Mohamed H. Sayegh

Endothelial-to-mesenchymal transition contributes to cardiac fibrosis

Cardiac fibrosis, associated with a decreased extent of microvasculature and with disruption of normal myocardial structures, results from excessive deposition of extracellular matrix, which is mediated by the recruitment of fibroblasts. The source of these fibroblasts is unclear and specific anti-fibrotic therapies are not currently available. Here we show that cardiac fibrosis is associated with the emergence of fibroblasts originating from endothelial cells, suggesting an endothelial-mesenchymal transition (EndMT) similar to events that occur during formation of the atrioventricular cushion in the embryonic heart. Transforming growth factor-β1 (TGF-β1) induced endothelial cells to undergo EndMT, whereas bone morphogenic protein 7 (BMP-7) preserved the endothelial phenotype. The systemic administration of recombinant human BMP-7 (rhBMP-7) significantly inhibited EndMT and the progression of cardiac fibrosis in mouse models of pressure overload and chronic allograft rejection. Our findings show that EndMT contributes to the progression of cardiac fibrosis and that rhBMP-7 can be used to inhibit EndMT and to intervene in the progression of chronic heart disease associated with fibrosis.

Identification of cells initiating human melanomas.

Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies and solid cancers. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5+ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5+ melanoma cells possess greater tumorigenic capacity than ABCB5- bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5+ subpopulations for self-renewal and differentiation, because ABCB5+ cancer cells generate both ABCB5+ and ABCB5- progeny, whereas ABCB5- tumour populations give rise, at lower rates, exclusively to ABCB5- cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5+ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy.

Tissue expression of PD-L1 mediates peripheral T cell tolerance

Programmed death 1 (PD-1), an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. PD-1 has two ligands: PD-1 ligand 1 (PD-L1), which is expressed broadly on hematopoietic and parenchymal cells, including pancreatic islet cells; and PD-L2, which is restricted to macrophages and dendritic cells. To investigate whether PD-L1 and PD-L2 have synergistic or unique roles in regulating T cell activation and tolerance, we generated mice lacking PD-L1 and PD-L2 (PD-L1/PD-L2−/− mice) and compared them to mice lacking either PD-L. PD-L1 and PD-L2 have overlapping functions in inhibiting interleukin-2 and interferon-γ production during T cell activation. However, PD-L1 has a unique and critical role in controlling self-reactive T cells in the pancreas. Our studies with bone marrow chimeras demonstrate that PD-L1/PD-L2 expression only on antigen-presenting cells is insufficient to prevent the early onset diabetes that develops in PD-L1/PD-L2−/− non-obese diabetic mice. PD-L1 expression in islets protects against immunopathology after transplantation of syngeneic islets into diabetic recipients. PD-L1 inhibits pathogenic self-reactive CD4+ T cell–mediated tissue destruction and effector cytokine production. These data provide evidence that PD-L1 expression on parenchymal cells rather than hematopoietic cells protects against autoimmune diabetes and point to a novel role for PD-1–PD-L1 interactions in mediating tissue tolerance.

Delayed graft function in kidney transplantation

Delayed graft function is a form of acute renal failure resulting in post-transplantation oliguria, increased allograft immunogenicity and risk of acute rejection episodes, and decreased long-term survival. Factors related to the donor and prerenal, renal, or postrenal transplant factors related to the recipient can contribute to this condition. From experimental studies, we have learnt that both ischaemia and reinstitution of blood flow in ischaemically damaged kidneys after hypothermic preservation activate a complex sequence of events that sustain renal injury and play a pivotal part in the development of delayed graft function. Elucidation of the pathophysiology of renal ischaemia and reperfusion injury has contributed to the development of strategies to decrease the rate of delayed graft function, focusing on donor management, organ procurement and preservation techniques, recipient fluid management, and pharmacological agents (vasodilators, antioxidants, anti-inflammatory agents). Several new drugs show promise in animal studies in preventing or ameliorating ischaemia-reperfusion injury and possibly delayed graft function, but definitive clinical trials are lacking. The goal of monotherapy for the prevention or treatment of is perhaps unattainable, and multidrug approaches or single drug targeting multiple signals will be the next step to reduce post-transplantation injury and delayed graft function.

Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance

The mechanisms of allograft tolerance have been classified as deletion, anergy, ignorance and suppression/regulation. Deletion has been implicated in central tolerance, whereas peripheral tolerance has generally been ascribed to clonal anergy and/or active immunoregulatory states. Here, we used two distinct systems to assess the requirement for T-cell deletion in peripheral tolerance induction. In mice transgenic for Bcl-xL, T cells were resistant to passive cell death through cytokine withdrawal, whereas T cells from interleukin-2-deficient mice did not undergo activation-induced cell death. Using either agents that block co-stimulatory pathways or the immunosuppressive drug rapamycin, which we have shown here blocks the proliferative component of interleukin-2 signaling but does not inhibit priming for activation-induced cell death, we found that mice with defective passive or active T-cell apoptotic pathways were resistant to induction of transplantation tolerance. Thus, deletion of activated T cells through activation-induced cell death or growth factor withdrawal seems necessary to achieve peripheral tolerance across major histocompatibility complex barriers.

The Programmed Death-1 (PD-1) Pathway Regulates Autoimmune Diabetes in Nonobese Diabetic (NOD) Mice

Programmed death-1 (PD-1) receptor, an inhibitory costimulatory molecule found on activated T cells, has been demonstrated to play a role in the regulation of immune responses and peripheral tolerance. We investigated the role of this pathway in the development of autoimmune diabetes. PD-1 or PD-L1 but not PD-L2 blockade rapidly precipitated diabetes in prediabetic female nonobese diabetic (NOD) mice regardless of age (from 1 to 10-wk-old), although it was most pronounced in the older mice. By contrast, cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) blockade induced disease only in neonates. Male NOD mice also developed diabetes after PD-1–PD-L1 pathway blockade, but NOR mice, congenic to NOD but resistant to the development of diabetes, did not. Insulitis scores were significantly higher and frequency of interferon γ–producing GAD-reactive splenocytes was increased after PD-1–PD-L1 pathway blockade compared with controls. Interestingly, PD-L1 but not PD-L2 was found to be expressed on inflamed islets of NOD mice. These data demonstrate a central role for PD-1–PD-L1 interaction in the regulation of induction and progression of autoimmune diabetes in the NOD mouse and provide the rationale to develop new therapies to target this costimulatory pathway in this disease.

Allogeneic bone marrow transplantation with co-stimulatory blockade inducesmacrochimerism and tolerance without cytoreductive host treatment

Allogeneic bone marrow transplantation (in immunocompetent adults) has always required cytoreductive treatment of recipients with irradiation or cytotoxic drugs to achieve lasting engraftment at levels detectable by non-PCR-based techniques (‘macrochimerism’ or ‘mixed chimerism’). Only syngeneic marrow engraftment at such levels has been achieved in unconditioned hosts. This requirement for potentially toxic myelosuppressive host pre-conditioning has precluded the clinical use of allogeneic bone marrow transplantation for many indications other than malignancies, including tolerance induction. We demonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatched allogeneic bone marrow, followed by one injection each of monoclonal antibody against CD154 and cytotoxic T-lymphocyte antigen 4 immunoglobulin, resulted in multi-lineage hematopoietic macrochimerism (of about 15%) that persisted for up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145 days) and demonstrated ongoing intrathymic deletion of donor-reactive T cells. A protocol of high-dose bone marrow transplantation and co-stimulatory blockade can thus achieve allogeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier to the more widespread use of allogeneic bone marrow transplantation. Although efforts have been made to minimize host pre-treatment for allogeneic bone marrow transplantation for tolerance induction, so far none have succeeded in eliminating pre-treatment completely. Our demonstration that this can be achieved provides the rationale for a safe approach for inducing robust transplantation tolerance in large animals and humans.

ABCB5-Mediated Doxorubicin Transport and Chemoresistance in Human Malignant Melanoma

Enhanced drug efflux mediated by ABCB1 P-glycoprotein and related ATP-binding cassette transporters is one of several mechanisms of multidrug resistance thought to impair chemotherapeutic success in human cancers. In malignant melanoma, its potential contribution to chemoresistance is uncertain. Here, we show that ABCB5, which functions as a determinant of membrane potential and regulator of cell fusion in physiologic skin progenitor cells, is expressed in clinical malignant melanoma tumors and preferentially marks a subset of hyperpolarized, CD133+ stem cell phenotype-expressing tumor cells in malignant melanoma cultures and clinical melanomas. We found that ABCB5 blockade significantly reversed resistance of G3361 melanoma cells to doxorubicin, an agent to which clinical melanomas have been found refractory, resulting in a 43% reduction in the LD50 from 4 to 2.3 micromol/L doxorubicin (P < 0.05). Our results identified ABCB5-mediated doxorubicin efflux transport as the underlying mechanism of resistance, because ABCB5 blockade significantly enhanced intracellular drug accumulation. Consistent with this novel ABCB5 function and mechanism in doxorubicin resistance, gene expression levels of the transporter across a panel of human cancer cell lines used by the National Cancer Institute for drug screening correlated significantly with tumor resistance to doxorubicin (r = 0.44; P = 0.016). Our results identify ABCB5 as a novel drug transporter and chemoresistance mediator in human malignant melanoma. Moreover, our findings show that ABCB5 is a novel molecular marker for a distinct subset of chemoresistant, stem cell phenotype-expressing tumor cells among melanoma bulk populations and indicate that these chemoresistant cells can be specifically targeted via ABCB5 to enhance cytotoxic efficacy.

Quantifying the Frequency of Alloreactive T Cells In Vivo: New Answers to an Old Question

Alloreactive T cell precursor frequency was measured in vivo using fluorescent dye labeling in combination with novel models based on lymphocyte activation and recovery. CFSE-labeled C57BL/6 (H-2b) spleen and lymph node cells were adoptively transferred to C57BL/6×DBA F1 (H-2b/d) recipients, a parent→F1 MHC mismatch in which only donor cells respond. Recipients were sacrificed at serial time points to assess engraftment efficiency, and the extent of donor cell activation and proliferation. These data were used to calculate alloreactive T cell frequencies that varied 30-fold (0.71 ± 0.31% to 21.05 ± 3.62%), depending upon whether it was assumed that all donor cells injected became established and were capable of responding, or that only those present at later time points (24–72 h) were available to respond. By measuring the number of cells established in the recipient 24 h after transfer, before proliferation, we calculated an in vivo alloreactive frequency of ∼7%. Using CD69 expression at 48 h to quantify activation, we found that 40–50% of the alloactivated CD4+ donor T cells do not divide. Studies of cotransferred congenic and allogeneic cells demonstrated that bystander proliferation does not occur. We conclude that accurate calculations of alloreactive precursor frequency must account for both proliferation and cell engraftment. When this is done, a high percentage of alloreactive T cells exists across an MHC mismatch, but not all alloreactive cells proliferate in vivo. Bystander proliferation is negligible, revealing exquisite specificity to the alloresponse. These data provide a novel approach to quantify alloreactive T cell responses during specific immunomodulatory strategies in vivo.

Anaritide in Acute Tubular Necrosis

BACKGROUND: Atrial natriuretic peptide, a hormone synthesized by the cardiac atria, increases the glomerular filtration rate by dilating afferent arterioles while constricting efferent arterioles. It has been shown to improve glomerular filtration, urinary output, and renal histopathology in laboratory animals with acute renal dysfunction. Anaritide is a 25-amino-acid synthetic form of atrial natriuretic peptide. METHODS: We conducted a multicenter, randomized, double-blind, placebo-controlled clinical trial of anaritide in 504 critically ill patients with acute tubular necrosis. The patients received a 24-hour intravenous infusion of either anaritide (0.2 microgram per kilogram of body weight per minute) or placebo. The primary end point was dialysis-free survival for 21 days after treatment. Other end points included the need for dialysis, changes in the serum creatinine concentration, and mortality. RESULTS: The rate of dialysis-free survival was 47 percent in the placebo group and 43 percent in the anaritide group (P = 0.35). In the prospectively defined subgroup of 120 patients with oliguria (urinary output, < 400 ml per day), dialysis-free survival was 8 percent in the placebo group (5 of 60 patients) and 27 percent in the anaritide group (16 of 60 patients, P = 0.008). Anaritide-treated patients with oliguria who no longer had oliguria after treatment benefited the most. Conversely, among the 378 patients without oliguria, dialysis-free survival was 59 percent in the placebo group (116 of 195 patients) and 48 percent in the anaritide group (88 of 183 patients, P = 0.03). CONCLUSIONS: The administration of anaritide did not improve the overall rate of dialysis-free survival in critically ill patients with acute tubular necrosis. However, anaritide may improve dialysis-free survival in patients with oliguria and may worsen it in patients without oliguria who have acute tubular necrosis.