Ivan Todorov

TGF-beta activates Akt kinase through a microRNA-dependent amplifying circuit targeting PTEN.

Akt kinase is activated by transforming growth factor-β1 (TGF-β) in diabetic kidneys, and has important roles in fibrosis, hypertrophy and cell survival in glomerular mesangial cells. However, the mechanisms of Akt activation by TGF-β are not fully understood. Here we show that TGF-β activates Akt in glomerular mesangial cells by inducing the microRNAs (miRNAs) miR-216a and miR-217, both of which target PTEN (phosphatase and tensin homologue), an inhibitor of Akt activation. These miRNAs are located within the second intron of a non-coding RNA (RP23-298H6.1-001). The RP23 promoter was activated by TGF-β and miR-192 through E-box-regulated mechanisms, as shown previously. Akt activation by these miRs led to glomerular mesangial cell survival and hypertrophy, which were similar to the effects of activation by TGF-β. These studies reveal a mechanism of Akt activation through PTEN downregulation by two miRs, which are regulated by upstream miR-192 and TGF-β. Due to the diversity of PTEN function, this miR-amplifying circuit may have key roles, not only in kidney disorders, but also in other diseases.

Reciprocal differentiation and tissue-specific pathogenesis of Th1, Th2, and Th17 cells in graft-versus-host disease

In acute graft-versus-host disease (GVHD), naive donor CD4(+) T cells recognize alloantigens on host antigen-presenting cells and differentiate into T helper (Th) subsets (Th1, Th2, and Th17 cells), but the role of Th subsets in GVHD pathogenesis is incompletely characterized. Here we report that, in an MHC-mismatched model of C57BL/6 donor to BALB/c recipient, WT donor CD4(+) T cells predominantly differentiated into Th1 cells and preferentially mediated GVHD tissue damage in gut and liver. However, absence of interferon-gamma (IFN-gamma) in CD4(+) T cells resulted in augmented Th2 and Th17 differentiation and exacerbated tissue damage in lung and skin; absence of both IL-4 and IFN-gamma resulted in augmented Th17 differentiation and preferential, although not exclusive, tissue damage in skin; and absence of both IFN-gamma and IL-17 led to further augmentation of Th2 differentiation and idiopathic pneumonia. The tissue-specific GVHD mediated by Th1, Th2, and Th17 cells was in part associated with their tissue-specific migration mediated by differential expression of chemokine receptors. Furthermore, lack of tissue expression of the IFN-gamma-inducible B7-H1 played a critical role in augmenting the Th2-mediated idiopathic pneumonia. These results indicate donor CD4(+) T cells can reciprocally differentiate into Th1, Th2, and Th17 cells that mediate organ-specific GVHD.

Absence of donor Th17 leads to augmented Th1 differentiation and exacerbated acute graft versus host disease

Th17 is a newly identified T-cell lineage that secretes proinflammatory cytokine IL-17. Th17 cells have been shown to play a critical role in mediating autoimmune diseases such as EAE, colitis, and arthritis, but their role in the pathogenesis of graft-versus-host disease (GVHD) is still unknown. Here we showed that, in an acute GVHD model of C57BL/6 (H-2(b)) donor to BALB/c (H-2(d)) recipient, IL-17(-/-) donor T cells manifested an augmented Th1 differentiation and IFN-gamma production and induced exacerbated acute GVHD. Severe tissue damage mediated by IL-17(-/-) donor T cells was associated with increased Th1 infiltration, up-regulation of chemokine receptors by donor T cells, and enhanced tissue expression of inflammatory chemokines. Administration of recombinant IL-17 and neutralizing IFN-gamma in the recipients given IL-17(-/-) donor cells ameliorated the acute GVHD. Furthermore, the regulation of Th1 differentiation by IL-17 or Th17 may be through its influence on host DCs. Our results indicate that donor Th17 cells can down-regulate Th1 differentiation and ameliorate acute GVHD in allogeneic recipients, and that treatments neutralizing proinflammatory cytokine IL-17 may augment acute GVHD as well as other inflammatory autoimmune diseases.

Insulin gene expression is regulated by DNA methylation.

Background Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression. Methodology/Principal Findings Genomic DNA samples from several tissues were bisulfite-treated and sequenced which revealed that cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in insulin-producing pancreatic beta cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2). Examination of the Ins2 gene in mouse embryonic stem cell cultures revealed that it is fully methylated and becomes demethylated as the cells differentiate into insulin-expressing cells in vitro. Conclusions/Significance Our findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.

Post-transcriptional Up-regulation of Tsc-22 by Ybx1, a Target of miR-216a, Mediates TGF-β-induced Collagen Expression in Kidney Cells

Increased accumulation of extracellular matrix proteins and hypertrophy induced by transforming growth factor-β1 (TGF-β) in renal mesangial cells (MC) are hallmark features of diabetic nephropathy. Although the post-transcriptional regulation of key genes has been implicated in these events, details are not fully understood. Here we show that TGF-β increased microRNA-216a (miR-216a) levels in mouse MC, with parallel down-regulation of Ybx1, a miR-216a target and RNA-binding protein. TGF-β also enhanced protein levels of Tsc-22 (TGF-β-stimulated clone 22) and collagen type I α-2 (Col1a2) expression in MC through far upstream enhancer E-boxes by interaction of Tsc-22 with an E-box regulator, Tfe3. Ybx1 colocalized with processing bodies in MC and formed a ribonucleoprotein complex with Tsc-22 mRNA, and this complex formation was reduced by TGF-β, miR-216a mimics, or Ybx1 shRNA to increase Tsc-22 protein levels but enhanced by miR-216a inhibitor oligonucleotides. Chromatin immunoprecipitation (ChIP) assays revealed that TGF-β could increase the occupancies of Tsc-22 and Tfe3 on enhancer E-boxes of Col1a2. Co-immunoprecipitation assays revealed that TGF-β promoted the interaction of Tsc-22 with Tfe3. These results demonstrate that post-transcriptional regulation of Tsc-22 mediated through Ybx1, a miR-216a target, plays a key role in TGF-β-induced Col1a2 in MC related to the pathogenesis of diabetic nephropathy.

Recognition and Killing of Brain Tumor Stem-Like Initiating Cells by CD8+ Cytolytic T Cells

Solid tumors contain a subset of stem-like cells that are resistant to the cytotoxic effects of chemotherapy/radiotherapy, but their susceptibility to cytolytic T lymphocyte (CTL) effector mechanisms has not been well characterized. Using a panel of early-passage human brain tumor stem/initiating cell (BTSC) lines derived from high-grade gliomas, we show that BTSCs are subject to immunologic recognition and elimination by CD8(+) CTLs. Compared with serum-differentiated CD133(low) tumor cells and established glioma cell lines, BTSCs are equivalent with respect to expression levels of HLA class I and ICAM-1, similar in their ability to trigger degranulation and cytokine synthesis by antigen-specific CTLs, and equally susceptible to perforin-dependent CTL-mediated cytolysis. BTSCs are also competent in the processing and presentation of antigens as evidenced by the killing of these cells by CTL when antigen is endogenously expressed. Moreover, we show that CTLs can eliminate all BTSCs with tumor-initiating activity in an antigen-specific manner in vivo. Current models predict that curative therapies for many cancers will require the elimination of the stem/initiating population, and these studies lay the foundation for developing immunotherapeutic approaches to eradicate this tumor population.

Inhibition of p38 Pathway Suppresses Human Islet Production of Pro-Inflammatory Cytokines and Improves Islet Graft Function

Nonspecific inflammation is associated with primary graft nonfunction (PNF). Inflammatory islet damage is mediated at least partially by pro‐inflammatory cytokines, such as interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) produced by resident islet macrophages. The p38 pathway is known to be involved in cytokine production in the cells of the monocyte–macrophage lineage. Therefore, inhibition of the p38 pathway may prevent pro‐inflammatory cytokine production by resident islet macrophages and possibly reduce the incidence of PNF. Our present study has demonstrated that inhibition of the p38 pathway by a chemical p38 inhibitor, SB203580, suppresses IL‐1β and TNF‐α production in human islets exposed to lipopolysaccharide (LPS) and/or inflammatory cytokines. Although IL‐1β is predominantly produced by resident macrophages, ductal cells and islet vascular endothelial cells were found to be another cellular source of IL‐1β in isolated human islets. SB203580 also inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in the treated islets. Furthermore, human islets treated with SB203580 for 1 h prior to transplantation showed significantly improved graft function. These results suggest that inhibition of the p38 pathway may become a new therapeutic strategy to improve graft survival in clinical islet transplantation.

Mesenchymal Stem Cells Facilitate the Induction of Mixed Hematopoietic Chimerism and Islet Allograft Tolerance without GVHD in the Rat

Induction of hematopoietic chimerism and subsequent donor‐specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type‐1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft‐versus‐host disease (GVHD). Streptozotocin‐diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co‐infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor‐specific immune tolerance, shown by the engraftment of donor skin and second‐set islet transplants and acute rejection of a third‐party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2‐week peritransplant administration of 15‐deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (0/15), whereas it occurred in 4/11 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.

HDAC inhibitor reduces cytokine storm and facilitates induction of chimerism that reverses lupus in anti-CD3 conditioning regimen.

In allogeneic hematopoietic cell transplantation (HCT), donor T cell-mediated graft versus host leukemia (GVL) and graft versus autoimmune (GVA) activity play critical roles in treatment of hematological malignancies and refractory autoimmune diseases. However, graft versus host disease (GVHD), which sometimes can be fatal, remains a major obstacle in classical HCT, where recipients are conditioned with total body irradiation or high-dose chemotherapy. We previously reported that anti-CD3 conditioning allows donor CD8+ T cells to facilitate engraftment and mediate GVL without causing GVHD. However, the clinical application of this radiation-free and GVHD preventative conditioning regimen is hindered by the cytokine storm syndrome triggered by anti-CD3 and the high-dose donor bone marrow (BM) cells required for induction of chimerism. Histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA) are known to induce apoptosis of cancer cells and reduce production of proinflammatory cytokines by nonmalignant cells. Here, we report that SAHA inhibits the proliferative and cytotoxic activity of anti-CD3-activated T cells. Administration of low-dose SAHA reduces cytokine production and ameliorates the cytokine storm syndrome triggered by anti-CD3. Conditioning with anti-CD3 and SAHA allows induction of chimerism with lower doses of donor BM cells in old nonautoimmune and autoimmune lupus mice. In addition, conditioning with anti-CD3 and SAHA allows donor CD8+ T cell-mediated GVA activity to reverse lupus glomerulonephritis without causing GVHD. These results indicate that conditioning with anti-CD3 and HDAC inhibitors represent a radiation-free and GVHD-preventative regimen with clinical application potential.

MCM2 - a promising marker for premalignant lesions of the lung: a cohort study

BackgroundBecause cells progressing to cancer must proliferate, marker proteins specific to proliferating cells may permit detection of premalignant lesions. Here we compared the sensitivities of a classic proliferation marker, Ki-67, with a new proliferation marker, MCM2, in 41 bronchial biopsy specimens representing normal mucosa, metaplasia, dysplasia, and carcinoma in situ.MethodsParallel sections were stained with antibodies against MCM2 and Ki-67, and the frequencies of staining were independently measured by two investigators. Differences were evaluated statistically using the two-sided correlated samples t-test and Wilcoxon rank sum test.ResultsFor each of the 41 specimens, the average frequency of staining by anti-MCM2 (39%) was significantly (p < 0.001) greater than by anti-Ki-67 (16%). In metaplastic lesions anti-MCM2 frequently detected cells near the epithelial surface, while anti-Ki-67 did not.ConclusionsWe conclude that MCM2 is detectable in 2-3 times more proliferating premalignant lung cells than is Ki-67. The promise of MCM2 as a sensitive marker for premalignant lung cells is enhanced by the fact that it is present in cells at the surface of metaplastic lung lesions, which are more likely to be exfoliated into sputum. Future studies will determine if use of anti-MCM2 makes possible sufficiently early detection to significantly enhance lung cancer survival rates.