Genetics
Abstract
Hailey-Hailey disease (HHD) is an autosomal dominant trait characterized by erythematous and oozing skin lesions preponderantly involving the body folds. In the present unusual case, however, unilateral segmental areas along the lines of Blaschko showing a rather severe involvement were superimposed on the ordinary symmetrical phenotype. Based on this observation and similar forms of mosaicism as reported in other autosomal dominant skin disorders, we postulated that in such cases, 2 different types of segmental involvement can be distinguished. Accordingly, the linear lesions as noted in the present case would exemplify type 2 segmental HHD. In the heterozygous embryo, loss of heterozygosity occurring at an early developmental stage would have given rise to pronounced linear lesions reflecting homozygosity or hemizygosity for the mutation. By analyzing DNA and RNA derived from blood and skin samples as well as keratinocytes of the index patient with various molecular techniques including RT-PCR, real-time PCR, and microsatellite analysis, we found a consistent loss of the paternal wild-type allele in more severely affected segmental skin regions, confirming this hypothesis for the first time, to our knowledge, at the molecular and cellular level.
Authors
Pamela Poblete-Gutiérrez, Tonio Wiederholt, Arne König, Frank K. Jugert, Yvonne Marquardt, Albert Rübben, Hans F. Merk, Rudolf Happle, Jorge Frank
Abstract
Although inflammation and protease/antiprotease imbalance have been postulated to be critical in cigarette smoke–induced (CS-induced) emphysema, oxidative stress has been suspected to play an important role in chronic obstructive pulmonary diseases. Susceptibility of the lung to oxidative injury, such as that originating from inhalation of CS, depends largely on its upregulation of antioxidant systems. Nuclear factor, erythroid-derived 2, like 2 (Nrf2) is a redox-sensitive basic leucine zipper protein transcription factor that is involved in the regulation of many detoxification and antioxidant genes. Disruption of the Nrf2 gene in mice led to earlier-onset and more extensive CS-induced emphysema than was found in wild-type littermates. Emphysema in Nrf2-deficient mice exposed to CS for 6 months was associated with more pronounced bronchoalveolar inflammation; with enhanced alveolar expression of 8-oxo-7,8-dihydro-2′-deoxyguanosine, a marker of oxidative stress; and with an increased number of apoptotic alveolar septal cells — predominantly endothelial and type II epithelial cells — as compared with wild-type mice. Microarray analysis identified the expression of nearly 50 Nrf2-dependent antioxidant and cytoprotective genes in the lung that may work in concert to counteract CS-induced oxidative stress and inflammation. The responsiveness of the Nrf2 pathway may act as a major determinant of susceptibility to tobacco smoke–induced emphysema by upregulating antioxidant defenses and decreasing lung inflammation and alveolar cell apoptosis.
Authors
Tirumalai Rangasamy, Chung Y. Cho, Rajesh K. Thimmulappa, Lijie Zhen, Sorachai S. Srisuma, Thomas W. Kensler, Masayuki Yamamoto, Irina Petrache, Rubin M. Tuder, Shyam Biswal
Abstract
Poly(ADP-ribosyl)ation is rapidly formed in cells following DNA damage and is regulated by poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is known to be involved in various cellular processes, such as DNA repair, genomic stability, transcription, and cell death. During apoptosis, PARP-1 is cleaved by caspases to generate 89-kDa and 24-kDa fragments, a hallmark of apoptosis. This cleavage is thought to be a regulatory event for cellular death. In order to understand the biological significance of PARP-1 cleavage, we generated a PARP-1 knockin (PARP-1KI/KI) mouse model, in which the caspase cleavage site of PARP-1, DEVD214, was mutated to render the protein resistant to caspases during apoptosis. While PARP-1KI/KI mice developed normally, they were highly resistant to endotoxic shock and to intestinal and renal ischemia-reperfusions, which were associated with reduced inflammatory responses in the target tissues and cells due to the compromised production of specific inflammatory mediators. Despite normal binding of NF-κB to DNA, NF-κB–mediated transcription activity was impaired in the presence of caspase-resistant PARP-1. This study provides a novel insight into the function of PARP-1 in inflammation and ischemia-related pathophysiologies.
Authors
Virginie Pétrilli, Zdenko Herceg, Paul O. Hassa, Nimesh S.A. Patel, Rosanna Di Paola, Ulrich Cortes, Laura Dugo, Helder-Mota Filipe, Christoph Thiemermann, Michael O. Hottiger, Salvatore Cuzzocrea, Zhao-Qi Wang
Abstract
Complex I deficiency, the most common respiratory chain defect, is genetically heterogeneous: mutations in 8 nuclear and 7 mitochondrial DNA genes encoding complex I subunits have been described. However, these genes account for disease in only a minority of complex I–deficient patients. We investigated whether there may be an unknown common gene by performing functional complementation analysis of cell lines from 10 unrelated patients. Two of the patients were found to have mitochondrial DNA mutations. The other 8 represented 7 different (nuclear) complementation groups, all but 1 of which showed abnormalities of complex I assembly. It is thus unlikely that any one unknown gene accounts for a large proportion of complex I cases. The 2 patients sharing a nuclear complementation group had a similar abnormal complex I assembly profile and were studied further by homozygosity mapping, chromosome transfers, and microarray expression analysis. NDUFS6, a complex I subunit gene not previously associated with complex I deficiency, was grossly underexpressed in the 2 patient cell lines. Both patients had homozygous mutations in this gene, one causing a splicing abnormality and the other a large deletion. This integrated approach to gene identification offers promise for identifying other unknown causes of respiratory chain disorders.
Authors
Denise M. Kirby, Renato Salemi, Canny Sugiana, Akira Ohtake, Lee Parry, Katrina M. Bell, Edwin P. Kirk, Avihu Boneh, Robert W. Taylor, Hans-Henrik M. Dahl, Michael T. Ryan, David R. Thorburn
Abstract
Gene-based delivery can establish a sustained supply of therapeutic proteins within the nervous system. For diseases characterized by extensive CNS and peripheral nervous system (PNS) involvement, widespread distribution of the exogenous gene may be required, a challenge to in vivo gene transfer strategies. Here, using lentiviral vectors (LVs), we efficiently transduced hematopoietic stem cells (HSCs) ex vivo and evaluated the potential of their progeny to target therapeutic genes to the CNS and PNS of transplanted mice and correct a neurodegenerative disorder, metachromatic leukodystrophy (MLD). We proved extensive repopulation of CNS microglia and PNS endoneurial macrophages by transgene-expressing cells. Intriguingly, recruitment of these HSC-derived cells was faster and more robust in MLD mice. By transplanting HSCs transduced with the arylsulfatase A gene, we fully reconstituted enzyme activity in the hematopoietic system of MLD mice and prevented the development of motor conduction impairment, learning and coordination deficits, and neuropathological abnormalities typical of the disease. Remarkably, ex vivo gene therapy had a significantly higher therapeutic impact than WT HSC transplantation, indicating a critical role for enzyme overexpression in the HSC progeny. These results indicate that transplantation of LV-transduced autologous HSCs represents a potentially efficacious therapeutic strategy for MLD and possibly other neurodegenerative disorders.
Authors
Alessandra Biffi, Michele De Palma, Angelo Quattrini, Ubaldo Del Carro, Stefano Amadio, Ilaria Visigalli, Maria Sessa, Stefania Fasano, Riccardo Brambilla, Sergio Marchesini, Claudio Bordignon, Luigi Naldini
Abstract
Excessive inflammatory responses can emerge as a potential danger for organisms’ health. Physiological balance between pro- and anti-inflammatory processes constitutes an important feature of responses against harmful events. Here, we show that cannabinoid receptors type 1 (CB1) mediate intrinsic protective signals that counteract proinflammatory responses. Both intrarectal infusion of 2,4-dinitrobenzene sulfonic acid (DNBS) and oral administration of dextrane sulfate sodium induced stronger inflammation in CB1-deficient mice (CB1–/–) than in wild-type littermates (CB1+/+). Treatment of wild-type mice with the specific CB1 antagonist N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide (SR141716A) mimicked the phenotype of CB1–/– mice, showing an acute requirement of CB1 receptors for protection from inflammation. Consistently, treatment with the cannabinoid receptor agonist R(-)-7-hydroxy-Δ6-tetra-hydrocannabinol-dimethylheptyl (HU210) or genetic ablation of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) resulted in protection against DNBS-induced colitis. Electrophysiological recordings from circular smooth muscle cells, performed 8 hours after DNBS treatment, revealed spontaneous oscillatory action potentials in CB1–/– but not in CB1+/+ colons, indicating an early CB1-mediated control of inflammation-induced irritation of smooth muscle cells. DNBS treatment increased the percentage of myenteric neurons expressing CB1 receptors, suggesting an enhancement of cannabinoid signaling during colitis. Our results indicate that the endogenous cannabinoid system represents a promising therapeutic target for the treatment of intestinal disease conditions characterized by excessive inflammatory responses.
Authors
Federico Massa, Giovanni Marsicano, Heike Hermann, Astrid Cannich, Krisztina Monory, Benjamin F. Cravatt, Gian-Luca Ferri, Andrei Sibaev, Martin Storr, Beat Lutz
Abstract
A major drawback of current approaches to antiangiogenic gene therapy is the lack of tissue-specific targeting. The aim of this work was to trigger endothelial cell–specific apoptosis, using adenoviral vector–mediated delivery of a chimeric death receptor derived from the modified endothelium-specific pre-proendothelin-1 (PPE-1) promoter. In the present study, we constructed an adenovirus-based vector that targets tumor angiogenesis. Transcriptional control was achieved by use of a modified endothelium-specific promoter. Expression of a chimeric death receptor, composed of Fas and TNF receptor 1, resulted in specific apoptosis of endothelial cells in vitro and sensitization of cells to the proapoptotic effect of TNF-α. The antitumoral activity of the vectors was assayed in two mouse models. In the model of B16 melanoma, a single systemic injection of virus to the tail vein caused growth retardation of tumor and reduction of tumor mass with central tumor necrosis. When the Lewis lung carcinoma lung-metastasis model was applied, i.v. injection of vector resulted in reduction of lung-metastasis mass, via an antiangiogenic mechanism. Moreover, by application of the PPE-1–based transcriptional control, a humoral immune response against the transgene was avoided. Collectively, these data provide evidence that transcriptionally controlled, angiogenesis-targeted gene therapy is feasible.
Authors
Shoshana Greenberger, Aviv Shaish, Nira Varda-Bloom, Keren Levanon, Eyal Breitbart, Iris Goldberg, Iris Barshack, Israel Hodish, Niva Yaacov, Livnat Bangio, Tanya Goncharov, David Wallach, Dror Harats
Abstract
Hemophilia is a bleeding disorder caused by mutations in the genes encoding coagulation Factor VIII (FVIII) or FIX. Current treatment is through intravenous infusion of the missing protein. The major complication of treatment is the development of neutralizing Ab’s to the clotting factor. Infusion of recombinant activated human Factor VII (rhFVIIa), driving procoagulant reactions independently of human FVIII (hFVIII) or hFIX, has been successful in such patients and could in theory provide hemostasis in all hemophilia patients. However, its high cost and short half-life have limited its use. Here, we report a novel treatment strategy with a recombinant adeno-associated virus vector delivering a modified FVII transgene that can be intracellularly processed and secreted as activated FVII (FVIIa). We show long-term expression, as well as phenotypic correction of hemophilia B mice following gene transfer of the murine FVIIa homolog, with no evidence of thrombotic complications at these doses. These data hold promise for a potential treatment for hemophilia and other bleeding disorders.
Authors
Paris Margaritis, Valder R. Arruda, Majed Aljamali, Rodney M. Camire, Alexander Schlachterman, Katherine A. High
Abstract
CBS domains are defined as sequence motifs that occur in several different proteins in all kingdoms of life. Although thought to be regulatory, their exact functions have been unknown. However, their importance was underlined by findings that mutations in conserved residues within them cause a variety of human hereditary diseases, including (with the gene mutated in parentheses): Wolff-Parkinson-White syndrome (γ2 subunit of AMP-activated protein kinase); retinitis pigmentosa (IMP dehydrogenase-1); congenital myotonia, idiopathic generalized epilepsy, hypercalciuric nephrolithiasis, and classic Bartter syndrome (CLC chloride channel family members); and homocystinuria (cystathionine β-synthase). AMP-activated protein kinase is a sensor of cellular energy status that is activated by AMP and inhibited by ATP, but the location of the regulatory nucleotide-binding sites (which are prime targets for drugs to treat obesity and diabetes) was not characterized. We now show that tandem pairs of CBS domains from AMP-activated protein kinase, IMP dehydrogenase-2, the chloride channel CLC2, and cystathionine β-synthase bind AMP, ATP, or S-adenosyl methionine,while mutations that cause hereditary diseases impair this binding. This shows that tandem pairs of CBS domains act, in most cases, as sensors of cellular energy status and, as such, represent a newly identified class of binding domain for adenosine derivatives.
Authors
John W. Scott, Simon A. Hawley, Kevin A. Green, Miliea Anis, Greg Stewart, Gillian A. Scullion, David G. Norman, D. Grahame Hardie
Abstract
Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU–conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus–transduced human SRCs following BG/BCNU treatment.
Authors
Steven P. Zielske, Jane S. Reese, Karen T. Lingas, Jon R. Donze, Stanton L. Gerson
Copyright © 2024 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)