ABSTRACT
Objective@#To investigate the pathogenic mechanism of two novel ITGB2 mutations in leukocyte adhesion defect type 1 (LAD1).@*Methods@#The clinical history and blood sample of an 11 years old patient admitted to Affiliated Hospital of Qingdao University in August 2014 were collected. Expression of CD18 (encoded by ITGB2) was analyzed by flow cytometry. Novel ITGB2 mutations were identified by next-generation sequencing technology and confirmed by Sanger sequencing. The functional effect of ITGB2 mutations was detected by PolyPhen2. Expression vectors of both wild type and mutant ITGB2 were constructed and transfected into mammalian cells for analysis of protein stability and subcellular location.@*Results@#The symptoms of the patient (recurrent infections, lowered alveolar ridge and hypodontia) supported the diagnosis of LAD1. Expression of CD18 on the leukocytes was significantly decreased (0.2%) compared with the control samples from the parents (paternal: 99.0%; maternal: 99.1%). The patient was identified to be compound heterozygous for ITBG2 c.954del G (novel mutation) and c.1802C>A (paternal originated). ITGB2 c.954 del G was confirmed to be a harmful frameshift mutation; ITGB2 c.1802C>A was also predicted to be harmful. In terms of protein stability. There was no significant difference between mutant D18 and wild type. However, subcellular location analysis showed the mutant D18 could not locate on cell membrane.@*Conclusion@#The compound heterozygous of ITGB2 mutations (c.954del G and c.1802C>A) decreases the expression and impairs the location of CD18 on leukocytes, which leads to LAD1.
ABSTRACT
Objective To investigate the effects of monocytes on phenotypic changes of human proximal tubular HK-2 cells and the mechanism.Methods Monocytes were co-cultured with HK-2 cells.Morphological changes of HK-2 cells were detected by inverted phase contrast microscope.Expressions of E-cadherin,α-SMA and fibronectin were assessed by RT-PCR,Western blotting and immunocytochemical staining.Flow cytometry techniques was applied to evaluate intercellular cell adhesion molecule-1 (ICAM-1) expression on HK-2 cells.The intracellular signal was investigated by gene microarr ay.Results The typical epithelial cell morphology of HK-2 cells disappeared after co-culture with monocytes,accompanied by decreased E-cadherin expression and increased α-SMA and fibronectin expression (all P＜0.05).The expression of ICAM-1 on HK-2 cells was increased by monocytes stimulation.Interestingly,administration of CD18 antibody directly inhibited the phenotypic change of HK-2 cells.Furthermore,NF-κB signaling might be critical in mediating this process,and blockade of this signaling pathway could inhibit 1CAM-1 expression and epithelial mesenchymal transition (EMT) formation.Conclusion Monocytes can directly induce EMT of HK-2 cells via up-regulating ICAM-1 through NF-κB signaling pathway.
ABSTRACT
Objective To observe the influence of the expression of CD18 on the neutrophile and the leukocyte adhesion to retinal vascular endothelium by hypoxia-inducible factor-1 alpha (HIF-1α) in early diabetic retinopathy rats. Methods Male Sprague-Dawley rats received intraperitoneal injection of streptozotocin to induce diabetes model. 18 diabetic rats were divided into 3 groups randomly after 2 months of diabetes induction, including diabetic group (group B), HIF-1α anti-sense oligonucleotides (ASODN) injection group (group C) and HIF-1α sense oligonueleotides (SODN) injection group (group D), the age and weigh matched health rats were chosen as control group (group A), with 6 rats in each group. Then group A and B rats received 5 % glucose solution caudalis veins injection, group C and group D rats received HIF-1α ASODN and HIF-1α SODN caudalis veins injection, respectively(0.25 mg/kg). The level of CD18 on the neutrophil isolated from the peripheral blood was measured by flow cytometry. Retinal leukostasis was quantified with acridine orange leukocyte fluorography. Results The percentage of CD 18 positive neutrophil cell was (44.93±3. 60)% in group B, (18.66±1.52)% in group A, (31.66±4.72)% in group C,(51.00±5.66)% in group D. Compared with each other groups,the differences are statistically significant (F= 42. 46, P<0.001). The number of positive staining cells of retinal leukocyte was (46.16±10.68)in group A, (133.83±20.43)in group B, (99.83±9.28)in group C, (121.33±10. 23) in group C. Compared group B with group C,the number of positive staining cells raised about 2.89 times;compared group B with group C and D,the differences are statistically significant (P= 0.12, 95% confidence interval -3.69～28. 69 ). Conclusions In vivo, HIF-1α can decreased the expression of CD18 on neutrophils from diabetic rats' peripheral blood and the collection of retinal leukos- tasis in the diabetic animals. HIF-1α may serve as a therapeutic target for the treatment and/or prevention of early diabetic retinopathy.
ABSTRACT
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.