Ssy Yung

Impact of serum amyloid A on cellular cholesterol efflux to serum in type 2 diabetes mellitus.

OBJECTIVE Serum amyloid A (SAA) is an acute phase response protein and has apolipoprotein properties. Since type 2 diabetes is associated with chronic subclinical inflammation, the objective of this study is to investigate the changes in SAA level in type 2 diabetic patients and to evaluate the relationship between SAA and the capacity of serum to induce cellular cholesterol efflux via the two known cholesterol transporters, scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter G1 (ABCG1). METHODS 264 patients with type 2 diabetes mellitus (42% with normoalbuminuria, 30% microalbuminuria, and 28% proteinuria) and 275 non-diabetic controls were recruited. SAA was measured by ELISA. SR-BI and ABCG1-mediated cholesterol efflux to serum were determined by measuring the transfer of [(3)H]cholesterol from Fu5AH rat hepatoma cells expressing SR-BI and from human ABCG1-transfected CHO-K1 cells to the medium containing the tested serum respectively. RESULTS SAA was significantly increased in diabetic patients with incipient or overt nephropathy. Both SR-BI and ABCG1-mediated cholesterol efflux to serum were significantly impaired in all three groups of diabetic patients (p < 0.01). SAA inversely correlated with SR-BI-mediated cholesterol efflux (r = -0.36, p < 0.01) but did not correlate with ABCG1-mediated cholesterol efflux. Stepwise linear regression analysis showed that HDL, the presence or absence of diabetes, and log(SAA) were significant independent determinants of SR-BI-mediated cholesterol efflux to serum. CONCLUSION SAA was increased in type 2 diabetic patients with incipient or overt nephropathy, and SAA was associated with impairment of SR-BI-mediated cholesterol efflux to serum.

Serum immunoglobulin G level in patients with lupus nephritis and the effect of treatment with corticosteroids and mycophenolate mofetil

Background Reduced serum IgG level is associated with increased risk of infection. We investigated the circulating IgG level and its determining factors in active lupus nephritis patients treated with corticosteroids and mycophenolate mofetil (MMF). Methods This was a retrospective study on the longitudinal IgG profile in Class III/IV ± V lupus nephritis patients treated with prednisolone and MMF. Results 46 patients were included. At baseline, 34 (73.9%) patients (Group I) had normal or elevated IgG (1444.0 ± 600.5 mg/dL) while 12 (26.1%) (Group II) had IgG levels (567.8 ± 160.9 mg/dL) below the lower limit of normal. IgG levels at baseline, three, six and 12 months after treatment were 1215.4 ± 649.7 mg/dL, 843.9 ± 347.6 mg/dL, 914.5 ± 362.4 mg/dL and 1034.6 ± 452.5 mg/dL respectively. Treatment with prednisolone and MMF led to a significant drop in IgG after two weeks, reaching a nadir at eight weeks, followed by gradual normalization. Similar changes in IgG were observed in Group I patients but there was non-significant change in Group II within the first 24 weeks. Eighteen (39.1%) patients had low IgG by six months, and only one patient had IgG <300 mg/dL, at both three and six months. IgG level was negatively associated with proteinuria at six months (r = −0.711, p = 0.010). Five of 18 patients with low IgG had infections within the first year, while IgG levels below the lower limit of normal did not increase infection risk (relative risk 1.863; 95% confidence interval 0.466 to 6.818, p = 0.280). Conclusion Reduced IgG occurred in 26% of active lupus nephritis patients and the IgG levels are significantly influenced by the severity of proteinuria. Treatment with prednisolone and MMF does not result in clinically important suppression of IgG.

Serum level of proximal renal tubular epithelial cell-binding immunoglobulin G in patients with lupus nephritis

In vitro data showed that immunoglobulin G (IgG) from lupus nephritis (LN) patients could bind to proximal renal tubular epithelial cells (PTEC), but the clinical relevance of such binding remained unclear. Binding of IgG and subclasses to PTEC was measured by cellular ELISA (expressed as OD index) in 189 serial serum samples from 23 Class III/IV ± V LN patients who had repeated renal flares (48 during renal flares, 141 during low level disease activity (LLDA)), and compared with 64 patients with non-lupus glomerular diseases (NLGD) and 23 healthy individuals. Total IgG PTEC-binding index was 0.34 ± 0.16, 0.29 ± 0.16, 0.62 ± 0.27 and 0.83 ± 0.38 in healthy controls, NLGD, LN patients during LLDA, and LN patients during nephritic flare, respectively (p < 0.001, LLDA vs. renal flare; p < 0.001, healthy controls or NLGD vs. LN during LLDA or renal flare). PTEC-binding index for IgG1 was 0.09 ± 0.05, 0.16 ± 0.12, 0.44 ± 0.34 and 0.71 ± 0.46 for the corresponding groups (p < 0.001, LLDA vs. renal flare; p < 0.001, healthy controls or NLGD vs. LN during LLDA or renal flare). Sixteen of 48 episodes (33.3%) of nephritic flare showed persistent PTEC-binding IgG seropositivity for more than 9.4 ± 3.1 months, despite clinical response to immunosuppressive treatment. Total IgG and IgG1 PTEC-binding correlated with anti-dsDNA level (r = 0.34 and 0.52, respectively, p < 0.001 for both), and inversely with C3 level (r = –0.26 and –0.50, respectively, p = 0.002 and<0.001). Sensitivity/specificity of PTEC-binding index in detecting renal flares was 45.8%/80.1% for total IgG (ROC AUC 0.630, p = 0.007) and 87.5%/35.5% for IgG1 (ROC AUC 0.615, p = 0.018). IgG1 PTEC-binding index correlated with tubulo-interstitial inflammation score in renal biopsy from corresponding patients. Our data suggested that total IgG and IgG1 PTEC-binding index in serum of LN patients correlate with serological activity, and in combination could predict renal flares. The correlation between IgG1 PTEC-binding and tubulo-interstitial inflammation suggests potential pathogenetic significance.

208 Eda+ fibronectin in tubulo-interstitial injury in lupus nephritis

Background and aims Anti-dsDNA antibody plays a critical role in the pathogenesis of lupus nephritis and contributes to inflammatory and fibrotic processes in the kidney. EDA+ spliced variant of fibronectin (EDA+ FN), normally only weakly expressed, is markedly increased in pathological conditions. We investigated the effect of human polyclonal anti-dsDNA antibodies on the expression of EDA+ FN in proximal tubular epithelial cells (PTEC) and the functional consequence. Methods EDA+ FN expression in human renal biopsies of Class III/IV±V lupus nephritis was assessed by cytochemistry. Cultured PTEC were incubated with control IgG or IgG anti-dsDNA antibodies isolated from lupus nephritis patients for 24 hour and the expression of EDA+ FN was investigated. Recombinant human EDA peptide was used to investigate the functional role of EDA+ FN in PTEC. Results The results showed that EDA+ FN was absent from normal kidney tissue but was markedly increased in the tubulo-interstitium in lupus nephritis patients. Cultured PTEC constitutively expressed native FN but not EDA+ FN. Anti-dsDNA antibodies, compared with serum-free-medium and control IgG, increased EDA+ FN expression by 5.8- and 5.6-fold respectively (p<0.05 for both), and the induction was mediated through PI3K and mTOR activation. Exogenous IL-1β and TGF-β1, but not IL-6, IL-8 or MCP-1, induced EDA+ FN by 1.8- and 2.3-fold respectively. Recombinant EDA peptide increased native FN, collagen I, laminin and SNAIL expression, but decreased E-cadherin expression, in PTEC. Conclusions Our data demonstrated a role of EDA+ FN in the pathogenesis of tubulo-interstitial disease in lupus nephritis.

205 Annexin ii-binding immunoglobulin g level correlates with clinical and renal histological disease activity in lupus nephritis

Background and aims Annexin II mediates anti-dsDNA antibody binding to mesangial cells and downstream inflammatory and fibrotic processes. We investigated the relationship between annexin II-binding IgG and clinical or histological activity in lupus nephritis. Methods Serial serum samples from 28 patients with Class III/IV±V lupus nephritis were studied. Annexin II-binding IgG level was measured with an in-house ELISA. Glomeruli were isolated from NZBWF1 mice, gene and protein expression of annexin II and its binding protein p11 were investigated by real-time PCR and cytochemical staining respectively. Ultrastructural localization of annexin II was determined by electron microscopy and immunogold staining. Results Annexin II-binding IgG level was associated with anti-dsDNA level and disease activity in 42% of lupus nephritis patients. Annexin II-binding IgG level correlated with Activity Index (r=0.44, p=0.04), leukocyte infiltration score (r=0.52, p=0.02), and karyorrhexis/fibrinoid necrosis score (r=0.66, p=0.002) in renal biopsies, and also with the amount of mesangial electron-dense deposit scored semi-quantitatively (r=0.63, p=0.009). Glomerular annexin II and p11 expression increased with disease progression in NZBWF1 mice, and annexin II was found on the surface of mesangial cells and in the mesangial matrix, co-localising with electron-dense deposits. Conclusions Our data demonstrated an association between annexin II-binding IgG level and clinical/histological disease activity in proliferative lupus nephritis. Co-localization of annexin II with electron-dense deposits suggests a pathogenic role for annexin II.

Mycophenolic acid ameliorates anti-dsDNA antibody binding to proximal tubular epithelial cells and the subsequent induction of inflammatory and fibrotic processes

Objectives: We tested the hypothesis that lupus disease is caused by, or contributed to, so-called endogenous retroelements—genomic segments of ‘selfish’ DNA. Forty percent of our genome consist of such retroelements, many of them active. As an indication that the retroelements may cause lupus disease, patients that lack the enzyme Trex1 suffer from systemic lupus erythematosus, chilblain lupus or Aicardi-Goutières syndrome—an inflammation of the brain, with lupus disease developing in the course of the disease. Trex1 is a DNA exonuclease that degrades retroelement cDNA. Results and Conclusions: To show that unrestricted retroelements contribute to autoimmune disease, we increased the concentration of endogenous retroviral cDNA in B/W mice and, thereby, exacerbated lupus disease. Conversely, to ameliorate autoimmune disease, we introduced human APOBEC3 (abbreviated A3) enzymes into mice that lack Trex1. The lack in Trex1 makes the mice inefficient in retroelement restriction; the single mouse Apobec3 enzyme does not cover a wide range of retroelements, whereas the seven human A3 enzymes do. We will present data to show that human A3 enzymes are advantageous in Trex1deficient mice with autoimmune disease. This indicates that retroelements are indeed a cause for the disease.

Hyaluronan in the pathogenesis of lupus nephritis in NZB/W mice and the effect of 4-methylumbelliferone

Objectives: We tested the hypothesis that lupus disease is caused by, or contributed to, so-called endogenous retroelements—genomic segments of ‘selfish’ DNA. Forty percent of our genome consist of such retroelements, many of them active. As an indication that the retroelements may cause lupus disease, patients that lack the enzyme Trex1 suffer from systemic lupus erythematosus, chilblain lupus or Aicardi-Goutières syndrome—an inflammation of the brain, with lupus disease developing in the course of the disease. Trex1 is a DNA exonuclease that degrades retroelement cDNA. Results and Conclusions: To show that unrestricted retroelements contribute to autoimmune disease, we increased the concentration of endogenous retroviral cDNA in B/W mice and, thereby, exacerbated lupus disease. Conversely, to ameliorate autoimmune disease, we introduced human APOBEC3 (abbreviated A3) enzymes into mice that lack Trex1. The lack in Trex1 makes the mice inefficient in retroelement restriction; the single mouse Apobec3 enzyme does not cover a wide range of retroelements, whereas the seven human A3 enzymes do. We will present data to show that human A3 enzymes are advantageous in Trex1deficient mice with autoimmune disease. This indicates that retroelements are indeed a cause for the disease.

Annexin II on human mesangial cells mediates anti-dsDNA antibody binding

Objectives: We tested the hypothesis that lupus disease is caused by, or contributed to, so-called endogenous retroelements—genomic segments of ‘selfish’ DNA. Forty percent of our genome consist of such retroelements, many of them active. As an indication that the retroelements may cause lupus disease, patients that lack the enzyme Trex1 suffer from systemic lupus erythematosus, chilblain lupus or Aicardi-Goutières syndrome—an inflammation of the brain, with lupus disease developing in the course of the disease. Trex1 is a DNA exonuclease that degrades retroelement cDNA. Results and Conclusions: To show that unrestricted retroelements contribute to autoimmune disease, we increased the concentration of endogenous retroviral cDNA in B/W mice and, thereby, exacerbated lupus disease. Conversely, to ameliorate autoimmune disease, we introduced human APOBEC3 (abbreviated A3) enzymes into mice that lack Trex1. The lack in Trex1 makes the mice inefficient in retroelement restriction; the single mouse Apobec3 enzyme does not cover a wide range of retroelements, whereas the seven human A3 enzymes do. We will present data to show that human A3 enzymes are advantageous in Trex1deficient mice with autoimmune disease. This indicates that retroelements are indeed a cause for the disease.