| B7-1 molecule is an important co-stimulatory molecule expressed on the surface of antigen-presenting cell(APC),and its combination to the receptor CD28 can generate costimulatory signals,which is essential to the primary immune response.Without this costimulatory signal,T cells will enter to a state of anergy,tolerance,even apoptosis.The hyperreaction of B7/CD28 signal is closely related to the occurrence of autoimmune diseases.Systemic lupus erythematosus(SLE)is a complex autoimmune disease characterized by T cell and B cell activation,the presence of autoantibodies against self-antigens,and immune complex(IC)deposition within multiple systems.This disease affects virtually any organ in the body.A wide spectmrum of severity,ranging from relatively mild manifestations(e.g.,skin rash or arthritis)to seriously disabling or even life-threatening complications,such as lupus nephritis,neuropsychiatric disorders and other major organs,could be involved.Some researches had revealed that the onset of SLE was associated with T lymph cells activities.Investigations revealed that the B7-1 and B7-2 melocules on SLE patients' cells were overexpression.Moreover,the autoimmune diseases were closely related to the over activity of B7/CD28 signal.Blockade the B7/CD28 signal could induce specific immune tolerance,which was beneficial for autoimmune diseases' prevention and treatment.In the present study,the non-human primate cynomolgus monkeys were established as SLE animal model induced by Pristane(2,6,10,14-tetramethylpentadecane)in part from the closest genetic relationship to human beings(98.5%homology).The Lupus-specific biomarkers and manifestations over a 246-day period were observed at multi-level.To visualize and quantify kidney function in real time,contrast-enhanced ultrasound was used.The indicative biomarkers and manifestations fulfilled major diagnosis criteria according to the "Criteria of Lupus" of the American College of Rheumatology.Based upon the model,we apply the mouse anti-human B7-1 monoclonal antibody to the model in order to evaluate its reverse effect and molecular mechanism by blocking B7/CD28 signal pathway,which could provide some experimental and theoretical data,even find a biologic intervention which is specific,efficient and harmfulless for SLE.Part I Establishment and Characterization of a PRISTANE-inducedlupus-associated model in the nonhuman primate cynomolgus monkeyObject:To establish and Characterization of a Pristane-induced lupus-associated model by the "Criteria of Lupus" of the American College of Rheumatologyin the nonhuman primate cynomolgus monkey.Methods:6 femal cynomolgus monkey divided randomLy into 2 groups.In modelgroup,3 monkeys were given Pristane via intraperitoneal injection(3.5 mL/kg,volume/weight)on day 1,the same dose of Pr:istane was given by intraperitoneal injection again on day 120 in this study.In control group,the remaining 3 monkeys were given a sham injection of saline solution(0.9%NaCl).All the clinical changes were observed and recorded daily.The urinary protein and hematuria concentrations were determined by colorimetric analyses using an Albustix assay.Blood serum was separated and the blood urea nitrogen(BUN)level was examined by blood biochemical examination and blood clotting tests.Autoantibodies in the sera were detected using the indirect immunofluorescence to measure the levels of antinuclear antibody(ANA)and anti-double-stranded DNA(anti-dsDNA)antibody at a 1:10?1:100 dilution.On day 210(week 30),all the surviving animals received CEUS examinations using the VINNO M80 Ultrasound system to visualize and quantify kidney function in real time.Splenocytes from 2 different groups were prepared then the immune cells markers were analyzed by flow cytometry.The renal tissue was stained with hematoxylin-eosin(H&E)via light microscopy.The severity of glomerular,tubular,and vascular lesions was scored in a blinded manner.Frozen kidney sections were incubated with anti-human IgG to detect the immune complexes.Renal specimens was under scanning electron microscopy(SEM)analysis.Results:1.Clinical manifestations were observed from week 28 in the experimental group including cutaneous manifestations,i.e.raised red patchesacross the cheeks and nose,arthritis,neurologic disorders and other symptoms.The extent of lesions increased gradually.2.Proteinuria and hematuria in the experimental group monkeys were detected at day 180(week 26;positive ratio 1/3 with 30 mg/dL)and day 210(positive ratio 2/3 with 300 mg/dL).Both the urinary protein and hematuria concentration gradually increased until the 225th day(up to 1000 mg/dL and 0.2 mg/dL).3.The blood urea nitrogen(BUN)level increased with time from week 32,The BUN level of Pristane-treated monkey 1 was significantly higher at the study endpoint.Blood clotting tests showed the prothrombin time(PT)and the international normalized ratio(INR)in the experimental group were markedLy increased(P<0.05).4.ANA and ds-DNA antibodies,in one Pristane-induced monkey's serum,could be detected beginning at the 27th week.At 35-week,ANA and ds-DNA antibodies were present in all of the experimental group monkeys,with cytoplasmic ANA staining patterns.5.The contrast agent perfused rapidly from the renal sinus to the renal capsule in the control group,A similar contrast agent enhancement pattern was observed in the Pristane-treated group.However,the speed of renal cortical enhancement was markedly lower than that of the control group,with reduced enhancement intensity and prolonged contrast agent disappearance.The TICs in the control group displayed a rapid ascent,reached peak intensity quickly and then slowed gradually.In comparison,the modelgroup reached a lower peak intensity,with a slow increase and decrease,and displayed an extended peak time.The indexes of AUC,a,and PI showed significant differences(compared with the control group,P<0.05,P<0.05,P<0.01,respectively).6.The expressional percentage of CD 11 bn and CD3tCD4+significantly increased in the model group(P<0.05),CD19+ and CD19+CD21+were significantly up-regulated in the model group(P<0.01).7.The Pristane-treated group monkeys presented white tough punctiforms or lumps on the mesentery and abdominal wall.8.The model monkeys exhibitecd diffuse proliferative glomerulonephritis affecting more than 50%of glomeruli,with enlarged glomerular volume from moderate to severe.The glomerular capillaries were significantly congested with an influx of leukocytes.The renal injury fulfilled lupus nephritis class IV based on the 1997 revised criteria for the classification of SLE under the auspices of the World Health Organization.9.Lupus cells accompanied by a large number of inflammatory cells along with bronchioles in pulmonary of model monkey No.3.10.Immunofluorescence revealed immune complex deposits in the renal glomeruli of the experimental monkeys.11.SEM showed electron-dense deposits in the renal glomerular endothelial cells of all the experimental monkeys.Conclusion:we successfully induced lupus associated model with systemic lupus syndrome.This primate model can be a valuable translational model for fturther pathogenesis and symptomology studies and for exploring therapeutic candidates.Part II Preparation and study on the function of mouse anti-humanB7-1 monoclonal antibody in vitroObject:To obtain B7-1 monoclonal antibody,analyze its recognition of membrane B7-1 molecule and its biological function in vitro.Methods:Hybridoma of secretion anti-human B7-1(clone No.4E5,established by our lab)were intraperitoneal injected into Balb/c-nude mice,the ascites were harvested and purified with ProteinG affinity chromatography method.The Ig isotype of purified monoclonal antibody was identified with the rapid test paper.Flow cytometry assay was used to test the ability of recognization the membranes B7-1 molecule on different cell strains.The effects of blockade costimulatory signals was detected by MTT assay in vitro.Results:The positive rate of ascites formation in mice was about 82%.Ascites harvest was 7.4 mL from each BALB/c-nude mouse on average.The purified monoclonal antibody from ascites was about 2.1 mg/mL.The monoclonal antibody could specifically recognize membrane B7-1 on cells of Daudi?929/B7-1?spleen cells of monkey but not L929/MOCK.the positive rate of recognization were 95%?92%?58.2%?1.14%,respectively.In addition,this monoclonal antibody could inhibit the growth and proliferation of L929/B7-1 cells and PBTCs via blocking the B7-1 costimulatory signals.Conclusions:Mouse anti-human B7-1 monoclonal antibody which could efficiently block the B7-1 costimulatory signals was successfully obtained.Part III Study on the reverse effect and molecular mechanism of blockadeB7/CD28 signal pathway in lupus disease in cynomolgus monkeyObject:To evaluate the reverse effect of pathological injury and its molecular mechanism by blockade B7/CD28 signal pathway in the lupus-associate cynomolgus monkey model,as well as to explore the biological targeted therapy strategy for lupus disease.Methods:6 femal cynomolgus monkeys were given Pristane via intraperitoneal injection(3.5 mL/kg,volume/weight)on day 1,then same dose of Pristane was given by intraperitoneal injection again on day 120.All the monkeys were divided randomly into 2 groups,namely 4E5 monoclone antibody-treated group(n=3)and mouse IgG treated group(n=3).The 4E5 monoclone antibody was given to 4E5-treated group on day287(41stweek)?289(41Stweek)?292(41Stweek)?295(41ndweek)?302(43thweek),317(45thweek)?347(49thweek)?377(53rdweek)by superficial vein injection.Mean while,the IgG antibody was sham-injection in IgG treated group.All the survivals were sacrificed on day 510(week 72).The skin lesions were scored and the survival rate was caculated.The urrinary protein and hematuria concentrations were determined by colorimetric analyses using an Albustix assay.Blood serum was separated and the blood urea nitrogen(BUN)and creatine levels were examined by blood biochemical examinations.Autoantibodies in sera were detected using the indirect immunofluorescence assay to measure the titer of antinuclear antibody(ANA)and anti-double-stranded DNA(anti-dsDNA)at a 1:10?1:3000 dilution.On week-40 and week-55,all the survival animals received CEUS examinations using the VINNO M80 ultrasound system to visualize and quantify kidney function in real time.The indexes of time-intense curse(TIC)were comparied,including slope rate of ascending curve,slope rate of descending curve,area under curve,AUC,peak intensity,time to peak,etc.PBMCs from 2 groups were separated by Ficoll density gradient centrifiugation and the Tim-1/Tim-3 on PBMCs were detected.Splenocytes from 2 different groups were prepared then the immune cells markers were analyzed by flow cytometry.The spleen were measured after dissection from 2 groups.The renal tissue was stained with hematoxylin-eosin(H&E)via light microscopy.The severity of glomerular,tubular,and vascular lesions was scored in a blinded manner.Frozen kidney sections were incubated with anti-human IgG to detect the immune complexes.Renal specimens was under scanning electron microscopy(SEM)analysis.Blood serum from week 1,5,9 and week 45,50,55 were separated,then the IL-4 and IFN-y levels were investigated by human IL-4 ELISA ready-set-go and human IFN-ELISA ready-set-go separately.Results:l.the clinical manifestations were observed beginning from 26th week,then the antibodies were treated on each group.The survival time of 4E5 treated group was markedly longer than IgG treated group(68.6713.51 weeks vs 50.67±5.86 weeks,P=0.01).2.Skin lesions in IgG treated group were deteriorated with time.Contrarily,4E5 treated group was alleviated from 50th week.The scores of 2 group showed significant difference on week 55(P=0.03).3,From 0?28 week,the BUN level fluctuated in the normal range.It went up with time until 43`aweek then moved down in 4E5-treated group.On contrary,the BUN level was always in high state.Similarly,the creatine level went up from 33rd week in both group,the 4E5-treated group moved down from week-41 after 4E5 antibody intervened.On contrary,the creatine level was still in high state during all the period.4.Urinary protein was observed from 25th week,positive rate 83%(5/6)in both group,30~100 mg/dL_Hematuria positive ratel7%(1/6)with 0.2 mg/dL.5.ANA in monkey's serum could be detected beginning at the 29th week with titer 1:500-1:1000.At week-45,all the monkeys showed positive results in ANA indirect-immunofluorescence assay with titer 1:1500~1:3000.From 55th week,the ANA titer deacreased to 1:500-1:1000 in 4E5-treated group,however,the one in IgG-treated group inceased(P<0.05).On the other hand,the anti ds-DNA antibodies in both group were detected from 29th week with titer 1:10 of positive rate 4/6.The titer of anti ds-DNA antibodies increased with 1:10?1:100 at week 45.Then the anti ds-DNA antibodies in 4E5-treated monkeys5 serum decreased into 1:10?1:50 at 55th week,it showed lower than IgG treated group.6.The real-time perfusions in 4E5 treated group(40thveek,55thweek)and IgG treated group(40thweek,55thweek)by CEUS examinations showed kidney perfusion function in both groups impaired,the 4E5 treated group relieved after anti-B7-1 antibody intervened,but peak intense decreased comparing with the 40-week.The parameters of TICs in IgG treated group exhibited a slowly ascend and slowly descend with lower peak.AUC(P<0.05)?MTT(P<0.05)and PI(P<0.01)of IgG treated group were much lower than 4E5-treated group.7.The expression of immuno molecular markers(Membranous molecules of microphages,monocytes,DCs,B lymph cells and T lymph cells)showed down-regulated in 4E5-treated group,especially,the expression of CDllb+and CD3+,CD4+,CD3+CD4+showed significant lower than IgG treated group(P<0.05).Meanwhile,the CD4+Tim-+and CD4+Tim-3+ also decreased in PBMCs from 4E5-treated group,but CD4+ up-regulated.8.Gross anatomy assay presented enlargement spleen in IgG treated group with scattering points on the splenic surface.9.The IgG treated group monkeys exhibited diffuse proliferative glomerulonephritis affecting more than 50%of glomeruli,with enlarged glomerular volume from moderate to severe,scoring 2.66±0.47.The glomerular capillaries were significantly congested with an influx of leukocytes with score 1.677±0.47.However,the glomerulonephritis alliviated markedly in 4E5-treated group with score 1.3310.47(comparing the IgG-treated group,P<0.05)as well as the glomerular capillaries repaired with score 0.67J10.47(comparing the IgG-treated group,P>0.05).10.Immunofluorescence revealed immune complex deposits in the renal glomeruli of the 4E5-treated monkeys decreased.11.SEM showed electron-dense deposits in the renal glomerular endothelial cells of 4E5-treated monkeys were much better than Ig G group monkeys.12.The IFN-y levels in all the serum samples from 2 groups ascended highly significantly(P<0.01)in each group during the whole period,but IFN-y level of 4E5-treated group presented markedly lower than IgG group at the end satage(5.22±0.50 vs 6.71 ±0.82 pg/mL,P<0.05).The IL-4 level fluctuated during whole experimental period.After the antibodies intervention,the IL-4 level down-regulated a little in 4E5-treated group(comparing with Ig G group,P>0.05).Contusion:The anti-B7-1 antibody(4E5)could blockade the B7/CD28 signal,which could reduce the activation of immune cells and even relieve/reverse the pathological damage of cynomolgus monkey models.The blockade of B7/CD28 signal could be a promising biological target to intervent the onset of SLE,and anti-B7-1 antibody could be a one of candidate for therapeutic strategy.Part IV Preliminary study on preclinical pharmacokinetics of antiB7-1 monoclonal antibody(4E5)Object:To study anti B7-1 monoclonal antibody(4E5)pharmacokinetics by single intravenous in beagle dogs and cynomolgus monkeys,seperatly,for further developmentvof anti B7-1 monoclonal antibody(4E5)application.Methods:Standard curve was established by double sandwich ELISA method using mouse IgG,then recovery rate was calculated.Beagle dogs(n=3)were given anti B7-1 monoclonal antibody(4E5)by a single intravenous injection with 1.6mg/kg,cynomolgus monkeys(n=3)were given with 2.5 mg/kg,seperatly.Blood samples were collected at different time and serum concentrations were measured.Pharmacokinetic parameters were calculated by WinNolin software under non compartment analysis.Results:2 hours later after administration,the peak level reached 10.5610.71 ?g/mL in Beagle dogs with clearance(CL)1.86±0.23 mL/h/kg,MRTlast 121.43±3.85 h,Vss 232.66±23.19 mL/kg.The half-time t1/2 was 64.77±6.01 h.However,5 min later after administration,the peak level reached 193.562± 11.68 ?g/mL in cynomolgus monkeys with CL 0.18±0.016 mL/h/kg.MRT was 53.6366±1.45 h,Vss 9.73±1.17 mL/kg.The half-time t1/2 was 33.76±0.87 h,which was much shorter than that in beagle dogs.Contusions:The metabolism of mouse B7-1 monoclonal antibody(clone No.4E5)in the cynomolgus monkey was much faster than in beagle dogs.The half-time and the average residence time were both shorter incynomolgus monkeys.The pharmacokinetic investigations could provide reference datas for further application and durg development. |
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