Correlation between single nucleotide polymorphism and protein expression of FcγRⅡb in Hashimoto′s thyroiditis / 中华内分泌代谢杂志

Objective:To investigate whether FcγRⅡb rs775 single nucleotide polymorphism confers susceptibility to Hashimoto′s thyroiditis and its impact on expression of FcγRⅡb protein on B cell surface.Methods:A total of 187 Hashimoto′s thyroiditis patients(HT group) were enrolled, including 46 males(24.60%) and 141 females(75.40%), with a median age of 43(32, 53) years, and 187 healthy controls(conrol group), including 62 males(33.16%) and 125 females(66.84%), with a median age of 41(31, 51) years. The peripheral blood of two groups were sequenced, genotype and allele frequencies distribution of FcγRⅡb rs775 T>C were compared with clinical parameters as strata between the two groups. At the same time, the expression of inhibitory receptor FcγRⅡb on B cell surface was detected using flow cytometry.Results:Compared with control group, the mutant homozygous CC genotype was obviously enrichment in HT group( OR=3.321, 95% CI 1.175-9.386, P=0.018), and the proportion of CC genotype increased in male of HT group( P=0.076). However, there is no significant difference in genotype and allele frequencies between control group and HT group after stratification by sex. In addition, the percentage of FcγRⅡb on B cell surface decreased significantly in HT group( P=0.029). Conclusion:There was no significant correlation between FcγRⅡb polymorphism and the down-regulation of FcγRⅡb protein on B cell surface in Hashimoto′s thyroiditis patients, and FcγRⅡb can be a predisposed factor for Hashimoto′s thyroiditis.

Establishment and validation of OX40/FcγR-humanized mice for the study of agonistic anti-OX40 antibody / 上海交通大学学报（医学版）

Objective • To establish a rapid method to evaluate the activity of agonistic antibody using OX40 (tumor necrosis factor receptor superfamily member 4)/FcγR (Fcγ receptor)-humanized mice. Methods • Bone marrow cells from OX40-humanized mice and FcγR-humanized mice were collected and mixed with equal ratio. Then the mixed bone marrow cells were administrated into irradiated wild-type mice through the tail veins. The reconstruction efficiency of the immune system was confirmed by detecting the expression of hOX40 and hFcγR in the immune cells of chimera mice. After the chimera mice were generated successfully, they were used to evaluate the immunostimulatory activity of anti-hOX40 antibodies to CD4+ or CD8+ T cells. The results of flow cytometry were statistically analyzed. The unpaired t-test was used to compare the means between the two groups, and oneway ANOVA was used to compare the means between multiple groups. Results • Flow cytometry analysis showed that wild-type recipient mice were efficiently reconstituted with hFcγR expressing cells and hOX40 expressing cells to generate OX40/FcγR-humanized bone marrow chimera mice. In these mice, B cells and myeloid cells expressed hFcγRs (P<0.05), and T cells expressed hOX40 upon in vitro stimulation (P<0.05). When these mice were used to evaluate the immunostimulatory activity of anti-hOX40 antibody, significant expressions of IFN-γ and hOX40 were observed (P<0.05). Conclusion • OX40/FcγR-humanized bone marrow chimera mice are generated based on hFcγR expressing cells and hOX40 expressing cells, suggesting a rapid method to build a mouse model with both hFcγR and hOX40 expression. These mice are suitable for evaluating the immunostimulatory activity of agonistic human anti-hOX40 antibodies.

Knockout of IgG receptor FcγRⅡB aggravates systemic and adipose tissue inflammatory responses in mice / 基础医学与临床

Objective To investigate the effect of knockout of IgG receptor( FcγRIIB) on systemic and adipose tis-sue inflammation and fat tissue lipid metabolism treated with high-fat diet ( HFD) . Methods We used 10 male mice with IgG receptor FcγRIIB knockout ( FcγRIIB-/-) and another 10 male FcγRIIB+/+mice as control, and trea-ted them with HFD. At the end of the 17th week, mice were weighed, the blood was taken by cardiac puncture af-ter sacrifice. Adipose tissue was collected to measure inflammation and lipid metabolism. Results Compared with FcγRIIB+/+mice, FcγRIIB-/-mice had significantly increased levels of inflammatory cytokines, IL-6, TNF-α, and IFN-γ in the serum, and increased macrophage infiltration in adipose tissue. M1 polarization gene MCP-1 and TNF-α increased ( P＜0.05) and M2 polarization gene ARG and IL-10 did not differ significantly. However, there was no significant difference in body mass, adipocyte size and expression of lipid metabolism related genes PPARG, CEBPα, FASn, HSL, ATGL, UCP-1 and GLUT4. Conclusions Under HFD treatment, knocking out the IgG re-ceptor FcγRIIB aggravates the inflammatory response of the whole body and adipose tissue, but cannot influence lipid metabolism of adipose tissue and body weight.

Alteration in Leukocyte Subsets and Expressions of FcγR and Complement Receptors among Female Ragpickers in Eastern India

BACKGROUND: There are a million ragpickers in India who gather and trade recyclable municipal solid wastes materials for a living. The objective of this study was to examine whether their occupation adversely affects their immunity. METHODS: Seventy-four women ragpickers (median age, 30 years) and 65 age-matched control housemaids were enrolled. Flow cytometry was used to measure leukocyte subsets, and leukocyte expressions of Fcγ receptor I (CD64), FcγRIII (CD16), complement receptor 1 (CD35) and CR3 (CD11b/CD18), and CD14. Serum total immunoglobulin-E was estimated with enzyme-linked immunosorbent assay. RESULTS: Compared with the controls, ragpickers had significantly (p < 0.0001) higher levels of CD8+T-cytotoxic, CD16+CD56+natural killer, and CD4+CD45RO+memory T-cells, but depleted levels of CD19+B-cells. The percentage of CD4+T-helper-cells was lower than the control group (p < 0.0001), but their absolute number was relatively unchanged (p = 0.42) due to 11% higher lymphocyte counts in ragpickers. In ragpickers, the percentages of CD14+CD16+intermediate and CD14dim CD16+nonclassical monocyte subsets were elevated with a decline in CD14+CD16-classical monocytes. The expressions of CD64, CD16, CD35, and CD11b/CD18 on both monocytes and neutrophils, and CD14 on monocytes were significantly higher in ragpickers. In addition, ragpickers had 2.7-times more serum immunoglobulin-E than the controls (p < 0.0001). After controlling potential confounders, the profession of ragpicking was positively associated with the changes. CONCLUSION: Ragpicking is associated with alterations in both innate (neutrophils, monocytes, and natural killer cell numbers and expression of complement and Fcγ receptors) and adaptive immunity (numbers of circulating B cells, helper, cytotoxic, and memory T cells).

Prokaryotic expression of the extracellular portion of mouse FcγRⅡb and the functions of the ex-pressed proteins in a mouse model of SLE / 中华微生物学和免疫学杂志

Objective To clone and express the extracellular portion of mouse Fcγreceptor Ⅱ-b ( FcγRⅡb) and to analyze the functions of the expressed proteins in a mouse model of systemic lupus ery-thematosus ( SLE) .Methods The gene fragment encoding FcγRⅡb was amplified by PCR, and then in-serted into the prokaryotic expression vector pET-32a(+) to construct the recombinant expression plasmid pET-FcγRⅡb.The expression plasmid was identified with restriction enzymes and then sent to the Shanghai Bio-Engineering Co.LTD for further sequencing analysis.The transformed Escherichia coli ( E.coli) BL21 ( DE3) strains carrying the recombinant expression plasmid pET-FcγRⅡb were induced by isopropylβ-D-1-thiogalactoside ( IPTG) .The expressed fusion proteins were analyzed by Western blot assay and purified with purification kits.The immune complex ( IC)-binding ability of FcγRⅡb was measured by ELISA.MRL/lpr mice with SLE in both the prevention (12 weeks old, n=40) and the treatment groups (19 weeks old, n=40) were randomly divided into four groups including 60 μl (4.8 μg) treatment group, 120 μl (9.6 μg) treatment group, 180μl (14.4μg) treatment group and PBS treatment group with 10 mouse in each group. The MRL/lpr mice with SLE were injected with the fusion protein through tail vein once a week for four con-secutive weeks.Serum samples were collected from each mouse after one week of observation.The levels of FcγRⅡb in soluble form in mice form both the prevention and treatment groups as well as the levels of anti-double stranded DNA antibodies were detected by ELISA.Results The gene encoding FcγRⅡb was ampli-fied and the recombinant expression plasmid pET-FcγRⅡb was successfully constructed.The recombinant proteins were expressed in the prokaryotic expression system, and then successfully purified.The recombi-nant proteins could bind to IC.Compared with the corresponding PBS control group, the levels of FcγRⅡb in soluble form were increased in mice from both prevention and treatment groups after treating with various concentrations of the recombinant protein (P<0.05).Significant differences in the levels of FcγRⅡb were found among mice of the same age after treating with different concentrations of the recombinant protein ( P<0.05).Compared with the corresponding PBS control group, the levels of anti-double stranded DNA anti-bodies were decreased in mice from both prevention and treatment groups after treating with various concen-trations of the recombinant protein (P<0.05).The levels of anti-double stranded DNA antibodies were grad-ually decreased along with the increasing dosage of protein (P<0.05).Conclusion The extracellular por-tion of murine FcγRⅡb in soluble form was successfully expressed.The recombinant proteins played a cer-tain role in the prevention and treatment of SLE in a mouse model.

Immunosuppression mediated by porcine FcγRⅢin PRRSV infection to PAMS / 中国免疫学杂志

Objective:To study immunosuppression mediated by the porcine FcγRⅢ in porcine reproductive and respiratory syndrome virus ( PRRSV ) infection to pulmonary alveolar macrophages ( PAMS ).Methods: In this study pulmonary alveolar macrophages cells were treated with containing 200 TCID50 PRRSV,lipopolysaccharide (LPS) (100 ng/ml) and purified mouse anti-pig FcγRⅢIgG (550 μg/ml) separately,simultaneously,PAM cells treated with purified mouse anti-pig FcγRⅢIgG (550 μg/ml) was infected by 200 TCID50 PRRSV ,untreated PAM cells as the control group.Each group were post-cultured 12,24,36,48,60,72 h, the cells and the supernatant were collected.The dynamic variation of PRRSV RNA copies in inoculation group were detected by using real-time fluorescence quantitative PCR method.mRNA level of IFN-αand TNF-αin each group were detected by using relative fluorescence quantitative PCR.Results:The result showed that mRNA level of IFN-αwas improved during PRRSV infection to PAMS 12-24 h,and mRNA level of IFN-αwas inhibited during 36-72 h,then mRNA level of IFN-αrecovered normally; mRNA level of TNF-αwas increased slightly post-infection 12-72 h.IFN-αand TNF-αmRNA levels of PAM cells treated with LPS were both up-regu-lated,using the purified mouse anti-pig FcγRⅢ IgG to treat the PAM cells,selective activation of porcine FcγRⅢ in the PAM cells down-regulated significantly mRNA levels of IFN-αand TNF-α.PRRSV infection assay mediated by selective activation FcγRⅢof the PAM cells inhibited antiviral cytokine ( IFN-αand TNF-α) mRNA levels.Conclusion:The results show selective activation of FcγRⅢinhibited significantly mRNA levels of the antiviral cytokine IFN-αand TNF-αof host cells,and innate antiviral immune response to PRRSV infection.

FcγR gene polymorphisms in Kawasaki disease susceptibility and the efficacy of intravenous immunoglobulin therapy / 国际儿科学杂志

Fcgamma receptors family conclude three groups(CD64、CD32、CD16),of which FcγR Ⅱa,Ⅱb,Ⅲb have Gene polymorphisms,for this reason they have an effect on affinity of FcγR to antibodies different,so FcγRⅡa,Ⅱb,Ⅲb are related to genetic susceptibility and IVIG treatment response.The Gene subtype of FcγR Ⅱ a is considered to be the susceptibility genes of KD.Polymorphisms of FcγRs were intimately connected to pathogenesis,coronary artery impairment and disease prevention of Kawasaki disease.

Alteration of Fc gamma receptors in childhood acute immune thrombocytopenia / 中华微生物学和免疫学杂志

ObjectiveTo investigate the alteration of Fc gamma receptors expressed on monocytes in childhood acute immune thrombocytopenia (aITP).Methods27 patients with aITP and 25 health controls were enrolled in this study.The expression of FcγR Ⅰ and FcγRⅢ on monocytes surface was determined by flow cytometry.Real-time PCR was performed to detect the levels of FcγR Ⅱ a and FcγR.Ⅱ b mRNA.The plasma concentration of IL-4 and IFN-γwas analyzed by ELISA.ResultsThe expression of FcγR I and FcγRⅢ on monocytes was significantly highet in ITP pationts than thatinhealth control group.Transcription levels of FcγR Ⅱ a mRNA and the ratio of FcγR Ⅱ a/Ⅱ b mRNA on monocytes were significantly elevated,while the inhibitory FcγR Ⅱ b mRNA was significant lower in aITP patients (P＜0.05) ; in comparison with healthy controls. After dexamethasone treatment,the stimulatory FcγR Ⅰ,FcγRⅢ and FcγR Ⅱ a mRNA were significantly decreased while inhibitory FcγR Ⅱ b mRNA was increased.(2)The higher levels of plasma IFN-y and lower IL-4 were to be found in aITP patients compared with healthy controls.Correlation analysis showed that the level of plasma IFN-γwas positively correlated with the elevated expression of FcγR Ⅱ a mRNA on monocytes,while the level of IL-4 was positively correlated with FcγRⅡ b mRNA.Conclusion The over expression of inhibitory FcγRs and the down-regulation of inhibitory FcγR Ⅱb mRNA on monocytes,and the imbalance of stimulatory and inhibitory FcγR might be involve in immunological pathogenesis of the acute ITP.The alteration levels of plasma pro-inflammatory cytokine IFN-γand anti-inflammatory cytokine IL-4 might be involved in imbalance expression of FcγR in acute ITP.Dexamethasone therapy could shift the balance between stimulatory and inhibitory FcγR toward inhibitory FcγR Ⅱ b in aITP patients.