Kusworini Handono

Analysis of urinary TGF-β1, MCP-1, NGAL, and IL-17 as biomarkers for lupus nephritis

This study was aimed to determine the diagnostic performance of transforming growth factor beta 1 (uTGF-β1), monocyte chemoattractant protein-1 (uMCP-1), neutrophil gelatinase-associated lipocalin (uNGAL) and interleukin 17 (uIL-17) in LN. Seventy participants were studied, categorized into three groups: 38 severe LN (class III-IV LN patients); 12 mild LN (class I-II LN patients); and 20 control (healthy volunteers). Diagnosis of SLE was based on the 1997 ARA criteria. Class NL classified according to ISN/RPS 2004. uTGF-β1, uMCP-1, uNGAL, uIL-17 levels were determined by ELISA, using spot urine. The level of uMCP-1 and uNGAL was significantly greater in severe or mild LN compared with control group (P<0.05). The level of uTGF-β1 and uIL-17 was significantly higher in severe LN than that controls group (P<0.05). The AUC of uTGF-β1, uMCP-1, uNGAL, uIL-17 was 66.50%; 86.90%; 87.50%; 71.70%, with the cut-off value of 27.13pg/ml; 1.54pg/ml; 446.30pg/ml; 36.62pg/ml. Only uNGAL showed a significant correlation with the activity (P=0.016; r=0.389) and chronicity indices (P=0.018; r=0.381). This study showed that uTGF-β1, uMCP-1, uNGAL, uIL-17 levels were increased in LN. The AUC values for each biomarker are indicating a good diagnostic value. Urinary NGAL had the best sensitivity and specificity followed by uMCP-1, uIL-17 and uTGF-β1. For combinations of two biomarkers, the best sensitivity and specificity were displayed by the combination of uTGF-β1 & u-NGAL, followed by uMCP-1 & uNGAL.

Treatment of low doses curcumin could modulate Th17/Treg balance specifically on CD4+ T cell cultures of systemic lupus erythematosus patients

Introduction The balance between T helper 17 (Th17) and regulatory T cells (Treg) is a new paradigm in the pathogenesis of systemic lupus erythematosus (SLE). Currently, there are no drugs that able to modulate Th17/Treg balance specifically in SLE. Curcumin is a bioactive agent that has a specific action against hyperproliferative cells. However, its role in modulating Th17/Treg balance in SLE is still unknown. This research aimed to investigate the role of curcumin in modulating Th17/Treg balance on CD4+ T cell cultures of SLE patients. Material and methods CD4+ T cells from SLE 6 untreated patients and 6 healthy subjects were collected, stimulated with Th17 differentiating factors, and curcumin 0.1 and 1 µg/ml was added on cultures. After 72 hours incubation, cells were harvested and measured for Th17 and Treg percentages using flow cytometry and interleukin-17A (IL-17A) and transforming growth factor-β1 (TGF-β1) levels using ELISA. Results Administration of low doses of curcumin (0.1 and 1 µg/ml) could decrease Th17 percentages (p = 0.000 and p = 0.000 compared to control), reduce IL-17A productions (p = 0.000 and p = 0.000 compared to control), increase Treg percentages (p = 0.001 and p = 0.000 compared to control), and increase TGF-β1 productions (p = 0.001 and p = 0.000 compared to control) on CD4+ T cells of SLE patients. Interestingly, these effects were not reproduced on CD4+ T cells cultures of healthy subjects. Conclusions These data suggest that curcumin can modulate Th17/Treg balance specifically on CD4+ T cells of SLE patients without affecting healthy subjects.

Changes to signal peptide and the level of transforming growth factor- β1 due to T869C polymorphism of TGF β1 associated with lupus renal fibrosis

Lupus Nephritis (LN) is a serious manifestation of lupus that can lead to End Stage Renal Disease (ESRD). Fibrosis is the main feature of ESRD, and it is likely influenced by Transforming Growth Factor Beta1 (TGFβ1). The T869C gene polymorphism of TGFβ1 is assumed to change the signal peptide, that has potential to interfere the urine production and renal protein expression of TGFβ1. The influence of T869C gene polymorphism on TGFβ1 production and renal fibrosis was evaluated in this study. Subjects were 45 patients LN with renal fibrosis and 45 participants without renal fibrosis as control, that were recruited from 2011 to 2013.Their urinary TGFβ1 levels and TGFβ1 gene polymorphisms were examined. All lupus patients underwent renal biopsy to assess their protein expression of TGFβ1 in the renal tissue by immunohistochemistry and their renal fibrosis by morphometry and chronicity index. Changes in the signal peptide interaction with Signal Recognition Particle (SRP) and translocon of endoplasmic reticulum were analyzed by Bioinformatics. Levels of urinary and protein expression of TGFβ1 increased in the LN with renal fibrosis group. There were significant differences in levels of urinary TGFβ1 in T, C allele and TT, TC, CC genotypes between case and control groups. Furthermore, patients with C allele are 3.86 times more at risk of renal fibrosis than T allele. The C allele encodes proline, which stabilizes the interaction of the TGFβ1 signal peptide with SRP and translocon, resulting in elevation of TGFβ1 secretion. Our results indicated that T869C gene polymorphism of TGFβ1 changes the signal peptide, that contributes to the production of urinary TGFβ1 and affects renal fibrosis in lupus nephritis.

Effect of Curcuma xanthorrhiza Supplementation on Systemic Lupus Erythematosus Patients with Hypovitamin D Which Were Given Vitamin D3 towards Disease Activity (SLEDAI), IL-6, and TGF-β1 Serum

Background Curcumin contained in Curcuma xanthorrhiza is an immunomodulator that has similar biological effect as vitamin D. Combination of curcumin and vitamin D3 is expected to work synergistically. Objective To determine the effect of Curcuma xanthorrhiza supplementation on vitamin D3 administration to SLEDAI, IL-6, and TGF-β1 serum in SLE patients with hypovitamin D. Methods This was a double-blind RCT conducted in Saiful Anwar Hospital, Malang, in January 2016–March 2017. Subjects were SLE active (SLEDAI > 3) with levels of 25(OH)D3 ≤ 30 ng/ml and divided into two groups: those receiving cholecalciferol 3 × 400 IU and placebo 3 × 1 tablets (group I) and those receiving 3 × 400 IU cholecalciferol and Curcuma xanthorrhiza 3 × 20 mg for 3 months (group II). SLEDAI, levels of vitamin D, IL-6, and TGF-β1 in serum were evaluated before and after the treatment. Results There were no significant differences in SLEDAI reduction, decreased serum levels of IL-6, and increased levels of TGF-β1 serum among groups after the treatment. Decreased levels of serum IL-6 have a positive correlation with SLEDAI reduction. Conclusion. Curcuma xanthorrhiza supplementation on vitamin D3 had no effects on SLEDAI and serum levels of IL-6 and TGF-β1. This clinical trial is registered with NCT03155477.

Low Fetal Weight is Directly Caused by Sequestration of Parasites and Indirectly by IL-17 and IL-10 Imbalance in the Placenta of Pregnant Mice with Malaria.

The sequestration of infected erythrocytes in the placenta can activate the syncytiotrophoblast to release cytokines that affect the micro-environment and influence the delivery of nutrients and oxygen to fetus. The high level of IL-10 has been reported in the intervillous space and could prevent the pathological effects. There is still no data of Th17 involvement in the pathogenesis of placental malaria. This study was conducted to reveal the influence of placental IL-17 and IL-10 levels on fetal weights in malaria placenta. Seventeen pregnant BALB/C mice were divided into control (8 pregnant mice) and treatment group (9 pregnant mice infected by Plasmodium berghei). Placental specimens stained with hematoxylin and eosin were examined to determine the level of cytoadherence by counting the infected erythrocytes in the intervillous space of placenta. Levels of IL-17 and IL-10 in the placenta were measured using ELISA. All fetuses were weighed by analytical balance. Statistical analysis using Structural Equation Modeling showed that cytoadherence caused an increased level of placental IL-17 and a decreased level of placental IL-10. Cytoadherence also caused low fetal weight. The increased level of placental IL-17 caused low fetal weight, and interestingly low fetal weight was caused by a decrease of placental IL-10. It can be concluded that low fetal weight in placental malaria is directly caused by sequestration of the parasites and indirectly by the local imbalance of IL-17 and IL-10 levels.

The Potential effect of G915C polymorphism in regulating TGF-β1 transport into Endoplasmic Reticulum for cytokine production.

The TGF-β1 cytokine concentration is known to be higher in nephritis with implied Lupus Nephritis severity. The production of TGF-β1 cytokine is associated with G915C polymorphism. Therefore, it is of interest to study G915C polymorphism. The G915C polymorphism changes codon 25 which encodes arginine into proline in the signal peptide of TGF-β1. The amino acid substitution affects signal peptide properties that may inhibit the transport of TGF-β1 into the endoplasmic reticulum and eventually decline the cytokine production. Hence, the effect of G915C polymorphism on the properties of the signal peptide, the ability of TGF-β1 transport into the endoplasmic reticulum and the concentrations of urinary TGF-β1 in Lupus Nephritis patients was studied. The arginine substitution into proline decreased the polarity of the signal peptide for TGF-β1. The increased hydrophobicity with increased binding energy of the signal peptide for TGF-β1 to Signal Recognition Particle (SRP) and translocon is shown. This implies decreased protein complex stability in potentially blocking the transport of TGF-β1 into the endoplasmic reticulum. This transport retention possibly hampers the synthesis and maturation of TGF-β1 leading to decreased cytokine production.

Regulatory T Cells Compensation Failure Cause the Dysregulation of Immune Response in Pristane Induced Lupus Mice Model

Introduction Regulatory T cells’ (Tregs’) role remains unclear in the pathogenesis of systemic lupus erythematosus (SLE). This study was aimed at monitoring the percentage of Tregs within 32 weeks and monitoring its relationship with the percentage of other T helper (Th) cell subsets and the levels of autoantibodies and pro-inflammatory cytokines in a murine SLE model induced by pristane. Methods Forty-eight female BALB/c mice were divided into a healthy control (HC) and a pristine-induced (PI) group. SLE was induced by a single 0.5 cc pristane intraperitoneal injection. Six from each group were sacrificed every eight weeks until 32 weeks post-pristane injection. Treg, Th1, Th2 and Th17 percentages from the spleen were measured using flowcytometry. ANA, IL-6 and IFN-α levels were measured from serum using ELISA. Results The Treg percentage from the PI group increased significantly at 16 weeks compared to the HC group, while Th1, Th2 and Th17 percentages decreased. Tregs in the PI group began to reduce from the 24th to 32nd weeks, followed by an elevation of the Th1, Th2 and Th17 percentages. Tregs were negatively correlated with Th1 and Th2. Tregs in the PI group had a negative correlation with ANA and IFN-α levels from serum, whereas Tregs had a positive correlation with IL-6 levels. Conclusion The compensation of Tregs observed at 16 weeks after pristane injection failed, marked by a decreasing number of Tregs, followed by an increase of Th subsets, pro-inflammatory cytokines and autoantibodies. This compensatory failure of Tregs could be affected by pro-inflammatory cytokines, such as IFN-α and IL-6.

Chondrocyte Intracellular Matrix Strain Fields of Articular Cartilage Surface in Hyperglycemia Model of Rat: Cellular Morphological Study

Introduction: Chondrocyte is one cell in articular cartilage was products many proteins, molecules, and other factors. The external influence of cartilage, such as: hyperglycemia was entering joint capsule and impact to the chondrocytes and the cartilage. Hyperglycemia caused modification of heparan sulfate proteoglycan 2 (perlecan) proteins through glycation process. Aim: The aim of this study was to analyze morphological changing of chondrocytes pericellular matrix by the influence of hyperglycemia. Material and Methods: Eighteen adult male rats were divided into six groups: control, rat treated with sugar intake was 0.5 mg/kg, 0.75 mg/ kg, 1mg/kg, 1.5 g/kg and 2 mg/kg of body weight. The animal model was dislocated and left knee was taken to observe changing of chondrocytes pericellular matrix strain fields by Scanning Electron Microscope (SEM) from perpendicular to femoral condylus cartilage. Results: Changing of chondrocytes intracellular matrix strain fields as changing of cell diameters and cell distances at group control and group I to group V, which cell diameters was lower level and cell distances was the highest level at over diet 2. This changing of intracellular matrix strain fields was corresponding to changing chondrocytes morphology in hyperglycemia condition, due to hypertrophic stage as adaptive responses. This research as based on next research for accomplish of hyperglycemia influence to morphology articular cartilage changing to prevent degeneration of cartilage towards osteoarthritis. Conclusions: Present study concludes that hyperglycemia influence to chondrocyte intracellular matrix strain fields changing.

INTERLEUKIN-4 DAN INTERFERON GAMMA DI NEFRITIS LUPUS: HUBUNGAN AKTIVITAS PENYAKIT SERTA KEKAMBUHAN

Sampling for urinalysis to see the activity and the degree of recurrence of Lupus Nephritis (LN) is very difficult. New biomarkers that are more simple, sensitive, specific and non-invasive in assessing the activity of the LN need to be investigated. Interleukin-4 (IL-4) and interferon gamma (IFN-γ) were implicated to LN process. Urine samples from 17 LN patients were taken every month for 6 (six) months to examine the level of uIL-4, uIFN-γ, activity and recurrence of LN. Significant differences were observed in the uIFN-γ levels between the active and inactive LN groups (p=0.012), but not in uIL-4 levels (p=0.187). Correlations between each biomarker and renal domain score were weak (r=0.201, p=0.042 for uIL-4; r=0.268, p=0.006 for uIFN-γ). Significant differences were also found in the uIL-4 and uIFN-γ levels against LN recurrence (p=0.033; p=0.017). The best cut off values to assess recurrences and activity of LN were 8.17 pg/mL for uIL-4 showed a sensitivity of 74%, specificity 71%, NPV 90%, PPV 42% to assess recurrences and to assess activity of LN showed sensitivity 46%, specificity 75%, NPV 48%, PPV 78%. The cut off 18.58 pg/mL for uIFN-γ to predict recurrent and assess the activity of LN showed sensitivity 68%%, specificity 70%, NPV 88%, PPV 40% to predict the recurrent and to assess the activity of LN showed sensitivity 57%, specificity 64%, NPV 49%, PPV 73%. Based on the research, uIL-4 and uIFN-γ are not good enough to predict recurrence and activity of LN

PETANDA BIOLOGIK TERKINI LUPUS NEFRITIS

Lupus Nephritis (LN) is one of the serious clinical manifestation of Systemic Lupus Erythematosus (SLE). Early detection and treatment of renal activity may spare patients from renal damage. Conventional biomarkers such as urine sediment, proteinuria, creatinine, antids DNA antibody and their complement levels are not specific and sensitive enough in detecting the ongoing disease activity in the lupus kidneys and early relapse of nephritis. Renal biopsy is the gold standard in providing information on the histopathology of LN, but is invasive and it should take a serial of biopsies making it impractical when monitoring LN. Thus, some novel biomarkers are necessary to enhance the diagnostic accuracy and sensitivity of lupus renal disease, prognostic stratification, monitoring of treatment response and detection of early renal flares as well. Some novel biomarkers have been studied in LN, however, validation on a large scale of patients with different ethnic backgrounds is still needed.