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Objective:On the basis of the use of chemical methods to establish mouse model of lupus nephritis and its biological identification , we investigate the reverse effect of pathological lesions of B 7-1 human-mouse chimeric antibody blockade against B7/D28 signaling pathway in mice with lupus nephritis model.Methods:Pristane was injected intraperitoneally to 6-week-old female C57BL/6J mice at dose of 0.5 ml per mouse in one go,and urine protein,ANA and renal pathological changes were detected on a monthly basis.Mice whose urine protein content reached ++and ANA fluorescence intensity reached ++were randomly devided into three groups ,five each.Antibody intervention group was sequentially injected with B 7-1-mouse chimeric antibody by orbital venous , positive control group was injected with immunosuppressant CTX , negative control group was injected with isotype control IgG.Urine protein and ANA were also detected on a monthly basis.Mice were sacrificed three months after intervention was executed.Kidney was used for H&E dying , IC detection and electric microscope observation.Results: After four-month Pristane induction , urine protein content of 80%mice reached +-+++,meanwhile,serum ANA fluorescence intensity reached ++-+++.Glomerulonephritis infiltrating cells were observed Mice with urine protein and ANA , glomerular inflammatory cell infiltration , tubular epithelial cell degeneration visible edema ,vascular congestion significantly ,fibrosis.After antibody intervention ,urine protein content in antibody intervention group gradually reduced from ++-+++to ±-+++,ANA ++-+++to +-++,and were significantly different from that in the negative control group ( P<0.01 ).Analysis of kidney H&E dying showed that antibody glomerular infiltration of inflammatory cells in the intervention group and tubular congestion and other symptoms were improved significantly.Immunofluorescence staining indicated that fluorescence intensity of IC was significantly reduced in the antibody intervention group.Electron dense deposits reduction and glomerular basement membrane uniformity were observed in antibody intervention group by electric microscope when compared with the negative control group.Conclusion:B7-1 antibodies could downregulate immune response through inhibiting B 7-1/CD28 signaling pathway , reducing the production of autoantibodies and reversing pathological damage caused by autoimmune response .
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The immune effect of CD4~+CD28~-T cells on Graves'ophthalmopathy(GO)was investigated.The expressions of interferon-γ(IFN-γ),interleukin-2(IL-2),and IL-4 in CD4~+ CD28~-T ceils were assayed by flow cytometry in GO patients,Graves'disease(GD)patients without ophthalmopathy,and healthy control subjects.The results showed that the percentage of CD4~+CD28~-T cells significantly increased in GO patients(P<0.05),with increased IFN-γ expression(P<0.05)and decreased IL-2 expression(P<0.05).These changes were closely correlated with clinical activity score(P<0.05).There were no significant differences in IL-4 expression among three groups.The resuh suggests that CIM~+ CD28~- T cells which hishly secrete IFN-γare related to the pathological lesion of GO.
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Objective To study the expression of co-stimulatory molecules, GL50-ICOS, in thyroid tissue of patients with Graves' disease (GD) and to explore their relationship with the immune pathogenesis of GD.Methods RT-PCR, Western blot, immunohistochemistry were applied to detect the expression of GL50-ICOS in thyroid of GD. Thyrocytes were cultured in the absence or presence of pro-inflammatory cytokines. The expression of GL50 on thyroid follicular cells (TFC) was further measured by flow cytometry. Results (1) In GD patients,the percentage of CD4+ CD28- T cells was significantly increased as compared with the control healthy individuals. The expression of co-stimulatory molecule ICOS was up-regulated. (2) The mRNA level of ICOS was significantly increased in GD patients than that in nontoxic goiter(NTG) patients(P＜0.01). (3)Compared with NTG control group, the GL50 protein expression was much higher in thyroid tissues of GD patients (P ＜0.01). (4)The results of immunohistochemistry showed that GL50 expression was observed in all GD thyroid tissues, while no expression of GL50 was detected in NTG thyroid tissues(P＜0. 01). (5) The expression of GL50on primary cultured thyroid follicular cells was significantly increased under the stimulatation of pro-inflammatory cytokines in vitro. Conclusion GL50-ICOS is expressed abnormally in thyroid tissue of patients with GD.
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<p><b>OBJECTIVE</b>To explore the effect of recombinant human soluble CD(40) ligand (rhsCD(40)L) and CD(40)L cDNA transfected cell (CD(40)L-TC) on the behavior of malignant B lymphocytes, and investigate the possibility of using rhsCD(40)L as a new bio-factor in tumor immunotherapy.</p><p><b>METHOD</b>rhsCD(40)L and CD(40)L-TC were obtained by gene recombinant techniques. Multiple myeloma cell lines, XG2, XG7, U266 and 8226, B-lymphoma cell lines, Raji and Daudi were selected to detect responses to rhsCD(40)L and CD(40)L-TC stimulation. Cell growth curve, cell cycle, early apoptosis as well as membrane surface molecules on these cell lines were analyzed.</p><p><b>RESULTS</b>(1) The expression levels of CD(40) molecule on malignant B lymphocytes showed heterogeneity. High level of CD(40) on XG2, moderate on 8266, Raji, and Daudi, and no expression on U266 and XG7 were detected. The rhsCD(40)L stimulation gave rise to a typical homo-type cell aggregation of XG2 and Daudi. Meanwhile, at least 10 to 20 of CD(40)(+) XG2 or CD(40)(+) Daudi cells were found adherent to one pre-treat ed CD(40)L-TC. (2) Co-incubation with rhsCD(40)L (5 micro g/ml), or CD(40)L-TC (tumor cell: CD(40) = 5:1) resulted in a significant inhibition of in vitro cell growth of XG2, Raji and Daudi, with G(1)-phase arrest for XG2 and G(2)-phase for Raji and Daudi. These two kinds of CD(40) stimulators induced XG2, Raji and Daudi cells to apoptosis in vitro. The apoptotic rate for XG2 was 23.3% (rhsCD(40)L) and 18.8% (CD(40)L-TC), for Daudi 14.2% and 15.9%, and for Raji 11.6% and 8.9% respectively. (3) Phenotype analysis showed that CD(95) expression levels were significantly up-regulated on XG2, Raji and Daudi after stimulation with rhsCD(40)L or CD(40)L-TC, and CD(80) and CD(18) expression levels on Raji were respectively enhanced and decreased.</p><p><b>CONCLUSION</b>The abilities to directly inhibit XG2, Daudi and Raji cell proliferation, to induce themapoptosis, as well as to up-regulate immune co-stimulator molecule CD(80) expression on Raji cells would make rhsCD(40)L a potential bio-factor for tumor immuno-therapy.</p>
Subject(s)
Humans , B-Lymphocytes , Metabolism , Pathology , CD40 Antigens , Metabolism , CD40 Ligand , Genetics , Metabolism , Pharmacology , Cell Division , Coculture Techniques , DNA, Complementary , Genetics , Lymphoma, B-Cell , Metabolism , Pathology , Recombinant Proteins , Pharmacology , Time Factors , Transfection , Tumor Cells, CulturedABSTRACT
Objective To study CD40 expression on melanocytes induced by IFN-?and its significance. Methods CD40 expression was detected by flow cytometry. The capacity of melanocytes to stimulate T lymphocytes was evaluated by mixed ly mphocyte reaction and the supernatant cytokine levels were determined by ELISA. ResultsHuman melanocytes(MC) cultured in vitro expressed low but detectable CD40 surf ace protein. The surface expression of CD40 was markedly up-regulated by stimul ation with interferon(IFN)-?with different concentrations for 24 hours,48 hour s and 72 hours, respectively. The expression of CD40 was correlated with IFN-? levels after 24 hour incubation. MC underwent a morphologic change with an incre ased capacity to stimulate allogenic lymphocytes to proliferate after IFN-?sti mulation. Optimal enhancement of stimulating index(SI) was observed at an IFN-?concentration of 300 IU/ml after 72 hour treatment. Meanwhile concentrations o f interleukin-12 but not interleukin-8 or 10 were obviously increased in the s upernatants of cultured MC. Furthermore, ligation of CD40 via soluble CD40 ligan d(SCD40L) could enhance CD80 and ICAM-1 expression, which could be blocked by s pecific monoclonal antibody to CD40L. Conclusions Since CD40-CD40L is a pair of important and special costimulating signal, it is of great value to elucidate t he fact that CD40 is functionally expressed on MC, thus for a better understandi ng of MC′s role in cellular immune responses. MC might activate cytotoxic T lym phocyte directly and not via CD4 positive lymphocyte.
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AIM: To prepare the efficient tumor-DC vaccines, dendritic cells(DC) derived from 6-8 weeks Balb/c mice bone marrow progenitor cells were pulsed by apoptotic SP2/0 tumor cells and induced maturation by SP2/0 tumor lysates supernatants. Then SP2/0 tumor burdening Balb/c mice were immunized by the tumor-DC vaccines to observe the therapeutic effects in vivo .METHODS: Immature DC were derived by recombinant murine GM-CSF and IL-4, then were pulsed by SP2/0 apoptotic cells. Tumor-DC vaccines were stimulated by LPS and SP2/0 tumor lysates supernatants prepared by four cycles repetitive freezing and thawing, respectively. -thymidine incorporation test and standard 4h [ 51 Cr] release assay were used to detect the proliferation and activation of cytotoxic T lymphocytes (CTL) stimulated by DC in vitro . (4-5)?10 5 DC were immunized in the right inguen of SP2/0 tumor burdening Balb/c mice and most mice received three cycles immunization every two weeks. Changes of the tumor and mice life-spans were recorded. RESULTS: In vitro proliferation and activation of CTL induced by the tumor-DC vaccines of tumor lysates supernatants or LPS stimulation group were more powerful than other groups ( P
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Objective:To prepare the monoclonal antibody(mAb) against human B7 1 and analyse its biological characteristics.Methods:The B lymphocytes hybridization technique was applied by using XG7 B7 cell,a multiple myeloma(MM) cell line transfected with human B7 1 gene,as immunogen;the specificity and the antigen binding activity of mAbs were identified by flow cytometry and Western blot analysis;its biological effects on human PBTC and human B lymphoma cell line were examined by 3H TdR incroporation and annexin V satining.Results:Four mouse anti human B7 1(B7 1) hybridoma(1F11,3H8,6H2,7B10) were obtained.They secrete continuosly and steadily specific anti human B7 1(B7 1) mAb and their subclasses belong to IgG1 and IgM respectively;three of four mAbs could inhibit the proliferation of response cells(the human peripheral blood T lymphocytes),stimulated by costimulatory molecule B7 1.Furthermore,it was found that these mAbs induced the apoptosis of human B lymphoma cell line,Raji,which express naturally human B7 1 molecule,by using annexin V staining analysis after 24 hours of mAb treatment.Conclusion:Sucessefully obtained four mouse anti human B7 1 functional monoclonal antibodies,which have a potential value in anti allogenetic graft rejection and in the therapeutic approach of B lymphoma.
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Objective:To prepare functional monoclonal antibodies against human 4-1BBL molecule and analysis of their biological characteristics.Methods:Female BALB/c mice of 6-8 weeks old were immunized with 4-1BBL transfectant (L929/4-1BBL) as immunogen.The spleen B cells of the mice were fused with sp2/0 and hybridoma cells were screened with 4-1BBL transfectant (L929/4-1BBL) by FCM.The biological characteristics of antibody were investigated by rapid isotyping analysis,karyotype analysis,competitive inhibition test etc.Furthermore,the growth of monocytes in vitro was determined by cell number counting and the cytokine concentration in the supernatants was assayed by ELISA.Results:One hybridoma cell line named 3E7 was obtained,which had the property of secreting anti-human 4-1BBL monoclonal antibody continuously and steadily.This mAb specifically recognized human 4-1BBL molecules.The experimental results manifested that mAb 3E7 could effectively enhance the growth of monocytes and the high level IL-6 and TNF-? secretion.Conclusion:One hybridoma cell line which can secret a functional mouse anti-human 4-1BBL mAb has been developed successfully.This mAb can specifically recognize human 4-1BBL and regulate growth and functions of monocytes in vitro.
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Objective:To establish and identify humanized-SCID mouse model(hu-SCID).Methods:SCID mouse was treated by CTX to inhibit the hemocytopoiesis. With successive 4-day injection, human peripheral blood mononuclear cells(PBMC) were engrafted into SCID mouse through intraperitoneal injection. After 4, 8 and 12 weeks of engraftment, peripheral blood, spleen and liver tissues of engrafted SCID mouse were harvested. Human CD3~+, CD19~+ cells in peripheral blood were analyzed by inflorescence microscopy and FCM, human CD3~+, CD19~+ cells in spleen and liver tissues were observed by immune histochemistry, and human IgG level in SCID mouse serum was measured by ELISA.Results:After engraftment of 4, 8 and 12 weeks, human CD3~+, CD19~+ cells in SCID peripheral blood were identified by inflorescence microscopy and the percents were 31% and 10% respectively by FCM analysis. And these cells could be evidenced after 12 weeks later. Through immune histochemistry human CD3~+、CD19~+ cells were detected in mouse spleen but not in liver tissue. Furthermore the titer of human IgG in mouse serum was 390,1 100 and 1 040 ?g/ml at each time point respectively.Conclusion:Our experimental results demonstrated that a bona fide humanized SCID model was established.