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Objective:To prepare a 68Ga labeled probe targeting integrin alpha M(CD11b) receptor, namely 68Ga-1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid-Glycine-Arginine-Glutamate-Arginine-Glutamate-polyethylene glycol 11-1, 2, 4, 5-terazine/CD11b antibody-F(ab′) 2-trans-cyclooctene ( 68Ga-NOTA-Polypeptide-PEG 11-Tz/anti-CD11b-F(ab′) 2-TCO), and to explore its feasibility as a molecular probe for CD11b receptor through microPET imaging. Methods:Immunofluorescence was used to detect the expression of CD11b on the surface of RAW264.7 cell. CD11b specific monoclonal antibody (M1/70) was conjugated with TCO, and anti-CD11b-F(ab′) 2-TCO fragment was obtained. The ligand NOTA-Polypeptide-PEG 11-Tz was labeled with 68Ga, and its specific activity and radiochemical purity were detected. Pre-targeted cell binding experiment was conducted to evaluate the binding ability of molecular probe. CT26 colon cancer bearing mouse models were established, and then pre-targeted biodistribution and imaging experiments were performed. Immunohistochemical experiment was used to verify the expression of CD11b receptor in tumor. The one-way analysis of variance was used to compare the data. Results:The results of immunofluorescence demonstrated CD11b receptor was highly expressed on the surface of RAW264.7 cell. Anti-CD11b-F(ab′) 2-TCO fragment was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 68Ga-NOTA-Polypeptide-PEG 11-Tz was successfully synthesized, with the labeling efficiency of 94.6%. The specific activity was 7.0-7.4 MBq/μg, and the radiochemical purity was higher than 95%. Pre-targeted cell binding experiment confirmed that the molecular probe bound to the CD11b receptor. The biodistribution and imaging experiments showed that the kidney radioactivity uptake was high at pre-targeted 4, 12 and 24 h intervals, which proved that probe was excreted through the urinary system. In addition, molecular probe had higher radioactive uptake at the tumor site, with the tumor/muscle ratios of 9.23±1.45, 12.53±1.36 and 10.74±1.11 ( F=848.8, P<0.05). When the radioligand was injected 1 h after the pre-positioned 12 h interval, the images contrast was the best, with the standardized uptake value (SUV) in tumor and muscle of 0.67±0.12, 0.09±0.04, respectively. Immunohistochemistry verified the highly expression of CD11b receptor in tumor. Conclusions:The pre-targeted molecular probe 68Ga-NOTA-Polypeptide-PEG 11-Tz/anti-CD11b-F(ab′) 2-TCO is successfully synthesized. The molecular probe has targeting ability for CD11b + colon cancer, and is expected to be used as a tracer targeting CD11b receptor in vivo.
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Objective To evaluate the changes in natural Killer (NK) cell subsets definetd by CD11b and CD27 in the spleen of septic mice.Methods A total of 168 pathogen-free healthy male C57BL/6 mice,weighing 20-30 g,aged 8-10 weeks,were divided into 2 groups (n=84 cach) using a random number table:sham operation group (Sham group) and sepsis group (Sep group).Sepsis was induced by cecal ligation and puncture in chloral hydrate-anesthetized mice.Thirty mice in each group were selected to assess the survival within 4 days after operation,and the survival rate was calculated.At 2,4,6,24,48 and 72 h after operation (T1-6),6 mice were randomly selected,and blood samples were taken from the eyeballs for determination of serum tumor necrosis factor-alpha and interleukin-10 concentrations by enzyme-linked immunosorbent assay.Six mice were randomly selected at T4-6 and sacrificed,and the spleens were removed for measurement of the percentage of NK cells and their subsets by flow cytometry.Results Compared with Sham group,the survival rate was significantly decreased at different time points,the serum necrosis factor-alpha concentration at T1-3,6 and serum interleukin-10 concentration at T3-6 were increased,the percentage of NK cells was decreased at T4-6,the percentage of CD27-CD11b+NK cells was decreased and the percentage of CD27+CD11b+ and CD27+CD11b-NK cells was increased at T5,the percentage of CD27-CD11b-NK ceils was decreased at T4,5,and the percentage of CD27-CD11b-NK cells was increased at T6 in Sep group (P＜0.05).Conclusion Changes in NK cell subsets defined by CD11b and CD27 in the spleen play an important role in the development of sepsis in mice.
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Objective To investigate the role of integrin CD11b in liver ischemia/reperfusion (I/R) injury and its possible mechanism.Method CD11b-/-and WT (C57BL/6) mice were used to establish a 70％ liver warm I/R by clamping the left and median liver lobes for 60 min with vascular micro clamp at 37℃,then the clamp was removed and the abdominal incision was sutured.The blood plasma and liver samples were obtained at different time points (1,3,6,12,24 and 48 h) postreperfusion to assess liver function and cellular injury.Serum ALT and AST levels were determined,and HE staining and TUNEL assay were performed to estimate the severity of liver damage.Tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were assayed by Reverse transcription polymerase chain reaction (RT-PCR).Kupffer cells were isolated from the live,and the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity and active oxygen species (ROS) production were assayed.Result CD11b-/-mice displayed a significantly preserved liver function as represented by lower alanine aminotransferase (ALT) and aspartic transaminase (AST) levels,less histological damage and apoptosis compared to WT mice.Furthermore,TNF-α was decreased and IL-10 mRNA expression was increased in CD11b-/-mice compared to WT mice.Finally,CD11b-/-mice showed decreased activity of NADPH oxidase and less ROS production.Conclusion Integrin CD11 b may regulate the levels of inflammatory (TNF-α) and anti-inflammatory (IL-10) cytokines,enhance the activity of NADPH oxidase in Kupffer cells and enrich the production of ROS,which aggravate liver I/R injury.
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Objective To explore the role of the change of the CD11b expression in hyperleukocytosis and acute promyelocytic leukemia(APL) differentiation syndrome, we observed the effects of all-trans retinoie acid(ATRA) and trioxide arsenic(ATO) alone or the combination together with daunorubicin (DNR) on CD11b expression of human APL cell line NB4 cells. Methods By using the mono-antibody of CD11b FITC-conjugated, we monitored the level of CD11b expression of NB4 cells by flow cytometry. Results With the length of the 1 μmol/L ATRA exposure,the CD11b expression was increased. Exposed to ATRA for 24 h, 48 h, 72 h and 168 h;the CD11b expression of NB4 cell was (33.34±3.15)%, (55.59±5.13)%, (86.08±5.12)% and (90.69±2.69)%, respectively which were higher than control group and the difference was statistically significant (P0.05), but was significantly lower than that in ATRA exposure(P0.05). Conclusion By avoiding the increase of CD11b expression of promyelocytic leukemia cells, ATRA combined with ATO or two drugs together with DNR could play a role in eliminating incidence of hyperleukocytosis and retinoic acid syndrome.
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Objective To explore the expression of neutrophil adhesion molecule CD11b,and plasma level of elastase in patients with anaphylactoid purpura during different clinical phases and their correlation with disease activity.Methods A total of 20 patients with anaphylactoid purpura were recruited into this study,along with 20 normal human controls.Two blood samples were collected from each patients at the first visit(active phase)and after 3～5 weeks of treatment(remission phase).The expression of CD11b was measured by flow cytometry in 12 patients and normal controls,and plasma levels of elastase by ELISA in 20 patients and normal controls.Results Increased CD11b expression and elastase level were noted in patients in active phase compared with those in patients in remission phase(3367.25±434.57 vs 2569.33±411.06.13.98±2.05 vs 4.29±0.80.both P＜0.01).No significant difference was found in CD11b expression between patients in remission phase and normal controls(P＞0.05).while the elastase level was higher in patients in remission phase than in normal controls(4.29±0.80 vs 3.67±0.54.P＜0.05).In active phase of anaphylactoid purpura,the expression of CD11b was positively correlated with the plasma level of elastase(r=0.73,P＜0.01),while no correlation was noticed between them in remission phase(r=0.20,P=0.54).Conclusion Peripheral neutrophils are activated in anaphylactoid purpura,which seems to be more obvious in active phase than in remission phase.
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Objective The person's Mesangial Proliferative Glomemlonephritis (MsPGN) were divided into 3 types based on clini- cal manifestations: nephroticsyndrome, hematuria and proteinuria. To investigate the expression of CD11a, CD11b, CD62L and its signifi- cance in the kidneys patients with MsPGN. Methods CD11a, CD11b and CD62L expression in blood of MsPGN patients (n=35) were investigated by flow eytometry method, and the changes of these proteins in kidney were surveyed. Results In MsPGN, CD11a and CD11b expression in blood were significandy lower and CD62L expression in blood were markedly higher than that in normal humans. And the renal glomeruli of MsPGN also expressed CD11a, CD11b and CD62L. Conclusion The expression of CD11a, CD11b and CD62L are abnormal in MsPGN, and apoptosis may play certain role in the pathogenesis of MsPGN.
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Objective To investigate the effects of ulinastatin on the expression of neutrophil CD_(11b) and CD_(18) during orthotopic liver transplantation(OLT).Methods Forty patients with liver diseases,ASAⅢorⅣ, undergoing OLT were randomly divided into two groups.Ulinastatin group(n=20)received intravenous infusion of ulinastatin 3?10~5 IU in 100 ml normal saline after skin incision and repeated every 4 hours thereafter.Control group received same amount of normal saline instead(n=20).Blood samples were taken immediately before skin incision,120 min after skin incision,30 min of anhepatic phase,60 min of neohepatic phase and at the end of operation for measurement of CD_(11b) and CD_(18) expression of neutrophil by flow cytometry.Results CD_(11b)/CD_(18) expression was increased significantly in control group 60 min of neohepatic phase and at the end of operation compared to the level before skin incision,but in ulinastatin group there was no significant change in CD_(11b)/CD_(18) expression during the whole procedures(P＞0.05).CD_(11b)/CD_(18)expression was significantly lower 60 min of neohepatic phase and at the end of operation in ulinastatin group than in control group(P＜0.01).Conclusion Ulinastatin can inhibit the increase in CD_(11b)/CD_(18)expression during OLT,and be helpful for reducing the inflammatory response.