood cells of systemic lupus erythematosus patients / O papel das proteínas reguladoras do complemento CD55/CD59 em células de sangue periférico de pacientes com lúpus eritematoso sistêmico

CD55 e CD59 são proteínas de membrana ancoradas por glicosilfosfatidilinositol que apresentam propriedades reguladoras da ativação da cascata do complemento. Essa regulação ocorre através da inibição da C3 convertase pelo CD55 e prevenção da etapa final de polimerização do complexo de ataque à membrana pelo CD59. Deficiência na expressão dessas proteínas pode estar associada a uma maior ativação do sistema complemento, inclusive do complexo de ataque à membrana, levando à morte celular. Pacientes com lúpus eritematoso sistêmico, com anemia hemolítica e linfopenia, parecem apresentar uma deficiência adquirida de CD55 e CD59. Contudo, os mecanismos que modulam essa diminuída expressão continuam desconhecidos e o seu impacto nas manifestações do lúpus eritematoso sistêmico precisa ser mais bem estudado.

CD55 and CD59 are glycosylphosphatidylinositol-anchored proteins with regulatory properties on the activating cascades of the complement system. This regulation occurs through inhibition of the C3-convertase formation by CD55, and prevention of the terminal polymerization of the membrane attack complex by CD59. Deficiency in the expression of these proteins can be associated with increased susceptibility to complement-mediated cell death. Systemic lupus erythematosus patients with hemolytic anemia and lymphopenia seem to have an acquired deficiency of CD55 and CD59 proteins. However, the mechanisms involved in this deficiency and its impact on the clinical manifestation of SLE needs to be further investigated.

Clone, prokaryotic expression and preparation of polyclonal antibody of hCD59 / 细胞与分子免疫学杂志

AIM: To construct full length hCD59 eukaryotic and extracellular domain of hCD59 (hsCD59) prokaryotic expression vectors and prepare polyclonal antibody of hCD59. METHODS: cDNA fragments encoding hCD59 and hsCD59 were amplified from human PBMCs by RT-PCR and cloned into the eukaryotic vector pVAX-1 and prokaryotic vector pGEX-KG, respectively. The recombinant fusion protein GST-hsCD59 was expressed in E. coil BL21 induced by IPTG. Then the fusion protein was purified and identified. Polyclonal antibody against hCD59 was prepared by immunizing rabbit with pVAX-1-hCD59 and boosting with GSThsCD59 fusion protein, and the titer was identified. RESULTS: The recombinant eukaryotic vector pVAX-1-hCD59 and prokaryotic vector pGEX-KG-hsCD59 were successfully constructed. The GST-hsCD59 fusion protein was over-expressed in E. coli BL21 and the relative molecular mass (M~r) of the expression product was identical with predicted size. The titer of the anti-hCD59 serum was 1:3 200. CONCLUSION: We got the recombinant eukaryotic vector pVAX-1-hCD59, prokaryotic vector pGEX-KG-hsCD59 and rabbit anti-hCD59 polyclonal antibody successfully. These work would be helpful for the further study of the biological function of human CD59.

Study of xenograft in nude mice on ovarian cancer cell targeted CD59 by siRNA / 中国免疫学杂志

Objective:To investigate the inhibitiory effect of CD59-siRNA on CD59 gene expression and the growth of xenografted ovarian cancer in vivo,and to explore the role of CD59 in tumor immune escape.Methods:A2780 cells transfected with siRNA plasmid (T group),A2780 cells transfected with blank plasmid (V group) and the wild A2780 cells (C group) were subcutaneously injected into nude mice,respectively.The inhibitiory effect of CD59-siRNA on tumor growth and CD59 expression was evaluated by tumor growth curves and in situ hybridization (ISH) and immunohistochemistry (IHC).Results:The tumor growth curves demonstrated that the growth of cells transfected with siRNA plasmid was significantly inhibited (P<0.05).ISH and IHC showed that the expression of CD59 mRNA and CD59 protein were decreased significantly compared with other groups (P<0.05).Conclusion:Studies of in vivo experiment demonstrate that CD59-siRNA significantly inhibites the expression of CD59,increases the sensitivity of A2780 cells to the complement attack and inhibites the tumor growth.The results may further indicate the role of CD59 in tumor immune escape.

Expression of membrane inhibitor of reactive lysis in non-small cell lung carcinoma and its clinical significance / 中国基层医药

Objective To survey membrane inhibitor of reactive lysis(MIRL) expression in non-small cell lung careinoma(NSCLC) and to analyze the relationship between MIRL expression and clinical staging, adjuvant chemotherapy and disease-free survial. Methods The expression of MIRL in 8 adjacent tissues and 36 NSCLC sam-pies were determined by immunohistochemistry. Furthermore, the relationship between MIRL expression and clinical stage ,adjuvant chemotherapy and disease-free survival was assayed by follow-up. Results Among 36 samples of non-small-cell lung cancer,there were 10(27.8%) samples expressing MIRE. Out of 18 samples of squamous carcinoma, 4(22.2%) expressed MIRL,while 6(37.5%) expressed it in 16 samples of adenocarcinoma,there was no statistical significance between them(P>0.05). There were no expression in 2 samples of large cell carcinoma. There was no correlation between MIRL expression and disease-free survival(P>0.05). MIRL positive expression rate in patients with preoperational adjuvant chemotherapy was significantly lower than that of those without preoperational adjuvant chemotherapy(P<0.05). Conclusions There is great percentage of MIRE expression in NSCLC. Our present study suggests that the immunological inhibition of MIRL should be blocked when monoclonal antibody is used in the treat-merit of NSCLC.

Role of Complement Regulatory Proteins in the Survival of Murine Allo-transplanted Sertoli Cells

Sertoli cells (SC) are known to contain immunoprotective properties, which allow them to survive as allografts without the use of immunosuppressive drugs. Experiments were designed to determine which factors are related to prolonged survival of allogeneic SC. Balb/c derived Sertoli (TM4) and colon cancer (CT-26) cell lines were implanted beneath the kidney capsule of non-immunosuppressed C57BL/6 mice and compared their survival as allografts. Compared to TM4 graft, which survived more than 7 days after transplantation, CT-26 showed massive infiltration of polymorphonuclear cells, necrosis and enlargement of draining lymph nodes. Cultured cell lines showed no differences in their expression patterns of FasL, TGF beta1, clusterin and two complement regulatory proteins (CRP, i.e., membrane cofactor protein, MCP; decay accelerating factor, DAF), but protectin (CD59), another member of CRP was expressed only on TM4. These results suggest that CD59 and unknown factors may contribute to the prolonged survival of SC in non-immunoprivileged sites.