Pathology of antibody-mediated rejection in renal allograft / 器官移植

Antibody-mediated rejection (AMR), also known as humoral rejection, is an immune injury caused by rejection involved with multiple humoral immune effectors, such as antibodies and complements, etc. AMR plays a pivotal role in hyperacute, acute and chronic rejection. In this article, the basic definition of AMR, the research progress and major achievements on AMR pathology according to Banff classification on allograft pathology (Banff classification), and main pathological characteristics of AMR in renal allograft were reviewed, aiming to provide reference for accurate diagnosis and timely treatment of AMR, and guarantee the long-term survival of renal graft and recipients.

Expression and activity identification of bispecific antibody targeting PD-1/CD19 / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To construct and purify the recombinant bispecific antibody (BsAb) targeting PD-1 and CD19 and evaluate its activity. Methods: With pCAR1 plasmid as the vector, the eukaryotic expression vector of anti-PD-1/CD19 BsAb was constructed by molecular cloning technology, and then transfected into mammalian cell line CHO-S by PEI reagent for transiently expressing antibody. The BsAb was purified by Affinity chromatography and then identified by SDS-PAGE and WB. The blocking activity of BsAb on PD-1/PD-L1 in vitro was detected by Luciferase reporter gene assay. The activity of antibody (BsAb)-dependent cell (PBMC)-mediated cytotoxicity (ADCC) in vitro was evaluated by lactate dehydrogenase (LDH) cytotoxicity assay. Results: The double plasmid eukaryotic expression vector pCAR1-19X3 was successfully constructed, and anti-PD-1/CD19 BsAb was successfully expressed in CHO-S cells, named pCAR1-19X3-TY. pCAR1-19X3-TY could effectively block the binding of PD-1 to its ligand PD-L1 in vitro, and the EC50 based on the dose-response curve was 0.306 μg/ml. ADCC results showed that pCAR1-19X3-TY could mediate the cytotoxicity of PBMC against Raji cells, and the curve showed a linear upward trend; when the effect/target ratio was 50∶1, the target cell lysis rate of pCAR1-19X3-TY was (38.9±0.3)%, which was not significantly different from that of the positive treatment group (46.7±4.9)% (P>0.05), but significantly higher than that of the negative control group (1.2±0.1)% (P<0.05). Conclusion: The recombinant anti-PD-1/CD19 BsAb can effectively block the binding of PD-1 and PD-L1 and activate PBMC mediated cytotoxicity against Raji cells. pCAR1-19X3-TY has the potential application value in the treatment of B-cell malignant tumor.

CD 3/KIF20A bispecific antibody mediated anti-tumor effects of T lymphocytes on KIF20A positive PANC1 cells / 中华胰腺病杂志

Objective:To investigate the induction of specific T lymphocyte by bispecific monoclonal antibody in pancreatic cancer and its killing effects on KIF20A positive pancreatic cancer PANC1 cell line.Methods:CD 3/KIF20A bispecific monoclonal antibody was prepared and concentrated by chemical cross-linking method and purified by Sephrose-25 gel chromatography. Peripheral blood samples of healthy volunteers were collected, and monocytes were isolated using lymphocyte separation solution, and then cultured as dendritic cells (DC) and T cells respectively, and then co-cultured as DC-T cells. Meanwhile vitamin C was used to treat DC-T cells (vcDC-T cells). The levels of IFN-γ, IL-2, IL-4 and IL-12 in the supernatants and T cell subsets were detected by flow cytometry. About 1×10 5 T cells, DC-T cells, and vcDC-T cells with 10, 50, 100 and 300 ng CD 3/KIF20A antibody loaded were cocultured with PANC1 cells (20∶1) for 2, 6 and 10 hours to determine the highest killing rate dosage of CD 3/KIF20A antibody loaded cells. DC-T cells and DC-T cells, vcDC-T cells loaded with the highest killing rate dosage of CD 3/KIF20A antibody were cocultured with PANC1 cells (20∶1) for 2, 6, 8, 10 and 12 hours. The aggregation effect of effector cells on target cells was observed under inverted microscope, the killing rate of tumor cells was detected by LDH method. Results:The molecular weight of CD 3/KIF20A antibody was 130 000 measured and validated by SDS gel electrophoresis. The ratio of CD 8+ CD 28+ and CD40L subsets of vcDC-T cells was increased [(47.6±15.8)% vs (38.2±7.6)%, (52.1±4.9)% vs (44.7±3.2)% ] compared with that of DC-T cells, the ratio of negative regulatory cells (Treg) was decreased [(4.3±0.8)% vs (8.3±1.1)%]; the release of IL-2, IFN-γ and IL-12 was increased [(201.2±17.3) ng/L, (163.4±13.1)ng/L, (303.3±22.6)ng/L vs 221.8±17.6)ng/L, (190.4±11.7)ng/L vs (80.3±8.6)ng/L]. All the differences were statistically significant ( P<0.01). 100 ng CD 3/KIF20A loaded T cells were observed under microscope, which obviously targeted KIF20A + pancreatic cancer PANC1 cells and had a strongest killing power. At the killing cells to targeting cells ratio of 20∶1 with 4-hour coculture, the killing rate of CD 3/KIF20A-vcDC-T cells on PANC1 cells was (88.6±2.6)%, which was significantly higher than (68.4±3.4)% and (39.2±2.1)% in the CD3/KIF20A-DC-T group and (39.2±2.1)% in the DC-T group, increasing by 20% and at lease 45%, respectively. Conclusions:DC-T cells loaded with CD 3/KIF20A antibody can significantly increase the killing rate of KIF20A positive pancreatic cancer PANC1 cells, and vitamin C intervention can further enhance the killing ability of antibody loaded T cells.

Development of a Novel Optimization and Validation Method Based on DOE for the ADCC Potency of Anti-PD-1/PD-L1 Monoclonal Antibodies Using Reporter Genes / 中国药学杂志

OBJECTIVE: To develop a novel optimization and validation method based on design of experiment(DOE) for the antibody-dependent cell-mediated cytotoxicity (ADCC) potency of anti-programmed cell death 1 and anti-programmed cell death-ligand 1 (PD-1/PD-L1) monoclonal antibodies using reporter genes. METHODS: Jurkat-hFcγRIIIa-NFAT transgenic cell line was used as effector cells, 293FT-PD-1 cell line and CHO-PD-L1 cell line were used as target cells, respectively. The ADCC potency for anti-PD-1/PD-L1 monoclonal antibodies was detected with Luciferase detection system (BrightGloTM Luciferase Assay system), then the method was optimized and validated based on DOE. RESULTS: The anti-PD-1/PD-L1 monoclonal antibodies showed a dose-response relationship and the determination result complied with the following four-parameter equation: y=(A-D)/+D. The method was optimized and the testing parameters were determined as follows: the working concentration of anti-PD-1 monoclonal antibody was 10 000 ng•mL-1 to 4.833 ng•mL-1 and that of anti-PD-L1 was 2 000 ng•mL-1 to 0.488 ng•mL-1, the ratio of effector cells and target cells for anti-PD-1/PD-L1 monoclonal antibodies were 6:1 and 3:1, and the induction time for both of these antibodies was 20 h. The method possessed good specificity. The recovery rate test samples in the four different dilution groups were determined for 3 times, and the results showed that the relative potencies of anti-PD-1 monoclonal antibody were (51.74±2.22)%, (77.12±3.14)%, (118.71±2.83)% and (156.20±12.99)%, and the recoveries of which were (103.49±4.44)%, (102.83±4.19)%, (94.96±2.26)% and (104.14±8.66)%, respectively. While as for anti-PD-L1 monoclonal antibody, the relative potencies were (54.32±4.75)%, (75.24±4.25)%, (127.40±2.43)%, (156.82±3.27)% and the recoveries were (108.64±9.51)%, (100.33±5.67)%, (101.92±1.94)% and (104.55±2.18)%, respectively. The RSDs of the above results were all less than 10%. CONCLUSION: A novel optimization and validation method based on DOE for detecting ADCC potency of anti-PD-1/PD-L1 mAb is successfully developed. This detecting method based on reporter gene shows high specificity, good reproducibility and high accuracy, and might be used in the evaluation of ADCC potency of anti-PD-1/PD-L1 mAb.

Intracellular cytokine staining (ICS) for detection of NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) / 中华微生物学和免疫学杂志

Objective To analyze the possibility of using intracellular cytokine staining (ICS) to evaluate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and to detect the changes in ADCC activity among patients with chronic HIV and/or HCV infection. Methods Flow cytometry was per-formed to determine the percentages of NK cells and the expression of NK cell receptors. ImageStreamX MarkⅡ system was used to identify the expression of CD3, CD56, CD16 and CD32 on CD56brightNK and CD56dimNK subsets. Degranulation process and cytokine production in NK cells were detected using an anti-gen-antibody complex model of P815/Ab in combination with ICS. Differences in NK cell-mediated ADCC were evaluated among patients infected with HIV and/or HIV and healthy subjects by flow cytometry. Re-sults The percentages of CD107a+and IFN-γ+NK cells were positively correlated with the decrease of mean fluorescence intensity (MFI) of CD16. ICS assay revealed a positive correlation between the secretion of CD107a and IFN-γ by NK cells. CD16 was highly expressed in CD56dimNK cells. The ADCC mediated by CD56dimNK cells was stronger than that mediated by CD56brightNK cells. The rate of target cell lysis detected by rapid fluorescence assay was positively correlated with the percentage of CD107a+/IFN-γ+NK cells meas-ured by ICS. NK cell-mediated ADCC was suppressed in patients with chronic HIV and/or HCV infection. Conclusion This study suggests that ICS assay could be used to evaluate NK cell-mediated ADCC. It also reveals that NK cell-mediated ADCC is suppressed in patients with chronic HIV and/or HCV infection.

Understanding the biology of HER3 receptor as a therapeutic target in human cancer

HER3 belongs to the human epidermal growth factor receptor (HER) family which also includes HER1/EGFR/erbB1, HER2/erbB2, and HER4/erbB4. As a unique member of the HER family, HER3 lacks or has little intrinsic tyrosine kinase activity. It frequently co-expresses and forms heterodimers with other receptor tyrosine kinases (RTKs) in cancer cells to activate oncogenic signaling, especially the PI-3K/Akt pathway and Src kinase. Elevated expression of HER3 has been observed in a wide variety of human cancers and associates with a worse survival in cancer patients with solid tumors. Studies on the underlying mechanism implicate HER3 expression as a major cause of treatment failure in cancer therapy. Activation of HER3 signaling has also been shown to promote cancer metastasis. These data strongly support the notion that therapeutic inactivation of HER3 and/or its downstream signaling is required to overcome treatment resistance and improve the outcomes of cancer patients.

The development of HIV vaccines targeting gp41 membrane-proximal external region (MPER): challenges and prospects

A human immunodeficiency virus type-1 (HIV-1) vaccine which is able to effectively prevent infection would be the most powerful method of extinguishing pandemic of the acquired immunodeficiency syndrome (AIDS). Yet, achieving such vaccine remains great challenges. The membrane-proximal external region (MPER) is a highly conserved region of the envelope glycoprotein (Env) gp41 subunit near the viral envelope surface, and it plays a key role in membrane fusion. It is also the target of some reported broadly neutralizing antibodies (bNAbs). Thus, MPER is deemed to be one of the most attractive vaccine targets. However, no one can induce these bNAbs by immunization with immunogens containing the MPER sequence(s). The few attempts at developing a vaccine have only resulted in the induction of neutralizing antibodies with quite low potency and limited breadth. Thus far, vaccine failure can be attributed to various characteristics of MPER, such as those involving structure and immunology; therefore, we will focus on these and review the recent progress in the field from the following perspectives: (1) MPER structure and its role in membrane fusion, (2) the epitopes and neutralization mechanisms of MPER-specific bNAbs, as well as the limitations in eliciting neutralizing antibodies, and (3) different strategies for MPER vaccine design and current harvests.

The Evaluation of Biological Activity of Anti-Rh (D) Antibodies / 中国医师杂志

Objective To evaluate the biological activity of anti-Rh(D) antibodies in vitro. Methods The titres of plasma Anti-Rh (D) antibodies in the RH(-) patients uncorrectly infused RH(+) blood were detected by various methods. The anti-D antibodies were evaluated for their capacity to sensitize erythrocytes, trigger monocyte and K-cell mediated antibody-dependent cellular cytotoxicity (ADCC), mediate binding to monocyte and lymphocyte Fc gamma R, and stimulate phagocytosis by monocytes. The synergistic effect of anti-D antibodies in promoting hemolysis was detected by complement fixation test (CFT). Results The titre of plasma anti-D antibodies measured by micro-column gel indirect anti-globulin technique(MGIAT) was the highest compared with the other detection methods. When red cells were sensitized with anti-D antibodies, the binding and phagocytosis of red cells by monocytes and the lysis of red cells by monocytes or lymphocytes were great, and the hemolysis of red cells by alexin was great too. Conclusion Anti-Rh (D) antibodies had great biological activity in vitro, and MGIAT had the highest sensitivity for detecting anti-Rh(D) antibodies.

Fcr Receptor and Mac-1 Expression and Functional Differentiation of HL-60 Cells by All-trans Retinoic Acid

PURPOSE: During differentiation of HL-60 cells by all-trans retinoic acid(ATRA), we analyzed the expression of Fcr receptors and Mac-1 by molecules of equivalent soluble fluorochromes(MESF) and functional studies. METHODS: HL-60 cells were induced to differentiate by adding 1micrometer ATRA. On the 4th and 7th day after stimulation as well as before stimulation, the cells were analyzed for phenotypic and functional differentiation. Phenotypic analysis was performed by flow cytometry after staining the cells with PE-conjugated anti-human CD64(FcrRI), CD32(FcrRII), CD16(FcrRIII), CD11b, CD18. The measured fluorescent intensity was transformed into MESF. Phagocytic activity was measured by flow cytometry after incubation of the cells with fluorochrome-conjugated beads. Respiratory burst was measured by chemiluminescence assay. ADCC was measured by hemoglobin release assay. Opsonophagocytic activity was measured by fungicidal assay. Correlation between MESF of FcrR and Mac-1 and function of HL-60 was measured. RESULTS: Percent positive cells and MESF of CD11b and FcrRI increased on the 4th day and decreased on the 7th day. Percent positive cells of CD18 was 99% regardless of differentiation. But MESF of CD18 was increased on the 4th day and decreased on the 7th day. Percent positive cells of FcrRII were above 90% regardless of differentiation. MESF of FcrRII showed no significant change. FcrRIII expression was not induced. Phagocytic activity of HL-60 cells was increased twofold. Chemiluminescence of HL-60 cells was increased up to 60-fold on the 7th day. ADCC of HL-60 cells was incerased up to 2.5-fold on the 7th day. Opsonophagocytic activity increased twice on the 4th day. ADCC and opsonophagocytic activity correlates with the expression of CD11b/CD18 and FcrRII. CONCLUSION: Differentiation of HL-60 cells with ATRA induces several functional maturations until 7 days with expression of FcrR and Mac-1.

Functional Differentiation of HL-60 Cells by Dimethylsulfoxide and Phorbol 12-myristate 13-acetate

PURPOSE: During hematopoietic differentiation of HL-60 cells by DMSO and PMA, we demonstrated functional changes of HL-60 cells-phagocytic activity, respiratory burst, antibody-dependent cell-mediated cytotoxicity(ADCC) and opsonophagocytic activity. METHODS: HL-60 cells(ATCC CCL-240), cultured in RPMI 1640 and supplemented with 10% FBS, were induced to differentiate by adding 1.0% DMSO or 16nM PMA. On the 4th and 7th day after stimulation as well as before stimulation, the cells were analyzed for functional differentiation. Phagocytic activity was measured by flow cytometry after incubation of the cells with fluorochrome-conjugated beads. Respiratory burst was measured by chemiluminescence assay. ADCC was measured by hemoglobin release assay. Opsonophagocytic activity was measured by fungicidal assay using Candida albicans. RESULTS: Phagocytic activity of HL-60 cells was not increased by differentiation with DMSO. But PMA induced increase of phagocytic activity on 7th day. Respiratory burst studied by chemiluminescence was increased up to 4-fold on 7th day by DMSO. PMA induced increase upto 3-fold on 4th day. ADCC was increased upto 3-fold on 4th day by DMSO, but PMA induced little increase in ADCC. Opsonophagocytic activity was increased upto 3-fold on 4th day by DMSO or PMA. On differentiation with DMSO, respiratory burst correlates with FcgammaRI and FcgammaRII. ADCC and opsonophagocytic activity correlate with CD11b/CD18. On differentiation with PMA, phagocytic activity correlates with FcgammaRII. Respiratory burst correlates with CD11b. ADCC and opsonophagocytic activity of HL-60 cells correlate with CD18. CONCLUSION: Treatment of HL-60 cells with DMSO or PMA induces functional maturation differently. Phagocytic activity, respiratory burst, ADCC and opsonophagocytic activity of HL-60 cells correlated with expresson of FcgammaR and Mac-1.

Antibody-dependent Cell-mediated Cytotoxitity as a Prognostic Indicator in the Medical Treatment of Graves' Disease / 대한내분비학회지

BACKGROUND: The several forms of treatment of Graves disease-thyroidectomy, antithyroid drugs and radioiodide therapy-are in wide use now. But which therapy is best is a matter of debate. Some authors reported that in patients who underwent thyroidectomy, higher titers of serum antimicrosomal antibody were associated with 1) higher formation rates of germinal centers, 2) more lymphocyte infiltration in the thyroid tissue, 3) higher incidence of hypothyroidism, and 4) lower incidence of recurrence. We were interested in the relationship of thyroid autoantibody titers, ADCC(antibody-dependent cell-mediated cytotoxicity) activity and the clinical response to antithyroid medication. METHODS: We measured ADCC activities from patients in Graves disease(n-48), Hashimoto thyroiditis(n=17) and normal control(n=9). The patients of Graves disease were followed up for more than 1 year, and they were grouped into A(n=17, well responsed group to antithyroid medication) and B(n=31, poorly responsed group). We examined ADCC activities of patients' sera by chromium release assay. RESULTS: 1) Mean age of patients with Graves disease was 34.4210.4 years and 15 patients were male(31%). 2) Results of thyroid function tests of the Graves' patients were T 585.9 +/- 255.3 ng/dL, T4 21.3 +/- 12.2 mg/dL, TSH 0.11 +/- 0.06mIU/mL. Concentrations of antimicrosomal antibody, antithyroglobulin antibody and thyrotropin binding inhibitory immunoglobulin were 1279.1 +/- 1486.7 IU/mL, 488.1 +/- 751.1 IU/mL, and 38.5 +/- 33.4U/L respectively. 3) There was no significant difference between levels of thyroid hormones or concentrations of thyroid autoantibodies and ADCC activities in graves patients. 4) The ADCC activity of the Graves patient group(24.49%) was significantly higher than that of the normal control group(3.76%), and significantly lower than that of the Hashimotos thyroiditis group(36.34%). 5) There was no significant difference in ADCC activity between group A(18.24 +/- 13.44%) and B(27.91 +20.02%). CONCLUSION: From this results, we suggested that ADCC activity seems to be no value as a prognostic factor in predicting the response to antithyroid drugs in Graves disease patients. But, further studies, larger number of patients and long-term follow up, are needed.

The Effect of Prostaglandin and its Inhibitor on the Antibody - dependent Cellular Cytotoxicity Against Human Squamous Cell Carcinoma of the Head and Neck / 대한면역학회지

The effects of chimeric monoclonal antibodies (cMAbs), prostaglandin E, (PGE,), and indomethacin (INDO) on antibody-dependent cellular cytotoxicity (ADCC) against human squamous cell carcinoma of head and neck (SCCHN) cell line were examined. Using the PCI-50 SCCHN cell line as target and normal human peripheral blood mononuclear cells as effector, ADCC was enhanced by the treatment of cMAbs (1.25 p,g/ml), but was inhibited by exogenous PGE (5 X 10' M). The effects of cMAb and PGE were dose-dependent. Maximal suppression of activity occured when PGE was present during the entire 4-hr 'Cr-release assay period, whereas pretreatment of effector cells with PGE had minimal inhibitory effect after washing. These results indicate that decreased ADCC seen with SCCHN targets treated with PGE is related to post-binding events, such as binding of effector and target cells. Pre-treatment of effector cells with INDO (1 ug/ml) resulted in restoration of NK activity which was inhibited by PGE. Our in vitro results suggest that INDO can increase tumor cell killing by the reversal of the suppression for many imrnune functions by PGE.

Correlation of Natural Killer(NK) Cell Activity,Antibody Dependent Cellular Cytotoxicity(ADCC), and Serum Zinc Level in Behçet's Disease

BACKGROUND: Zinc which is widely used to treat Behcet's disease, is known to be an important modulator in various aspects of immunity including cell mediated immunity (CMI). CMI is suspected of playing a major role in the pathogenesis of Behçet's disease. OBJECTIVE: This study was done to clarify the relationship of CMI and zinc in Behçet's disease. METHODS: Serum zinc level, NK cell activity, and ADCC were measured in 83 patients with Behçet's diseade. The results were analyzed using multiple regression analysis. RESULTS: ADCC and serum zinc level were found to be two significant variables that affect NK cell activity positively and negatively, respectively. CONCLUSION: Serum zinc is presumed to exert inhibitory effect on NK cell activity but does not affect ADCC in Behçet's disease patients.

Effect of Pregnancy on the Immune Response against Trichinella spiralis Infection in Mice / 中国寄生虫学与寄生虫病杂志

0.05).Serum absorbance value of progesterone injected virgin mice(0.299) was significantly higher than that of no-injectedvirgin mice(0.191)(t=2.955,P0.05).Conclusion Pregnancy has a synergetic effect on immune response of mice against T.spiralis infection,which may be related with the increased level of serum anti-Trichinella antibody and enhanced ability of sera in mediating the death of pre-encapsulated larvae in ADCC.

In Vitro Study of Nonspecific Cellular Immunity in Rheumatoid Arthritis

Nonspecific immune parameters such as natural killer(NK) activity, antibody-dependent cellular cytotoxicity(ADCC), production of leukocyte migration inhibitory factor(LlF) and levels of immune complex(IC) were assessed in 47 patients with rheumatoid arthritis (RA) 20 with degenerative arthritis (DA) and 40 healthy controls. Peripheral blood (PB) as well as synovial fluid (SF) were collected from both RA and DA patients before treatment. Mononuclear cell suspensions and sera were prepared and submitted for the in vitro tests; 4-hr chromium-release assays using human K562 and mouse L1210 cells as targets for NK and ADCC assays respectively, 2-step agarose assay for LIF and platelet aggregation test for IC. Results revealed that 1) LIF activity of PB lymphocytes (PBL) from both RA and DA patients showed a significant (P < 0.05) decrease as compared with that from healthy controls. 2) PB-NK activity from RA patients showed an insignificant decrease as compared with that from DA or healthy controls. However, mononuclear cells isolated from SF (SFL) of RA patients exhibited significantly(P < 0.02) lower NK activity than PBL from the same patients. 3) In ADCC assays with PBL no significant differencies were observed among the 3 groups. 4) Higher titers of IC were detected in both PB and SF from RA patients than DA, and a negative correlation was found between serum IC levels and PB-NK activity. These data are discussed in light of previous reports, and a hypothesis regarding a decreased nonspecific cell-mediated immunity in conjunction with an increased humoral immune response, particularly in local sites, is proposed as one of the mechanisms underlying the pathogenesis of RA.

NK AND ADCC CYTOTOXICITY ACTIVITY IN PATIENTS WITH BLADDER CANCER AFTER SURGERY AND THEIR REACTION TO HuIFN-? REGULATION IN VITRO / 第二军医大学学报

0.05), and the mean ADCC activity was 43.0?21.44% (P 0.05) .The NK activity in 15/24(62.5%)cases with low NK and ADCC activity in 4/25(16.0%)cases with low ADCC were enhanced to normal level by HuIFN- ?.The results suggest that HuIFN-? enhanced the NK activity more efficiently than the ADCC activity in bladder cancer patients after surgery.