Soluble ACE2-mediated cell entry of SARS-CoV-2 via interaction with proteins related to the renin-angiotensin system.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause acute respiratory disease and multiorgan failure. Finding human host factors that are essential for SARS-CoV-2 infection could facilitate the formulation of treatment strategies. Using a human kidney cell line-HK-2-that is highly susceptible to SARS-CoV-2, we performed a genome-wide RNAi screen and identified virus dependency factors (VDFs), which play regulatory roles in biological pathways linked to clinical manifestations of SARS-CoV-2 infection. We found a role for a secretory form of SARS-CoV-2 receptor, soluble angiotensin converting enzyme 2 (sACE2), in SARS-CoV-2 infection. Further investigation revealed that SARS-CoV-2 exploits receptor-mediated endocytosis through interaction between its spike with sACE2 or sACE2-vasopressin via AT1 or AVPR1B, respectively. Our identification of VDFs and the regulatory effect of sACE2 on SARS-CoV-2 infection shed insight into pathogenesis and cell entry mechanisms of SARS-CoV-2 as well as potential treatment strategies for COVID-19.

Alterations in exhausted and classical memory B cells in lupus nephritis - Relationship with disease relapse.

INTRODUCTION: B cell exhaustion is a functional abnormality of B lymphocytes observed in chronic infections and shows association with autoreactivity. The role of exhausted and classical memory B cells in maintaining disease stability of lupus nephritis (LN) remains unclear. METHODS: We measured classical memory (CD19+CD21+CD27+), exhausted B cells (CD19+CD21-CD27-), and related cytokines in LN patients with multiple relapses (MR) (n = 15) and no relapse (NR) (n = 15) during disease remission. The expression of inhibitory/adhesion molecules, cell proliferation and calcium mobilization in classical memory and exhausted B cells were also assessed. RESULTS: The MR group had higher proportion of circulating exhausted and classical memory B cells compared to the NR group and healthy controls (HC) (p all <0.05 for MR vs. NR or HC). Blood levels of IL-6, BAFF, IL-21, CD62L, CXCR3 and Siglec-6 were all higher in the MR group (p < 0.05, for all). Exhausted B cells from the MR group showed higher FcRL4, CD22, CD85j and CD183 but lower CD62L expression than NR and HC groups. Exhausted B cells from MR patients exhibited reduced proliferation compared to NR patients and HC, while classical memory B cell proliferation in MR group was higher than the other two groups. Exhausted B cells from both MR and NR patients showed impaired calcium mobilization. CONCLUSION: Alterations in exhausted and classical memory B cells are related to disease relapse in LN. These findings may help devise new strategies for monitoring disease activity and preventing relapse in LN.

Serum syndecan-1, hyaluronan and thrombomodulin levels in patients with lupus nephritis.

OBJECTIVES: We investigated circulating syndecan-1, HA and thrombomodulin levels in patients with biopsy-proven Class III/IV ± V LN and their clinico-pathological associations. Patients with non-renal SLE or non-lupus chronic kidney disease, and healthy subjects served as controls. METHODS: Serum syndecan-1, HA and thrombomodulin levels were determined by ELISAs. RESULTS: Syndecan-1, HA and thrombomodulin levels were significantly higher during active LN compared with remission (P < 0.01, for all), and correlated with the level of proteinuria, estimated glomerular filtration rate, anti-dsDNA antibodies, complement 3 and serum creatinine. Longitudinal studies showed that syndecan-1 and thrombomodulin levels increased prior to clinical renal flare by 3.6 months, while HA level increased at the time of nephritic flare, and the levels decreased in parallel with treatment response. Receiver operating characteristic curve analysis showed that syndecan-1 and thrombomodulin levels distinguished patients with active LN from healthy subjects, LN patients in remission, patients with active non-renal lupus and patients with non-lupus chronic kidney disease (receiver operating characteristic area under curve of 0.98, 0.91, 0.82 and 0.95, respectively, for syndecan-1; and area under curve of 1.00, 0.84, 0.97 and 0.79, respectively, for thrombomodulin). HA level distinguished active LN from healthy subjects, LN patients in remission and non-lupus chronic kidney disease (receiver operating characteristic area under curve of 0.82, 0.71 and 0.90, respectively) but did not distinguish between renal vs non-renal lupus. Syndecan-1 and thrombomodulin levels correlated with the severity of interstitial inflammation, while HA level correlated with chronicity grading in kidney biopsies of active LN. CONCLUSION: Our findings suggest potential utility of serum syndecan-1, thrombomodulin and HA levels in clinical management, and their potential contribution to LN pathogenesis.

Clinico-pathological associations of serum VCAM-1 and ICAM-1 levels in patients with lupus nephritis.

OBJECTIVE: We investigated the clinico-pathological associations of serum VCAM-1 and ICAM-1 levels in patients with biopsy-proven Class III/IV±V lupus nephritis (LN). METHODS: Serum VCAM-1 and ICAM-1 levels were determined by ELISAs. Sera from patients with non-renal SLE or non-lupus chronic kidney disease (CKD), and healthy subjects served as controls. RESULTS: Seropositivity rate for VCAM-1 and ICAM-1 was 93.10% and 37.93% respectively at the time of nephritic flare, and 44.83% and 13.79% respectively at remission, with both showing higher levels during flare (P < 0.05, for both). VCAM-1 level correlated with proteinuria, serum creatinine, and anti-dsDNA antibodies, and inversely correlated with C3. VCAM-1 level also correlated with leukocyte infiltration and fibrinoid necrosis/karyorrhexis scores in active LN kidney biopsies. ICAM-1 level correlated with proteinuria, but not anti-dsDNA or C3, nor histopathological features. VCAM-1 level increased 4.5 months before renal flare, while ICAM-1 increase coincided with flare, and both decreased after treatment. ROC analysis showed that VCAM-1 distinguished active LN from healthy subjects, LN in remission, active non-renal lupus, and CKD (ROC AUC of 0.98, 0.86, 0.93 and 0.90 respectively). VCAM-1 level in combination with either proteinuria or C3 was superior in distinguishing active LN from remission compared to the measurement of individual markers. Serum ICAM-1 level distinguished active LN from healthy subjects and LN patients in remission (ROC AUC of 0.75 and 0.66 respectively), but did not distinguish between renal versus non-renal lupus. ICAM-1 level in combination with markers of endothelial cell activation (syndecan-1, hyaluronan and thrombomodulin) was superior to proteinuria, anti-dsDNA, or C3 in distinguishing active LN from quiescent disease. CONCLUSION: Our findings suggest potential utility of serum VCAM-1 and ICAM-1 in clinical management. Monitoring VCAM-1 may facilitate early diagnosis of flare. Combining selected biomarkers may be advantageous in diagnosing active LN. VCAM-1 may have a pathogenic role in renal parenchymal inflammation in active LN.

Pharmacokinetics and pharmacogenomics of mycophenolic acid and its clinical correlations in maintenance immunosuppression for lupus nephritis.

BACKGROUND: There is little data on mycophenolic acid (MPA) pharmacokinetics and pharmacogenomics and optimal MPA exposure in lupus nephritis (LN) patients during long-term maintenance. METHODS: We measured blood MPA levels at 1, 2, 4, 8, 10 and 12-h post-dose (i.e. C1, C2, C4, C8, C10 and C12) in 88 stable LN patients receiving maintenance prednisolone and mycophenolate mofetil, repeated every 6 months. The relationship between MPA exposure and single nucleotide polymorphisms (SNPs) of adenosine triphosphate-binding cassette subfamily C member 2 (ABCC2; rs2273697, rs3740066, rs717620 and rs17222723), organic anion-transporting polypeptides (OATPs; rs7311358 and rs4149117) and uridine diphosphate glucuronosyltransferase (UGT; rs17863762, rs6714486, rs17868320 and rs72551330) was also investigated. RESULTS: C1, C2 and C12 were 8.3 ± 6.6 , 7.2 ± 5.2 and 2.0 ± 1.4 mg/L and all correlated with the 12-h area under the curve (AUC0-12; r = 0.51, 0.85 and 0.73; P = 0.02, <0.001 and <0.001, respectively). C12 inversely correlated with hemoglobin, immunoglobulins and leukocyte levels (P < 0.05 for all). Five renal flares, 11 episodes of infection and 10 episodes of anemia (hemoglobin <10 g/dL) occurred over 96 weeks, with a corresponding C12 of 1.3 ± 0.5, 4.3 ± 2.6 and 2.9 ± 1.5 mg/L, respectively (versus 2.4 ± 1.2, 1.8 ± 1.2 and 1.7 ± 1.1 mg/L in patients without these complications; P = 0.041, <0.001 and 0.004). SNP rs2273697 A/G in the ABCC2 gene was associated with lower MPA exposure compared with G/G (1075.9 ± 239.9 versus 1891.5 ± 918.9 mgh/L per g/kg; P = 0.003). SNPs of OATP and UGT were unrelated to MPA level. CONCLUSION: MPA C12 correlates with the AUC0-12 and is related to renal flare, infection and anemia. SNP rs2273697 A/G is associated with lower MPA exposure.

Effect of mycophenolate and rapamycin on renal fibrosis in lupus nephritis.

Lupus nephritis (LN) leads to chronic kidney disease (CKD) through progressive fibrosis. Mycophenolate inhibits inosine monophosphate dehydrogenase and is a standard treatment for LN. The mammalian or mechanistic target of rapamycin (mTOR) pathway is activated in LN. Rapamycin inhibits mTOR and is effective in preventing kidney transplant rejection, with the additional merits of reduced incidence of malignancies and viral infections. The effect of mycophenolate or rapamycin on kidney fibrosis in LN has not been investigated. We investigated the effects of mycophenolate and rapamycin in New Zealand Black and White first generation (NZB/W F1) murine LN and human mesangial cells (HMCs), focusing on mechanisms leading to kidney fibrosis. Treatment of mice with mycophenolate or rapamycin improved nephritis manifestations, decreased anti-double stranded (ds) DNA antibody titer and reduced immunoglobulin G (IgG) deposition in the kidney. Both mycophenolate and rapamycin, especially the latter, decreased glomerular mTOR Ser2448 phosphorylation. Renal histology in untreated mice showed mesangial proliferation and progressive glomerulosclerosis with tubular atrophy, and increased expression of transforming growth factor ß1 (TGF-ß1), monocyte chemoattractant protein-1 (MCP-1), α-smooth muscle actin (α-SMA), fibronectin (FN) and collagen. Both mycophenolate and rapamycin ameliorated the histopathological changes. Results from in vitro experiments showed that both mycophenolate and rapamycin decreased mesangial cell proliferation and their binding with anti-dsDNA antibodies. Mycophenolate and rapamycin also down-regulated mTOR and extracellular signal-regulated kinase (ERK) phosphorylation and inhibited fibrotic responses in mesangial cells that were induced by anti-dsDNA antibodies or TGF-ß1. Our findings suggest that, in addition to immunosuppression, mycophenolate and rapamycin may reduce fibrosis in LN, which has important implications in preventing CKD in patients with LN.

Blocking Stemness and Metastatic Properties of Ovarian Cancer Cells by Targeting p70S6K with Dendrimer Nanovector-Based siRNA Delivery.

Metastasis is the cause of most (>90%) cancer deaths and currently lacks effective treatments. Approaches to understanding the biological process, unraveling the most effective molecular target(s), and implementing nanotechnology to increase the therapeutic index are expected to facilitate cancer therapy against metastasis. Here, we demonstrate the potential advantages of bringing these three approaches together through the rational design of a small interfering RNA (siRNA) that targets p70S6K in cancer stem cells (CSCs) in combination with dendrimer nanotechnology-based siRNA delivery. Our results demonstrated that the generation 6 (G6) poly(amidoamine) dendrimer can be used as a nanovector to effectively deliver p70S6K siRNA by forming uniform dendriplex nanoparticles that protect the siRNA from degradation. These nanoparticles were able to significantly knock down p70S6K in ovarian CSCs, leading to a marked reduction in CSC proliferation and expansion without obvious toxicity toward normal ovarian surface epithelial cells. Furthermore, treatment with the p70S6K siRNA/G6 dendriplexes substantially decreased mesothelial interaction, migration and invasion of CSCs in vitro, as well as tumor growth and metastasis in vivo. Collectively, these results suggest that p70S6K constitutes a promising therapeutic target, and the use of siRNA in combination with nanotechnology-based delivery may constitute a new approach for molecularly targeted cancer therapy to treat metastasis.

Lupus nephritis: An update on treatments and pathogenesis.

Immunosuppressive therapies for lupus nephritis (LN) have improved significantly over the past few decades, resulting in growing number of choices for treatment individualization and improved renal and patient outcomes. Corticosteroids combined with mycophenolate or cyclophosphamide induces a satisfactory response in a high proportion of Asian and Caucasian patients, but the rate of improvement varies considerably between patients. Relatively low disease flare rate was observed in Chinese patients receiving low-dose prednisolone and mycophenolate maintenance. Short-term results with calcineurin inhibitors (CNI) are encouraging, attributed both to their immunosuppressive efficacy and the action of these agents on podocyte biology leading to more rapid proteinuria suppression. Additional data, especially on the avoidance of nephrotoxicity and metabolic side effects, is required to facilitate selection of patients appropriate for this treatment. Modifications of standard regimens such as reducing corticosteroid exposure or using enteric-coated mycophenolate might help reduce treatment-related toxicities without compromising efficacy. While clinical outcomes of patients have improved with recent therapeutic advances, individual and ethnic variations in disease manifestations and treatment response, as well as the prevention of infections and long-term complications still present challenges to frontline clinicians. Recent data from histological examination and translational studies also suggest that complement activation via the alternative pathway, immune deposition on renal tubular basement membrane, and local inflammatory responses involving resident kidney cells are of pathogenic relevance in LN. The progress of clinical and translational studies has improved not only the understanding of disease mechanisms but also clinical decision making in the management of LN.

Anti-dsDNA antibodies and resident renal cells - Their putative roles in pathogenesis of renal lesions in lupus nephritis.

Lupus nephritis affects up to 70% of patients with systemic lupus erythematosus and is an important treatable cause of kidney failure. Cardinal features of lupus nephritis include loss of self-tolerance, production of autoantibodies, immune complex deposition and immune-mediated injury to the kidney, resulting in increased cell proliferation, apoptosis, and induction of inflammatory and fibrotic processes that destroy normal nephrons. The production anti-dsDNA antibodies is a cardinal feature in lupus and their level correlates with disease activity. In addition to the formation of immune complexes thereby triggering complement activation, how anti-dsDNA antibodies home to the kidney and induce pathological processes in the renal parenchyma remain to be fully elucidated. Data from our laboratory and other investigators show that the properties of anti-dsDNA antibodies vary between patients and change over time, and that anti-dsDNA antibodies could bind directly to integral cell surface molecules such as annexin II or α-actinin, or indirectly through chromatin material deposited on the cell surface. The binding of anti-dsDNA antibodies to mesangial cells and proximal renal tubular epithelial cells triggers downstream inflammatory and fibrotic pathways, which include the activation of the PKC and MAPK signaling pathways, increased secretion of pro-inflammatory cytokines and matrix protein deposition that contribute to pathological processes in the renal parenchyma.

Annexin II-binding immunoglobulins in patients with lupus nephritis and their correlation with disease manifestations.

Annexin II on mesangial cell surface mediates the binding of anti-dsDNA antibodies and consequent downstream inflammatory and fibrotic processes. We investigated the clinical relevance of circulating annexin II-binding immunoglobulins (Igs) in patients with severe proliferative lupus nephritis, and renal annexin II expression in relation to progression of nephritis in New Zealand Black and White F1 mice (NZBWF1/J) mice. Annexin II-binding Igs in serum were measured by ELISA. Ultrastructural localization of annexin II was determined by electron microscopy. Seropositivity rates for annexin II-binding IgG and IgM in patients with active lupus nephritis were significantly higher compared with controls (8.9%, 1.3% and 0.9% for annexin II-binding IgG and 11.1%, 4.0% and 1.9% for annexin II-binding IgM for patients with active lupus nephritis, patients with non-lupus renal disease and healthy subjects respectively). In lupus patients, annexin II-binding IgM level was higher at disease flare compared with remission. Annexin II-binding IgG and IgM levels were associated with that of anti-dsDNA and disease activity. Annexin II-binding IgG and IgM levels correlated with histological activity index in lupus nephritis biopsy samples. In NZBWF1/J mice, serum annexin II-binding IgG and IgM levels and glomerular annexin II and p11 expression increased with progression of active nephritis. Annexin II expression was present on mesangial cell surface and in the mesangial matrix, and co-localized with electron-dense deposits along the glomerular basement membrane. Our results show that circulating annexin II-binding IgG and IgM levels are associated with clinical and histological disease activity in proliferative lupus nephritis. The co-localization of annexin II and p11 expression with immune deposition in the kidney suggests pathogenic relevance.

Binding of anti-dsDNA antibodies to proximal tubular epithelial cells contributes to renal tubulointerstitial inflammation.

Immune deposits are often observed along the tubular basement membrane in patients with lupus nephritis, but the role of anti-dsDNA antibody (Ab) deposition on tubulointerstitial inflammation remains to be investigated. We examined the effect of human polyclonal anti-dsDNA Abs on inflammatory processes in cultured proximal renal tubular epithelial cells (PTEC, HK-2 cells) and their association with serum levels of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) in patients. Binding of anti-dsDNA Abs to HK-2 cells was investigated by cellular ELISA, flow cytometry and immunohistochemistry. IL-6, IL-8 and MCP-1 secretion, mitogen-activated protein kinase (MAPK) activation and the effect of mycophenolic acid (MPA) were investigated by ELISAs and Western blot analysis. NZBWF1/J mice with active nephritis were randomized to receive either mycophenolate mofetil (MMF) (100 mg/kg per day) or vehicle for up to 12 weeks to study renal histopathology focusing on tubulointerstitial changes. Our results demonstrated that anti-dsDNA Abs bound to HK-2 cell surface and induced IL-6, IL-8 and MCP-1 secretion through distinct MAPK pathways. MPA inhibited anti-dsDNA Ab binding to HK-2 cells and suppressed apical and basolateral IL-6 and IL-8, but not MCP-1, secretion. Anti-dsDNA Ab level correlated with serum and tubulointerstitial expression of IL-6, IL-8 and MCP-1. MMF treatment in NZBWF1/J mice reduced anti-dsDNA Ab production and MAPK activation in the renal tubulointerstitium, together with decreased IL-6 and MCP-1 expression. Our data demonstrate that anti-dsDNA Abs contribute to inflammatory processes in the tubulointerstitium in lupus nephritis through their binding to proximal renal tubular epithelial cells and induction of pro-inflammatory mediators, and MPA ameliorates anti-dsDNA Ab induced IL-6 and IL-8 secretion in these cells.

The role of icaritin in regulating Foxp3/IL17a balance in systemic lupus erythematosus and its effects on the treatment of MRL/lpr mice.

Systemic lupus erythematosus (SLE) is a female predominant autoimmune disease characterized by multi-organ dysfunctions. However, current available therapies control the disease at the cost of many potential adverse effects. The development of safer and more effective therapies for SLE is a critical unmet need. Icaritin (ICT) is an active monomer extracted from Chinese herbals named the Epimedium genus. In this study, we found that ICT exhibited the capacity of regulating Foxp3/IL17a balance, enhancing Treg cell suppressive activities, and inhibiting over-activation of CD4(+)T cells from SLE. We also observed that ICT regulated Foxp3/IL17a balance by increasing STAT5b expression and histone methylation modification. Subsequent experiments further confirmed that ICT-treated mice exhibited amelioration of renal damages and suggested that ICT may be a potential new drug for the treatment of SLE.

Increased expression of TLR2 in CD4(+) T cells from SLE patients enhances immune reactivity and promotes IL-17 expression through histone modifications.

The innate immune system has been shown to play an important pathologic role in systemic lupus erythematosus (SLE). TLR2, a PRR, recognizes exogenous PAMPs, and endogenous damage-associated molecular patterns and has been implicated in the initiation and maintenance of the perpetuated inflammatory reactions in autoimmune diseases. Here, we report increased expression of TLR2 in CD4(+) and CD8(+) T cells, CD19(+) B cells, and CD14(+) monocytes from SLE patients. Conventional treatment, such as hydroxychloroquine and corticosteroids, showed no effect on TLR2 expression in CD4(+) T cells from SLE patients. In vitro stimulation of TLR2 in CD4(+) T cells from SLE patients increased CD40L and CD70 expression, as well as secretion of IL-6, IL-17A, IL-17F, and TNF-α, while Foxp3 transcription decreased. This effect was reversed by TLR2 siRNA. Moreover, TLR2 activation upregulated H3K4 tri-methylation and H4 acetylation levels while downregulated H3K9 tri-methylation level in the IL-17A promoter region. In addition, it also increased H4 acetylation levels and decreased H3K9 tri-methylation levels in the IL-17F promoter region. In summary, our findings demonstrate that increased expression of TLR2 contributes to immune reactivity and promotes IL-17A and IL-17F expression through histone modifications in SLE.

The effect of mycophenolic acid on epigenetic modifications in lupus CD4+T cells.

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease involving multiple organs and characterized by overproduction of autoantibodies and T and B cell abnormalities. The treatment for SLE has been restricted to immunosuppressants and corticosteroids. Mycophenolate mofetil (MMF), as a relatively new immunosuppressant, is now widely used in the treatment of SLE patients, particularly those with nephritis. However, it is unclear whether mycophenolic acid (MPA) could modulate the reported disorders of epigenetic status in CD4(+)T cells from SLE patients. In this study, we demonstrated that MPA can upregulate the histone H3/H4 global acetylation status by regulating HATs and HDACs in lupus CD4(+)T cells. Furthermore, we found that MPA also affected the histone H4 acetylation and histone H3K4 tri-methylation levels in CD40L promoter region that inhibited the expression of CD40L. These findings indicate the potential epigenetic mechanism of therapeutic effects of MPA in SLE.

Anti-dsDNA antibody induces soluble fibronectin secretion by proximal renal tubular epithelial cells and downstream increase of TGF-ß1 and collagen synthesis.

The level of anti-dsDNA antibodies correlates with disease activity in lupus nephritis, but their role in pathogenic mechanisms remains to be defined. We investigated the effect of anti-dsDNA antibodies isolated from lupus nephritis patients on fibronectin synthesis and downstream fibrogenesis in proximal renal tubular epithelial cells (PTEC). Kidney biopsies were obtained from patients with active severe proliferative lupus nephritis. In vitro studies with cultured PTEC were performed to investigate the effect of human polyclonal IgG anti-dsDNA antibodies and mycophenolic acid (MPA). The role of IL-6, IL-8, MCP-1, TNF-α, TGF-ß1, and MAPK and PKC signaling pathways on soluble and cell-associated fibronectin synthesis was investigated using neutralizing antibodies or specific inhibitors. The effect of exogenous endotoxin-free soluble fibronectin on downstream fibrotic processes was also examined. Fibronectin expression was markedly increased in the tubulo-interstitium of lupus nephritis renal biopsies and it co-localized with IgG deposition. Anti-dsDNA antibodies significantly increased both secreted and cell-associated fibronectin, through prior activation of ERK, p38 MAPK, JNK, PKC-α and PKC-ßII. There was downstream induction of IL-6, IL-8, MCP-1, TNF-α and TGF-ß1. MPA inhibited the induction of inflammatory and fibrotic processes by anti-dsDNA antibody. Exogenous soluble fibronectin induced TGF-ß1 secretion and type I collagen synthesis in PTEC in a dose-dependent manner. Our data demonstrate that anti-dsDNA antibody contributes to tubulo-interstitial fibrosis in lupus nephritis through its action on PTEC. Anti-dsDNA antibody induces both cell-associated and soluble fibronectin secretion in PTEC, the former adds to extracellular matrix deposition while the latter amplifies the fibrotic process through induction of TGF-ß1 and collagen type I. The pro-fibrotic effects of anti-dsDNA antibody are ameliorated by MPA.

Peripheral whole blood FOXP3 TSDR methylation: a potential marker in severity assessment of autoimmune diseases and chronic infections.

Immune dysregulation is a cardinal feature of autoimmune diseases and chronic microbial infections. In particular, regulatory T cells are downregulated in autoimmune diseases while upregulated in chronic microbial infections. FOXP3 is the master regulator of Treg development. Treg-specific demethylated region (TSDR) is a highly conserved locus on the FOXP3 gene that is fully demethylated in natural Tregs but methylated in effector T cells. In our study, we used high resolution melt-polymerase chain reaction (HRM-PCR) to determine the FOXP3 TSDR methylation status in autoimmune diseases and chronic microbial infections. We found that FOXP3 TSDR to have the highest mean melting temperature (highly methylated) in active SLE patients compared to all the other groups (p < 0.001). The psoriasis group also had a significantly high mean melting temperature (78.62 ± 0.20) when compared with the inactive SLE group (78.49 ± 0.29, p < 0.05) and control group (78.44 ± 0.25, p < 0.01). There was no significant difference in melting temperature between inactive SLE and healthy controls. Disease activity in SLE was directly associated with methylation of the FOXP3 TSDR. On the other hand, patients with chronic microbial infections had significantly lower FOXP3 TSDR mean melting temperature (demethylated) when compared with healthy controls (78.28 ± 0.21 vs 78.44 ± 0.25, p < 0.05). Our results suggest that the use of HRM-PCR to detect FOXP3 TSDR methylation status is a reliable and easy method to predict natural regulatory T cell levels in peripheral blood in different disease conditions. Determining FOXP3 TSDR methylation status can be a useful tool in diagnosis, and monitoring the severity of autoimmune diseases and chronic microbial infections.

DNA methylation and mRNA and microRNA expression of SLE CD4+ T cells correlate with disease phenotype.

Systemic lupus erythematosus (SLE) is an autoimmune disease well known for its clinical heterogeneity, and its etiology secondary to a cross-talk involving genetic predisposition and environmental stimuli. Although genome-wide analysis has contributed greatly to our understanding of the genetic basis of SLE, there is increasing evidence for a role of epigenetics. Indeed, recent data have demonstrated that in patients with SLE, there are striking alterations of DNA methylation, histone modifications, and deregulated microRNA expression, the sum of which contribute to over-expression of select autoimmune-related genes and loss of tolerance. To address this issue at the level of clinical phenotype, we performed DNA methylation, mRNA and microRNA expression screening using high-throughput sequencing of purified CD4+ T cells from patients with SLE, compared to age and sex matched controls. In particular, we studied 42 patients with SLE and divided this group into three clinical phenotypes: a) the presence of skin lesions without signs of systemic pathology; b) skin lesions but also chronic renal pathology; and c) skin lesions, chronic renal pathology and polyarticular disease. Interestingly, and as expected, sequencing data revealed changes in DNA methylation in SLE compared to controls. However, and more importantly, although there were common methylation changes found in all groups of SLE compared to controls, there was specific DNA methylation changes that correlated with clinical phenotype. These included changes in the novel key target genes NLRP2, CD300LB and S1PR3, as well as changes in the critical pathways, including the adherens junction and leukocyte transendothelial migration. We also noted that a significant proportion of genes undergoing DNA methylation changes were inversely correlated with gene expression and that miRNA screening revealed the existence of subsets with changes in expression. Integrated analysis of this data highlights specific sets of miRNAs controlled by DNA methylation, and genes that are altered by methylation and targeted by miRNAs. In conclusion, our findings suggest select epigenetic mechanisms that contribute to clinical phenotypes and further shed light on a new venue for basic SLE research.

Entecavir treatment in kidney transplant recipients infected with hepatitis B.

Although nucleotide/side analogs improve the clinical outcome of hepatitis B surface antigen-positive (HBsAg+) kidney transplant recipients (KTR), a significant proportion of subjects have developed resistance to lamivudine (LAM). We retrospectively analyzed the efficacy and tolerability of entecavir (ETV) in HBsAg+ KTR at Queen Mary Hospital during 2005-2013. Twenty-one patients (10 treatment-naïve, 11 with LAM resistance) were included (duration of ETV treatment 34.7 ± 22.9 months, range 6-75 months). ETV treatment led to a decline of hepatitis B virus (HBV) DNA titer compared to baseline and is more significant in the treatment-naïve group (treatment-naïve: p = 0.028, <0.001 and <0.001; LAM-resistant p = 0.273, 0.180, and 0.109 after 12, 24, and 36 months). The cumulative rate of HBV DNA undetectability at 12, 24, and 36 months was 60%, 100%, and 100% for treatment-naïve group, and 27%, 45%, and 45% for LAM-resistant group, respectively. Time-to-HBV DNA undetectability and time-to-alanine transaminase (ALT) normalization were 15.7 ± 4.6 and 12.6 ± 3.7 months for treatment-naïve patients, and 24.5 ± 4.2 and 28.2 ± 3.5 months for those with LAM resistance. Genotypic resistance to ETV emerged after 20.0 ± 3.5 months with increase in ALT and HBV DNA in two patients with LAM resistance, but was not observed in the treatment-naïve group. Allograft dysfunction, de novo cirrhosis, or hepatocellular carcinoma did not occur during follow-up.

Targeting gonadotropin-releasing hormone receptor inhibits the early step of ovarian cancer metastasis by modulating tumor-mesothelial adhesion.

Ovarian cancer has a clear predilection to metastasize to the peritoneum, which represents one of the most important prognostic factors of poor clinical outcome. Gonadotropin-releasing hormone (GnRH) receptor is significantly overexpressed during the malignant progression of human ovarian cancer. Here, using lentiviral-based small interfering RNA (siRNA) technology to downregulate GnRH receptor in metastatic ovarian cancer cells, we show that GnRH receptor is an important mediator of ovarian cancer peritoneal metastasis. GnRH receptor downregulation dramatically attenuated their adhesion to the peritoneal mesothelium. By inhibiting the expression of GnRH receptor, we showed decreased expression of α2ß1 and α5ß1 integrin and adhesion to specific extracellular matrix (ECM) proteins. This was also associated with a reduction of P-cadherin. Furthermore, adhesion of ovarian cancer cells to different ECMs and the mesothelium were abrogated in response to ß1 integrin and P-cadherin reduction, confirming that the effects were ß1 integrin- and P-cadherin-specific. Using a mouse model of human ovarian cancer metastasis, we found that the inhibition of GnRH receptor, ß1 integrin, and P-cadherin significantly attenuated tumor growth, ascites formation, and the number of metastatic implants. These results define a new role for GnRH receptor in early metastasis and offer the possibility of novel therapeutic targets.