Intragraft memory-like CD127hiCD4+Foxp3+ Tregs maintain transplant tolerance.

CD4+Foxp3+ regulatory T cells (Tregs) play an essential role in suppressing transplant rejection, but their role within the graft and heterogeneity in tolerance are poorly understood. Here, we compared phenotypic and transcriptomic characteristics of Treg populations within lymphoid organs and grafts in an islet xenotransplant model of tolerance. We showed Tregs were essential for tolerance induction and maintenance. Tregs demonstrated heterogeneity within the graft and lymphoid organs of tolerant mice. A subpopulation of CD127hi Tregs with memory features were found in lymphoid organs, presented in high proportions within long-surviving islet grafts, and had a transcriptomic and phenotypic profile similar to tissue Tregs. Importantly, these memory-like CD127hi Tregs were better able to prevent rejection by effector T cells, after adoptive transfer into secondary Rag-/- hosts, than naive Tregs or unselected Tregs from tolerant mice. Administration of IL-7 to the CD127hi Treg subset was associated with a strong activation of phosphorylation of STAT5. We proposed that memory-like CD127hi Tregs developed within the draining lymph node and underwent further genetic reprogramming within the graft toward a phenotype that had shared characteristics with other tissue or tumor Tregs. These findings suggested that engineering Tregs with these characteristics either in vivo or for adoptive transfer could enhance transplant tolerance.

Interleukin-33 Exacerbates IgA Glomerulonephritis in Transgenic Mice Overexpressing B Cell Activating Factor.

BACKGROUND: The cytokine IL-33 is an activator of innate lymphoid cells 2 (ILC2s) in innate immunity and allergic inflammation. B cell activating factor (BAFF) plays a central role in B cell proliferation and differentiation, and high levels of this protein cause excess antibody production, including IgA. BAFF-transgenic mice overexpress BAFF and spontaneously develop glomerulonephritis that resembles human IgA nephropathy. METHODS: We administered IL-33 or PBS to wild-type and BAFF-transgenic mice. After treating Rag1-deficient mice with IL-33, with or without anti-CD90.2 to preferentially deplete ILC2s, we isolated splenocytes, which were adoptively transferred into BAFF-transgenic mice. RESULTS: BAFF-transgenic mice treated with IL-33 developed more severe kidney dysfunction and proteinuria, glomerular sclerosis, tubulointerstitial damage, and glomerular deposition of IgA and C3. Compared with wild-type mice, BAFF-transgenic mice exhibited increases of CD19+ B cells in spleen and kidney and ILC2s in kidney and intestine, which were further increased by administration of IL-33. Administering IL-33 to wild-type mice had no effect on kidney function or histology, nor did it alter the number of ILC2s in spleen, kidney, or intestine. To understand the role of ILC2s, splenocytes were transferred from IL-33-treated Rag1-deficient mice into BAFF-transgenic mice. Glomerulonephritis and IgA deposition were exacerbated by transfer of IL-33-stimulated Rag1-deficient splenocytes, but not by ILC2 (anti-CD90.2)-depleted splenocytes. Wild-type mice infused with IL-33-treated Rag1-deficient splenocytes showed no change in kidney function or ILC2 numbers or distribution. CONCLUSIONS: IL-33-expanded ILC2s exacerbated IgA glomerulonephritis in a mouse model. These findings indicate that IL-33 and ILC2s warrant evaluation as possible mediators of human IgA nephropathy.

Antigen Specific Regulatory T Cells in Kidney Transplantation and Other Tolerance Settings.

Kidney transplantation is the most common solid organ transplant and the best current therapy for end-stage kidney failure. However, with standard immunosuppression, most transplants develop chronic dysfunction or fail, much of which is due to chronic immune injury. Tregs are a subset of T cells involved in limiting immune activation and preventing autoimmune disease. These cells offer the potential to provide tolerance or to allow reduction in immunosuppression in kidney transplants. The importance of Tregs in kidney transplantation has been shown in a number of seminal mouse and animal studies, including those with T cell receptors (TCRs) transgenic Tregs (TCR-Tregs) or Chimeric Antigen Receptor (CAR) Tregs (CAR-Tregs) showing that specificity increases the potency of Treg function. Here we outline the animal and human studies and clinical trials directed at using Tregs in kidney transplantation and other tolerance settings and the various modifications to enhance allo-specific Treg function in vivo and in vitro.

Conventional Type 1 Dendritic Cells (cDC1) in Human Kidney Diseases: Clinico-Pathological Correlations.

Background: cDC1 is a subset of conventional DCs, whose most recognized function is cross-presentation to CD8+ T cells. We conducted this study to investigate the number and location of cDC1s in various human kidney diseases as well as their correlation with clinico-pathological features and CD8+ T cells. Methods: We analyzed 135 kidney biopsies samples. Kidney diseases included: acute tubular necrosis (ATN), acute interstitial nephritis (AIN), proliferative glomerulonephritis (GN) (IgA nephropathy, lupus nephritis, pauci-immune GN, anti-GBM disease), non-proliferative GN (minimal change disease, membranous nephropathy) and diabetic nephropathy. Indirect immunofluorescence staining was used to quantify cDC1s, CD1c+ DCs, and CD8+ T cells. Results: cDC1s were rarely present in normal kidneys. Their number increased significantly in ATN and proliferative GN, proportionally much more than CD1c+ DCs. cDC1s were mainly found in the interstitium, except in lupus nephritis, pauci-immune GN and anti-GBM disease, where they were prominent in glomeruli and peri-glomerular regions. The number of cDC1s correlated with disease severity in ATN, number of crescents in pauci-immune GN, interstitial fibrosis in IgA nephropathy and lupus nephritis, as well as prognosis in IgA nephropathy. The number of CD8+ T cells also increased significantly in these conditions and cDC1 number correlated with CD8+ T cell number in lupus nephritis and pauci-immune GN, with many of them closely co-localized. Conclusions: cDC1 number correlated with various clinic-pathological features and prognosis reflecting a possible role in these conditions. Their association with CD8+ T cells suggests a combined mechanism in keeping with the results in animal models.

Comparison of 4 sinus augmentation techniques for implant placement with residual alveolar bone height ≤3 mm.

This study compared implant outcomes following maxillary sinus floor augmentation (MSFA) in edentulous patients with a residual alveolar bone height ≤3 mm. Four techniques were evaluated: 1-stage bone-added osteotome sinus floor elevation procedure (BAOSFE) with simultaneous implant placement; 2-stage BAOSFE with delayed implant placement; 1-stage lateral window sinus floor elevation with simultaneous implant placement; and 2-stage lateral window sinus floor elevation with delayed implant placement. Patients were followed for 18 to 72 months (mean: 52.5 months) after prosthesis placement. Data were analyzed with cone-beam computed tomography. A total of 96 implants from 71 patients were analyzed; pretreatment, there were no significant differences between patients. Total implant survival was 98.9%. The mean residual bone height was significantly higher in the 1-stage BAOSFE group than the other groups (P < .01); 1 implant in this group failed at 3 months. There was no significant difference in total bone height gain between groups. However, the bone height gain of 1st sinus lifting with 2-stage BAOSFE was significantly lower than the 2-stage lateral window procedure (P < .01). There was no prosthesis failure. The favorable implant outcomes suggest these 1-stage and 2-stage MSFA procedures should be considered as alternative treatment options for patients with extremely atrophic posterior maxilla.

Indirectly Activated Treg Allow Dominant Tolerance to Murine Skin-grafts Across an MHC Class I Mismatch After a Single Donor-specific Transfusion.

BACKGROUND: Tolerance induced in stringent animal transplant models using donor-specific transfusions (DST) has previously required additional immunological manipulation. Here, we demonstrate a dominant skin-allograft tolerance model induced by a single DST across an major histocompatibility class I mismatch in an unmanipulated B6 host. METHODS: C57BL/6 (H-2) (B6) mice were injected intravenously with splenocytes from B6.C.H-2 (H-2k) (bm1) or F1 (B6 × bm1) mice before skin transplantation. Mice were transplanted 7 days postinjection with donor (bm1 or F1) and third-party B10.BR (H-2) skin grafts. RESULTS: B6 hosts acutely rejected skin grafts from B6.C.H-2 (bm1) and F1 (B6 × bm1) mice. A single transfusion of F1 splenocytes into B6 mice without any additional immune modulation led to permanent acceptance of F1 skin grafts. This graft acceptance was associated with persistence of donor cells long-term in vivo. The more rapid removal of DST bm1 cells than F1 cells was reduced by natural killer-cell depletion. Tolerant grafts survived an in vivo challenge with naive splenocytes. Both CD4CD25 and CD4CD25 T cells from F1 DST treated B6 mice suppressed alloproliferation in vitro. Tolerance was associated with expansion of peripheral Foxp3CD4CD25 regulatory T cells (Treg) and increased forkhead box P3 (Foxp3) expression in tolerant grafts. In tolerant mice, Foxp3 Treg arises from the proliferation of indirectly activated natural Foxp3 Treg (nTreg) and depletion of Foxp3 Treg abrogates skin-graft tolerance. CONCLUSIONS: This study demonstrates that the persistence of transfused semiallogeneic donor cells mismatched at major histocompatibility class I can enhance tolerance to subsequent skin allografts through indirectly expanded nTreg leading to dominant tolerance without additional immunological manipulation.

Regulatory innate lymphoid cells suppress innate immunity and reduce renal ischemia/reperfusion injury.

Innate lymphoid cells are a recently recognized group of immune cells with critical roles in tissue homeostasis and inflammation. Regulatory innate lymphoid cells are a newly identified subset of innate lymphoid cells, which play a suppressive role in the innate immune response, favoring the resolution of intestinal inflammation. However, the expression and role of regulatory innate lymphoid cells in kidney has not been reported. Here, we show that regulatory innate lymphoid cells are present in both human and mouse kidney, express similar surface markers and form a similar proportion of total kidney innate lymphoid cells. Regulatory innate lymphoid cells from kidney were expanded in vitro with a combination of IL-2, IL-7 and transforming growth factor-ß. These cells exhibited immunosuppressive effects on innate immune cells via secretion of IL-10 and transforming growth factor-ß. Moreover, treatment with IL-2/IL-2 antibody complexes (IL-2C) promoted expansion of regulatory innate lymphoid cells in vivo, and prevent renal ischemia/reperfusion injury in Rag-/- mice that lack adaptive immune cells including Tregs. Depletion of regulatory innate lymphoid cells with anti-CD25 antibody abolished the beneficial effects of IL-2C in the Rag-/- mice. Adoptive transfer of ex vivo expanded regulatory innate lymphoid cells improved renal function and attenuated histologic damage when given before or after induction of ischemia/reperfusion injury in association with reduction of neutrophil infiltration and induction of reparative M2 macrophages in kidney. Thus, our study shows that regulatory innate lymphoid cells suppress innate renal inflammation and ischemia/reperfusion injury.

Cone-beam computed tomography assessment of Schneiderian membranes: Non-infected and infected membranes, and membrane resolution following tooth extraction: A retrospective clinical trial.

BACKGROUND: Cone-beam computed tomography (CBCT) presurgical assessment on the maxillary sinus can reduce the possibility of Schneiderian membrane perforation. This study examined Schneiderian membrane thickness (SMT) and its relationship with neighboring hard tissues for patients with and without membrane thickening. For patients with sinus infections, we evaluated dimensional changes of the SMT post-extraction relative to pre-extraction SMT and residual bone height (RBH). METHODS: CBCT images from 93 patients needing single-tooth implant reconstruction without (n = 83) and with (n = 14) odontogenic infected maxillary sinuses were assessed. SMT, RBH, and lateral wall thickness (LWT) were measured. Causes of extraction, RBH in the infection site, and retrospective post-extraction record of SMT were recorded for the thickened SMT group. RESULTS: Mean SMT for normal SMT group was 1.13 ± 0.43 mm, RBH was 6.26 ± 2.38 mm; upper and lower LWT was 1.85 ± 0.95 mm, and 3.07 ± 2.26 mm, respectively. RBH and LWT had no significant relationships with SMT. For thickened SMT group, mean values for SMT and RBH prior to extraction were 4.53 ± 2.46 mm and 1.97 ± 1.43 mm, respectively. Pre-extraction SMT had a moderately negative correlation with pre-extraction RBH. SMT resolution in thickened SMT group was observed by 2.80 ± 1.37 months post-extraction; post-extraction SMT was not significantly different from normal SMT group (p = .187). CONCLUSIONS: Within the limitation of the sample size, thickened SMT induced by odontogenic infection subsides about 3 months following tooth extraction, and further sinus lifting implant surgery may be considered.

Promotion of ß-catenin/Foxo1 signaling ameliorates renal interstitial fibrosis.

Transforming growth factor ß (TGF-ß) is the key cytokine involved in causing fibrosis through cross-talk with major profibrotic pathways. However, inhibition of TGF-ß to prevent fibrosis would also abrogate its anti-inflammatory and wound-healing effects. ß-catenin is a common co-factor in most TGF-ß signaling pathways. ß-catenin binds to T-cell factor (TCF) to activate profibrotic genes and binds to Forkhead box O (Foxo) to promote cell survival under oxidative stress. Using a proximity ligation assay in human kidney biopsies, we found that ß-catenin/Foxo interactions were higher in kidney with little fibrosis, whereas ß-catenin/TCF interactions were upregulated in the kidney of patients with fibrosis. We hypothesised that ß-catenin/Foxo is protective against kidney fibrosis. We found that Foxo1 protected against rhTGF-ß1-induced profibrotic protein expression using a CRISPR/cas9 knockout of Foxo1 or TCF1 in murine kidney tubular epithelial C1.1 cells. Co-administration of TGF-ß with a small molecule inhibitor of ß-catenin/TCF (ICG-001), protected against kidney fibrosis in unilateral ureteral obstruction. Collectively, our human, animal and in vitro findings suggest ß-catenin/Foxo as a therapeutic target in kidney fibrosis.

Dendritic cell-targeted CD40 DNA vaccine suppresses Th17 and ameliorates progression of experimental autoimmune glomerulonephritis.

The CD40-CD40L costimulatory pathway is critical for T cell activation in autoimmune disease. We have previously found that blocking the CD40-CD40L pathway using a dendritic cell-targeted CD40 DNA (DEC-CD40) vaccine prevented the development of Heymann nephritis. In this study, we explored the effect of a DEC-CD40 vaccine in the treatment of experimental autoimmune glomerulonephritis (EAG), an animal model of human Goodpasture's disease induced by antigen α3IV-NC1. DEC-CD40 vaccine given at week 3 and week 6 after 3IV-NC1 injection reduced kidney structural and functional injury significantly in EAG. DEC-CD40 vaccination suppressed Th17 cell numbers and Th17 immune responses in kidney and spleen, but did not alter Th1 cells number and responses. Serum derived from rats with DEC-CD40 vaccination suppressed Th17 differentiation, but not Th1 differentiation in vitro. Furthermore, B cell activation, driven by Th17 cytokines, was suppressed by serum from rats vaccinated with DEC-CD40. A DNA vaccine encoding CD40 and targeting dendritic cell, ameliorates kidney injury in both early and late stages in EAG rats, indicating DEC-CD40 vaccination has a therapeutic role in EAG. Its effect is associated with the reduction of Th17 differentiation and Th17-mediated B cell activation.

Flt3 inhibition alleviates chronic kidney disease by suppressing CD103+ dendritic cell-mediated T cell activation.

BACKGROUND: Chronic kidney disease (CKD) is a global public health problem, which lacks effective treatment. Previously, we have shown that CD103+ dendritic cells (DCs) are pathogenic in adriamycin nephropathy (AN), a model of human focal segmental glomerulosclerosis (FSGS). Fms-like tyrosine kinase 3 (Flt3) is a receptor that is expressed with high specificity on tissue resident CD103+ DCs. METHODS: To test the effect on CD103+ DCs and kidney injury of inhibition of Flt3, we used a selective Flt3 inhibitor (AC220) to treat mice with AN. RESULTS: Human CD141+ DCs, homologous to murine CD103+ DCs, were significantly increased in patients with FSGS. The number of kidney CD103+ DCs, but not CD103- DCs or plasmacytoid DCs, was significantly decreased in AN mice after AC220 administration. Treatment with AC220 significantly improved kidney function and reduced kidney injury and fibrosis in AN mice. AC220-treated AN mice had decreased levels of inflammatory cytokines and chemokines, tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, CCL2 and CCL5 and reduced kidney infiltration of CD4 T cells and CD8 T cells. The protective effect of AC220 was associated with its suppression of CD103+ DCs-mediated CD8 T cell proliferation and activation in AN mice. CONCLUSION: Flt3 inhibitor AC220 effectively reduced kidney injury in AN mice, suggesting that this inhibitor might be a useful pharmaceutical agent to treat CKD.

DEC205-DC targeted DNA vaccine against CX3CR1 protects against atherogenesis in mice.

Studies disrupting the chemokine pathway CX3CL1 (fractalkine)/ CX3CR1 have shown decreased atherosclerosis in animal models but the techniques used to interrupt the pathway have not been easily translatable into human trials. DNA vaccination potentially overcomes the translational difficulties. We evaluated the effect of a DNA vaccine, targeted to CX3CR1, on atherosclerosis in a murine model and examined possible mechanisms of action. DNA vaccination against CX3CR1, enhanced by dendritic cell targeting using DEC-205 single chain variable region fragment (scFv), was performed in 8 week old ApoE-/- mice, fed a normal chow diet. High levels of anti-CX3CR1 antibodies were induced in vaccinated mice. There were no apparent adverse reactions to the vaccine. Arterial vessels of 34 week old mice were examined histologically for atherosclerotic plaque size, macrophage infiltration, smooth muscle cell infiltration and lipid deposition. Vaccinated mice had significantly reduced atherosclerotic plaque in the brachiocephalic artery. There was less macrophage infiltration but no significant change to the macrophage phenotype in the plaques. There was less lipid deposition in the lesions, but there was no effect on smooth muscle cell migration. Targeted DNA vaccination to CX3CR1 was well tolerated, induced a strong immune response and resulted in attenuated atherosclerotic lesions with reduced macrophage infiltration. DNA vaccination against chemokine pathways potentially offers a potential therapeutic option for the treatment of atherosclerosis.

Potentiating Tissue-Resident Type 2 Innate Lymphoid Cells by IL-33 to Prevent Renal Ischemia-Reperfusion Injury.

The IL-33-type 2 innate lymphoid cell (ILC2) axis has an important role in tissue homeostasis, inflammation, and wound healing. However, the relative importance of this innate immune pathway for immunotherapy against inflammation and tissue damage remains unclear. Here, we show that treatment with recombinant mouse IL-33 prevented renal structural and functional injury and reduced mortality in mice subjected to ischemia-reperfusion injury (IRI). Compared with control-treated IRI mice, IL-33-treated IRI mice had increased levels of IL-4 and IL-13 in serum and kidney and more ILC2, regulatory T cells (Tregs), and anti-inflammatory (M2) macrophages. Depletion of ILC2, but not Tregs, substantially abolished the protective effect of IL-33 on renal IRI. Adoptive transfer of ex vivo-expanded ILC2 prevented renal injury in mice subjected to IRI. This protective effect associated with induction of M2 macrophages in kidney and required ILC2 production of amphiregulin. Treatment of mice with IL-33 or ILC2 after IRI was also renoprotective. Furthermore, in a humanized mouse model of renal IRI, treatment with human IL-33 or transfer of ex vivo-expanded human ILC2 ameliorated renal IRI. This study has uncovered a major protective role of the IL-33-ILC2 axis in renal IRI that could be potentiated as a therapeutic strategy.

Redirecting TGF-ß Signaling through the ß-Catenin/Foxo Complex Prevents Kidney Fibrosis.

TGF-ß is a key profibrotic factor, but targeting TGF-ß to prevent fibrosis also abolishes its protective anti-inflammatory effects. Here, we investigated the hypothesis that we can redirect TGF-ß signaling by preventing downstream profibrotic interaction of ß-catenin with T cell factor (TCF), thereby enhancing the interaction of ß-catenin with Foxo, a transcription factor that controls differentiation of TGF-ß induced regulatory T cells (iTregs), and thus, enhance anti-inflammatory effects of TGF-ß In iTregs derived from EL4 T cells treated with recombinant human TGF-ß1 (rhTGF-ß1) in vitro, inhibition of ß-catenin/TCF transcription with ICG-001 increased Foxp3 expression, interaction of ß-catenin and Foxo1, binding of Foxo1 to the Foxp3 promoter, and Foxo transcriptional activity. Moreover, the level of ß-catenin expression positively correlated with the level of Foxo1 binding to the Foxp3 promoter and Foxo transcriptional activity. T cell fate mapping in Foxp3gfp Ly5.1/5.2 mice revealed that coadministration of rhTGF-ß1 and ICG-001 further enhanced the expansion of iTregs and natural Tregs observed with rhTGF-ß1 treatment alone. Coadministration of rhTGF-ß1 with ICG-001 also increased the number of Tregs and reduced inflammation and fibrosis in the kidney fibrosis models of unilateral ureteric obstruction and ischemia-reperfusion injury. Notably, ICG-001 prevented the fibrosis in distant organs (lung and liver) caused by rhTGF-ß1. Together, our results show that diversion of ß-catenin from TCF- to Foxo-mediated transcription inhibits the ß-catenin/TCF-mediated profibrotic effects of TGF-ß while enhancing the ß-catenin/Foxo-mediated anti-inflammatory effects. Targeting ß-catenin/Foxo may be a novel therapeutic strategy in the treatment of fibrotic diseases that lead to organ failure.

Evaluation of the Mandibular Lingual Canal and Anterior Loop Length to Minimize Complications Associated With Anterior Mandibular Surgeries: A Cone-Beam Computed Tomography Study.

PURPOSE: This retrospective study evaluated the localization, incidence, and dimensions of the mandibular lingual canal and the anterior loop in the Taiwanese population using the simulation and visual interpretation of cone-beam computed tomography to minimize complications during symphysis block surgical procedures. MATERIALS AND METHODS: The sample population consisted of 215 patients (105 men and 110 women; mean age, 57 yr). The median lingual canal, symphysis bone thickness, and anterior loop length were defined and calculated using cone-beam computed tomography and 3-dimensional reconstructed images. The correlation of all data for men and women was assessed and analyzed statistically using unpaired t tests. RESULTS: All patients exhibited at least 1 median lingual canal in the symphysis, and the diameter of the main branch ranged from 0.21 to 1.48 mm (mean, 0.85 mm), with relevant differences between genders (longer in men than in women). A harvesting depth of 4 mm for the distance from the buccal bone to the terminal end of the median lingual canal resulted in a risk of neurovascular injury (13.0%); this risk was notably higher in women (19.1%) than in men (6.7%). The right and left anterior loop lengths ranged from 0 to 5.46 mm (mean, 2.60 mm) and from 0 to 5.57 mm (mean, 2.61 mm), respectively, with no relevant differences between genders or sides. CONCLUSIONS: The results suggest that routine cone-beam computed tomographic examinations before surgical interventions in the symphysis region are necessary because of the numerous complicated anatomic variations.

Regulatory T cells in kidney disease and transplantation.

Regulatory T cells (Tregs) have been shown to be important in maintaining immune homeostasis and preventing autoimmune disease, including autoimmune kidney disease. It is also likely that they play a role in limiting kidney transplant rejection and potentially in promoting transplant tolerance. Although other subsets of Tregs exist, the most potent and well-defined Tregs are the Foxp3 expressing CD4(+) Tregs derived from the thymus or generated peripherally. These CD4(+)Foxp3(+) Tregs limit autoimmune renal disease in animal models, especially chronic kidney disease, and kidney transplantation. Furthermore, other subsets of Tregs, including CD8 Tregs, may play a role in immunosuppression in kidney disease. The development and protective mechanisms of Tregs in kidney disease and kidney transplantation involve multiple mechanisms of suppression. Here we review the development and function of CD4(+)Foxp3(+) Tregs. We discuss the specific application of Tregs as a therapeutic strategy to prevent kidney disease and to limit kidney transplant rejection and detail clinical trials in this area of transplantation.

Autophagy links ß-catenin and Smad signaling to promote epithelial-mesenchymal transition via upregulation of integrin linked kinase.

TGF-ß1 induces epithelial-mesenchymal transition (EMT) and autophagy in a variety of cells. However, the role of autophagy in TGF-ß1-induced EMT has not been clearly elucidated and the underlying mechanisms are unclear. In the present study, we found that TGF-ß1 induced both autophagy and EMT in mouse tubular epithelial C1.1 cells. Inhibition of autophagy by 3-methyladenine or siRNA knockdown of Beclin 1 reduced TGF-ß1-induced increase of vimentin and decreased E-cadherin expression. In contrast, rapamycin-associated enhancement of TGF-ß1-induced autophagy increased EMT of C1.1 cells. Serum rescue inhibited autophagy followed by reversal of EMT. Blocking of autophagosome-lysosomal but not proteosomal degradation reduced the decrease of E-cadherin, demonstrating a role for autophagy in degradation of E-cadherin during EMT. Autophagy promoted the activation of Src and Src-associated phosphorylation of ß-catenin at Y-654 leading to pY654-ß-catenin/p-Smad2 complex formation. Chromatin immunoprecipitation assay demonstrated binding by the pY654-ß-catenin/p-Smad2 complex to ILK promoter thus increasing ILK expression. Taken together, our results demonstrate that TGF-ß1-induced autophagy links ß-catenin and Smad signaling to promote EMT in C1.1 cells through a novel pY654-ß-catenin/p-Smad2/ILK pathway. The pathway delineated links disruption of E-cadherin/ß-catenin-mediated cell-cell contact to induction of EMT via upregulation of ILK.

Methyl-Guanine-Methyl-Transferase Transgenic Bone Marrow Transplantation Allows N,N-bis(2-chloroethyl)-Nitrosourea Driven Donor Mixed-Chimerism Without Graft-Versus-Host Disease, and With Donor-Specific Allograft Tolerance.

BACKGROUND: Transplant tolerance has been achieved by mixed chimerism in animal models and in a limited number of kidney transplant patients. However, these mixed-chimerism strategies were limited either by loss of long-term mixed chimerism or risk of graft-versus-host disease (GVHD). Selective bone marrow (BM) engraftment using marrow protective strategies are currently reaching clinical use. In this study, we tested the utility of methyl-guanine-methyl-transferase (MGMT)-transgenic-C57BL/6 BM into a major histocompatibility complex mismatched-BALB/c model followed by N,N-bis(2-chloroethyl)-nitrosourea (BCNU) treatment to enhance donor-cell engraftment and then evaluated transplant tolerance induction. METHODS: A single-dose of anti-CD8 antibody and busulfan was administered into BALB/c-host-mice at day 1. The BALB/c-mice also received costimulatory blockade through multiple-doses of anti-CD40L antibody. 10 × 10(6) BM-cells from MGMT-transgenic-mice were transplanted into host BALB/c mice at day 0. The BCNU was administered at 4 time points after BM transplantation (BMT). Heterotopic donor C57BL/6 cardiac allografts were performed at day 243 after BMT. Skin transplantation with third-party CBA, host BALB/c and donor C57BL/6 grafts was performed at day 358 after BMT. RESULTS: The BALB/c-mice showed long-term stable and high-level donor-cell engraftment with MGMT transgenic C57BL/6 BMT after BCNU treatment, demonstrating full reconstitution and donor cardiac-allograft tolerance and no GVHD with expanded donor and host Foxp3 T regulatory cells. Further, skin grafts from donor, host, and third party showed good immune function with rejection of third-party grafts from all mice and benefit from enhanced chimerism after BCNU with less cell infiltrate and no chronic rejection in the donor skin grafts of BCNU treated mice compared no BCNU treated mice. CONCLUSIONS: High-level mixed chimerism without GVHD can be achieved using MGMT transgenic BM in a mixed-chimerism model receiving BCNU across a major histocompatibility complex mismatch. Enhanced mixed chimerism leads to long-term donor-specific allograft tolerance.

Heymann nephritis in Lewis rats.

Human membranous nephritis is a major cause of end-stage kidney disease. Active Heymann nephritis (HN) is an auto-immune model of membranous nephritis induced in Lewis rats by immunization with a crude renal tubular antigen (Fx1A) or megalin (gp330). The pathogenesis of HN is through the binding of anti-Fx1A autoantibodies to the auto-antigen expressed on glomerular epithelial cells, resulting in severe glomerular injury and proteinuria. The pathological features of HN include immune deposits in glomeruli and infiltration of glomeruli and the tubulointerstitium by macrophages and T cells. This unit describes the method of the preparation of Fx1A and the induction of HN in Lewis rats by immunization with Fx1A.

Adriamycin nephropathy in BALB/c mice.

Chronic proteinuric renal injury is a major cause of end stage renal disease. Adriamycin nephropathy (AN) is a murine model of chronic proteinuric renal disease whereby chemical injury is followed by immune and structural changes that mimic human disease. This unit describes the method of AN induced by a single injection of adriamycin (ADR) in BALB/c mice. After the initial toxic injury, an immune-mediated chronic proteinuric renal disease that resembles human focal segmental glomerulosclerosis develops. The clinic pathological features of AN are nephrotic syndrome, focal glomerulosclerosis, tubular injury, and interstitial compartment expansion with mononuclear cell infiltrates that are composed largely of macrophages and T cells.