Jay D. Horton, Joseph L. Goldstein, Michael S. Brown
Patients with type 1 diabetes are identified after the onset of the disease, when β cell destruction is almost complete. β cell regeneration from islet cell precursors might reverse this disease, but factors that can induce β cell neogenesis and replication and prevent a new round of autoimmune destruction remain to be identified. Here we show that expression of IGF-I in β cells of transgenic mice (in both C57BL/6–SJL and CD-1 genetic backgrounds) counteracts cytotoxicity and insulitis after treatment with multiple low doses of streptozotocin (STZ). STZ-treated nontransgenic mice developed high hyperglycemia and hypoinsulinemia, lost body weight, and died. In contrast, STZ-treated C57BL/6–SJL transgenic mice showed mild hyperglycemia for about 1 month, after which they normalized glycemia and survived. After STZ treatment, all CD-1 mice developed high hyperglycemia, hypoinsulinemia, polydipsia, and polyphagia. However, STZ-treated CD-1 transgenic mice gradually normalized all metabolic parameters and survived. β cell mass increased in parallel as a result of neogenesis and β cell replication. Thus, our results indicate that local expression of IGF-I in β cells regenerates pancreatic islets and counteracts type 1 diabetes, suggesting that IGF-I gene transfer to the pancreas might be a suitable therapy for this disease.
Mónica George, Eduard Ayuso, Alba Casellas, Cristina Costa, Jean Christophe Devedjian, Fatima Bosch
Pseudophosphatases display extensive sequence similarities to phosphatases but harbor amino acid alterations in their active-site consensus motifs that render them catalytically inactive. A potential role in substrate trapping or docking has been proposed, but the specific requirements for pseudophosphatases during development and differentiation are unknown. We demonstrate here that Sbf1, a pseudophosphatase of the myotubularin family, is expressed at high levels in seminiferous tubules of the testis, specifically in Sertoli’s cells, spermatogonia, and pachytene spermatocytes, but not in postmeiotic round spermatids. Mice that are nullizygous for Sbf1 exhibit male infertility characterized by azoospermia. The onset of the spermatogenic defect occurs in the first wave of spermatogenesis at 17 days after birth during the synchronized progression of pachytene spermatocytes to haploid spermatids. Vacuolation of the Sertoli’s cells is the earliest observed phenotype and is followed by reduced formation of spermatids and eventual depletion of the germ cell compartment in older mice. The nullizygous phenotype in conjunction with high-level expression of Sbf1 in premeiotic germ cells and Sertoli’s cells is consistent with a crucial role for Sbf1 in transition from diploid to haploid spermatocytes. These studies demonstrate an essential role for a pseudophosphatase and implicate signaling pathways regulated by myotubularin family proteins in spermatogenesis and germ cell differentiation.
Ron Firestein, Peter L. Nagy, Megan Daly, Phil Huie, Marco Conti, Michael L. Cleary
Parathyroid hormone (PTH) is a potent pharmacologic inducer of new bone formation, but no physiologic anabolic effect of PTH on adult bone has been described. We investigated the role of PTH in fetal skeletal development by comparing newborn mice lacking either PTH, PTH-related peptide (PTHrP), or both peptides. PTH-deficient mice were dysmorphic but viable, whereas mice lacking PTHrP died at birth with dyschondroplasia. PTH-deficient mice uniquely demonstrated diminished cartilage matrix mineralization, decreased neovascularization with reduced expression of angiopoietin-1, and reduced metaphyseal osteoblasts and trabecular bone. Compound mutants displayed the combined cartilaginous and osseous defects of both single mutants. These results indicate that coordinated action of both PTH and PTHrP are required to achieve normal fetal skeletal morphogenesis, and they demonstrate an essential function for PTH at the cartilage-bone interface. The effect of PTH on fetal osteoblasts may be relevant to its postnatal anabolic effects on trabecular bone.
Dengshun Miao, Bin He, Andrew C. Karaplis, David Goltzman
Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders resulting from loss of acid sphingomyelinase (ASM) activity. We have used a knockout mouse model of NPD (ASMKO mice) to evaluate the effects of direct intracerebral transplantation of bone marrow–derived mesenchymal stem cells (MSCs) on the progression of neurological disease in this disorder. MSCs were transduced with a retroviral vector to overexpress ASM and were injected into the hippocampus and cerebellum of 3-week-old ASMKO pups. Transplanted cells migrated away from the injection sites and survived at least 6 months after transplantation. Seven of 8 treated mice, but none of the untreated controls, survived for ≥ 7 months after transplant. Survival times were greater in sex-matched than in sex-mismatched transplants. Transplantation significantly delayed the Purkinje cell loss that is characteristic of NPD, although the protective effect declined with distance from the injection site. Overall ASM activity in brain homogenates was low, but surviving Purkinje cells contained the retrovirally expressed human enzyme, and transplanted animals showed a reduction in cerebral sphingomyelin. These results reveal the potential of treating neurodegenerative lysosomal storage disorders by intracerebral transplantation of bone marrow–derived MSCs.
Hee Kyung Jin, Janet E. Carter, George W. Huntley, Edward H. Schuchman
The homeodomain transcription factor Pdx1 is required for pancreas development, including the differentiation and function of β cells. Mutations in Pdx1 or upstream hepatocyte nuclear factors cause autosomal forms of early-onset diabetes (maturity-onset diabetes of the young [MODY]). In mice, the Irs2 branch of the insulin/Igf signaling system mediates peripheral insulin action and pancreatic β cell growth and function. To investigate whether β cell failure in Irs2–/– mice might be related to dysfunction of MODY-related transcription factors, we measured the expression of Pdx1 in islets from young Irs2–/– mice. Before the onset of diabetes, Pdx1 was reduced in islets from Irs2–/– mice, whereas it was expressed normally in islets from wild-type or Irs1–/– mice, which do not develop diabetes. Whereas male Irs2–/–Pdx1+/+ mice developed diabetes between 8 and 10 weeks of age, haploinsufficiency for Pdx1 caused diabetes in newborn Irs2–/– mice. By contrast, transgenic expression of Pdx1 restored β cell mass and function in Irs2–/– mice and promoted glucose tolerance throughout life, as these mice survived for at least 20 months without diabetes. Our results suggest that dysregulation of Pdx1 might represent a common link between ordinary type 2 diabetes and MODY.
Jake A. Kushner, Jing Ye, Markus Schubert, Deborah J. Burks, Matthew A. Dow, Carrie L. Flint, Sanjoy Dutta, Christopher V.E. Wright, Marc R. Montminy, Morris F. White
Rotaviruses are the leading cause of severe diarrheal disease in young children. Intestinal mucosal IgA responses play a critical role in protective immunity against rotavirus reinfection. Rotaviruses consist of three concentric capsid layers surrounding a genome of 11 segments of double-stranded RNA. The outer layer proteins, VP4 and VP7, which are responsible for viral attachment and entry, are targets for protective neutralizing antibodies. However, IgA mAb’s directed against the intermediate capsid protein VP6, which do not neutralize the virus, have also been shown to protect mice from rotavirus infection and clear chronic infection in SCID mice. We investigated whether the anti-VP6 IgA (7D9) mAb could inhibit rotavirus replication inside epithelial cells and found that 7D9 acted at an early stage of infection to neutralize rotavirus following antibody lipofection. Using electron cryomicroscopy, we determined the three-dimensional structure of the virus-antibody complex. The attachment of 7D9 IgA to VP6 introduces a conformational change in the VP6 trimer, rendering the particle transcriptionally incompetent and preventing the elongation of initiated transcripts. Based on these observations, we suggest that anti-VP6 IgA antibodies confers protection in vivo by inhibiting viral transcription at the start of the intracellular phase of the viral replication cycle.
Ningguo Feng, Jeffrey A. Lawton, Joana Gilbert, Nelly Kuklin, Phuoc Vo, B.V. Venkataram Prasad, Harry B. Greenberg
Gaucher disease, the most common lysosomal storage disease, is caused by a deficiency of glucocerebrosidase resulting in the impairment of glucosylceramide degradation. The hallmark of the disease is the presence of the Gaucher cell, a macrophage containing much of the stored glucosylceramide found in tissues, which is believed to cause many of the clinical manifestations of the disease. We have developed adult mice carrying the Gaucher disease L444P point mutation in the glucocerebrosidase (Gba) gene and exhibiting a partial enzyme deficiency. The mutant mice demonstrate multisystem inflammation, including evidence of B cell hyperproliferation, an aspect of the disease found in some patients. However, the mutant mice do not accumulate large amounts of glucosylceramide or exhibit classic Gaucher cells in tissues.
Hiroki Mizukami, Yide Mi, Ryuichi Wada, Mari Kono, Tadashi Yamashita, Yujing Liu, Norbert Werth, Roger Sandhoff, Konrad Sandhoff, Richard L. Proia
The treatment of chronic inflammatory diseases is complicated by their unpredictable, relapsing clinical course. Here, we describe a new strategy in which an inflammation-regulated therapeutic transgene is introduced into the joints to prevent recurrence of arthritis. To this end, we designed a recombinant adenoviral vector containing a two-component, inflammation-inducible promoter controlling the expression of human IL-10 (hIL-10) cDNA. When tested in vitro, this system had a low-level basal activity and was activated four to five orders of magnitude by various inflammatory stimuli, including TNF-α, IL-1β, IL-6, and LPS. When introduced in joints of rats with recurrent streptococcal cell wall–induced arthritis, the IL-10 transgene was induced in parallel with disease recurrence and effectively prevented the influx of inflammatory cells and the associated swelling of the joints. Levels of inflammation-inducible hIL-10 protein within the joints correlated closely with the severity of recurrence. An endogenously regulated therapeutic transgene can thus establish negative feedback and restore homeostasis in vivo while minimizing host exposure to the recombinant drug.
A.V. Miagkov, A.W. Varley, R.S. Munford, S.S. Makarov
While the pathologic mechanisms responsible for organ-specific tissue damage in primary biliary cirrhosis (PBC) remain an enigma, it has been suggested that the pathology is mediated by autoreactive T cells infiltrating the intrahepatic bile ducts. Previously, we have documented that there is 100-fold enrichment in the frequency of CD4+ autoreactive T cells in the liver that are specific for peptides encoded by the E2 components of the pyruvate dehydrogenase complexes (PDC-E2). We have also recently characterized the first MHC class I–restricted epitope for PDC-E2, namely amino acid 159–167, a region very similar to the epitope recognized by MHC class II–restricted CD4+ cells and by autoantibodies. The effector functions of these PDC-E2159-167–specific CD8+ cytotoxic T lymphocytes (CTLs) are not well understood. We have taken advantage of tetramer technology and report herein that there is tenfold increase in the frequency of PDC-E2159-167–specific CTLs in the liver as compared with the blood in PBC. In addition, the precursor frequency of the CTLs in blood was significantly higher in early-stage PBC. Of interest was the fact that, upon stimulation with the peptide, the response of PDC-E2159-167 tetramer-positive cells is heterogeneous with respect to IFN-γ synthesis. These data, we believe for the first time, document the enrichment of autoantigen-specific CD8+ T cells in the PBC liver, suggesting that CD8+ T cells play a significant role in the immunopathogenesis of PBC.
Hiroto Kita, Shuji Matsumura, Xiao-Song He, Aftab A. Ansari, Zhe-Xiong Lian, Judy Van de Water, Ross L. Coppel, Marshall M. Kaplan, M. Eric Gershwin
Although hitherto considered as a strictly locally acting vasodilator, results from recent clinical studies with inhaled nitric oxide (NO) indicate that NO can exert effects beyond the pulmonary circulation. We therefore sought to investigate potential remote vascular effects of intra-arterially applied aqueous NO solution and to identify the mechanisms involved. On bolus application of NO into the brachial artery of 32 healthy volunteers, both diameter of the downstream radial artery and forearm blood flow increased in a dose-dependent manner. Maximum dilator responses were comparable to those after stimulation of endogenous NO formation with acetylcholine and bradykinin. Response kinetics and pattern of NO decomposition suggested that despite the presence of hemoglobin-containing erythrocytes, a significant portion of NO was transported in its unbound form. Infusion of NO (36 μmol/min) into the brachial artery increased levels of plasma nitroso species, nitrite, and nitrate in the draining antecubital vein (by < 2-fold, 30-fold, and 4-fold, respectively), indicative of oxidative and nitrosative chemistry. Infused N-oxides were inactive as vasodilators whereas S-nitrosoglutathione dilated conduit and resistance arteries. Our results suggest that NO can be transported in bioactive form for significant distances along the vascular bed. Both free NO and plasma nitroso species contribute to the dilation of the downstream vasculature.
Tienush Rassaf, Michael Preik, Petra Kleinbongard, Thomas Lauer, Christian Heiß, Bodo-Eckehard Strauer, Martin Feelisch, Malte Kelm
Older bone marrow transplantation (BMT) recipients are at heightened risk for acute graft-versus-host disease (GVHD) after allogeneic BMT, but the causes of this association are poorly understood. Using well-characterized murine BMT models we have explored the mechanisms of increased GVHD in older mice. GVHD mortality, morbidity, and pathologic and biochemical indices were all worse in old recipients. Donor T cell responses were significantly increased in old recipients both in vivo and in vitro when stimulated by antigen-presenting cells (APCs) from old mice, which also secreted more TNF-α and IL-12 after LPS stimulation. In a B6 → B6D2F1 model, CD4+ donor T cells but not CD8+ T cells mediated more severe GVHD in old mice. We confirmed the role of aged APCs in GVHD using B6D2F1 BM chimeras created with either old or young BM. Four months after chimera creation, allogeneic BMT from B6 donors caused significantly worse GVHD in old BM chimeras. APCs from these mice also stimulated greater responses from allogeneic cells in vitro. These data demonstrate a hitherto unsuspected mechanism of amplified donor T cell responses by aged allogeneic host APCs that increases acute GVHD in aged recipients in this BMT model.
Rainer Ordemann, Raymond Hutchinson, Jeffrey Friedman, Steven J. Burakoff, Pavan Reddy, Ulrich Duffner, Thomas M. Braun, Chen Liu, Takanori Teshima, James L.M. Ferrara