Siamak Agha-Mohammadi

An isogenetic myoblast expression screen identifies DUX4‐mediated FSHD‐associated molecular pathologies

Facioscapulohumeral muscular dystrophy (FSHD) is caused by an unusual deletion with neomorphic activity. This deletion derepresses genes in cis; however which candidate gene causes the FSHD phenotype, and through what mechanism, is unknown. We describe a novel genetic tool, inducible cassette exchange, enabling rapid generation of isogenetically modified cells with conditional and variable transgene expression. We compare the effects of expressing variable levels of each FSHD candidate gene on myoblasts. This screen identified only one gene with overt toxicity: DUX4 (double homeobox, chromosome 4), a protein with two homeodomains, each similar in sequence to Pax3 and Pax7. DUX4 expression recapitulates key features of the FSHD molecular phenotype, including repression of MyoD and its target genes, diminished myogenic differentiation, repression of glutathione redox pathway components, and sensitivity to oxidative stress. We further demonstrate competition between DUX4 and Pax3/Pax7: when either Pax3 or Pax7 is expressed at high levels, DUX4 is no longer toxic. We propose a hypothesis for FSHD in which DUX4 expression interferes with Pax7 in satellite cells, and inappropriately regulates Pax targets, including myogenic regulatory factors, during regeneration.

Tight control of transgene expression by lentivirus vectors containing second‐generation tetracycline‐responsive promoters

The goal of this study was to design improved regulatable lentivirus vector systems. The aim was to design tetracycline (tet)‐regulatable lentivirus vectors based on the Tet‐on system displaying low background expression in the absence of the doxycycline (DOX) inducer and high transgene expression levels in the presence of DOX.

Regulatable systems: applications in gene therapy and replicating viruses

Effective gene-based therapies require efficient delivery of therapeutic genes to targeted mammalian cells as well as regulatable gene expression. Advances in gene therapy have mainly focused on the development of viral systems — adenovirus, retrovirus, adeno-associated virus, herpes virus, autonomous parvoviruses — and nonviral systems, such as direct injection of naked or conjugated DNA, for delivering transgenes into mammalian cells. Most of the currently applied gene delivery systems rely on strong viral promoters to drive high levels of expression in a wide range of tissues, although promoters from the cellular genes for phosphoglycerate kinase, actin and histones, have also been used with varying degrees of success. These promoters exhibit constitutive expression of proteins, which can cause cellular toxicity and may select for the downregulation of the effector systems. Recent approaches to gene therapy have emphasized the need for gene delivery vectors that can efficiently introduce and control expression of foreign genes in a dose-dependent and reversible manner. Indeed, in certain disorders, only a specific range or dose of the therapeutic protein will achieve a successful outcome (1–3). In clinical applications, it will be beneficial to regulate the transgene expression in order to maintain protein concentrations within the therapeutic window and to optimize efficacy in response to the evolving nature of the disease. A regulatable system would be of value in modifying specific therapies if it could offer tight regulation in response to pharmacologic agents that can be safely and repetitively administered. For cancer gene therapy, therapeutic gene products that would be particularly valuable to control in this way include cytokines (see Agha-Mohammadi and Lotze, this Perspective Series, ref. 4), prodrug activating enzymes (see Springer and Niculescu-Duvaz, this Perspective Series, ref. 5), ribozymes, antibodies, tumoricidal genes, or antisense oligonucleotides. Regulatable systems also have great utility in controlling the expression of the vector delivering the therapeutic gene. If replicating viruses are to be used to deliver such genes or to lyse tumors directly, regulation of viral early promoters may be required to control the rate of viral replication and enhance the safety of the recombinant virus. First-generation regulatable systems, based on naturally occurring inducible promoters, generally suffer from high basal expression of the utilized promoter, weak induction of transgene expression, and reliance on inducible agents that exert pleiotropic effects on mammalian cells. Chimeric regulatable systems, devised to overcome these limitations, incorporate various prokaryotic and eukaryotic elements and offer greater specificity than can be achieved using natural inducible promoters. Transactivators in these chimeric systems are designed to interact specifically with sequences engineered into the vector. Recently developed chimeric systems are regulated by tetracycline (6), the progesterone antagonist RU486 (7), the insect hormone ecdysone (8), or rapamycin (FK506) (9). These drugs or hormones (or their analogs) act on modular transactivators composed of natural or mutant ligand binding domains and intrinsic or extrinsic DNA binding and transcriptional activation domains.

Postbariatric surgery breast reshaping: the spiral flap.

Introduction: After massive weight loss, the breasts have poor shape, projection, and skin elasticity. Breast reshaping is recognized as difficult and may require excess nearby tissues. As the senior authors approach evolved over the past 4 years, breast reshaping with the spiral flap became integral to an upper body lift. Materials and Methods: After the weight loss has stabilized, body contouring surgery has been performed on 53 patients over a 3-year period. Six patients had mastopexy and/or augmentation only. Eighteen patients had spiral flap breast reshaping as part of an upper body lift. This lift is a reverse abdominoplasty that ends along the inframammary fold incision of the Wise pattern mastopexy and continues laterally to along the back roll. Excess tissue from the epigastrium and lateral back roll is deepithelialized and used for augmentation. These flap extensions of the central breast pedicle are spiraled around the breast for augmentation, shaping, and suspension. When more tissue is needed, saline-filled silicone implants have been used, preferably during a second stage. Results: Follow up of this initial group ranged from 4 to 28 months with a mean of 11 months. In this initial effort, 14 of the 18 were pleased. In 3 patients, subsequent bilateral saline implants further augmented the breasts. Tip fat necrosis was evident by firmness of the tissues in 3 patients and resolved in all but 1. That 1 patient had operative debridement of the distal 50% of the flaps followed by saline-filled silicone implants. One patient was disappointed with the back scar. Two patients dislike the shape and fill of their breast and have not returned for revision. Conclusion: During 3 years of focused clinical activity, we have evolved the spiral flap reshaping with upper body lift into a comprehensive, effective, satisfying, and safe esthetic contouring of the breast and upper torso after massive weight loss performed with an upper body lift.

Nutritional deficiency of post-bariatric surgery body contouring patients: what every plastic surgeon should know.

Background: Bariatric surgery, particularly the Roux-en-Y gastric bypass, is currently the most effective method of sustainable weight loss for the morbidly obese patient population. Unfortunately, the nutritional adequacy of the postoperative diet has frequently been overlooked, and in the months to years that follow, many nutritional deficiencies have become apparent. Furthermore, once weight loss has reached a plateau, many patients become candidates for body contouring surgery and other aesthetic operations. Methods: The aim of this review was to highlight the nutritional deficiencies of post-bariatric surgery patients as related to planned body contouring surgery. This review was prepared by an extensive search of the bariatric surgery literature. Results: The current data indicate that many post-bariatric surgery patients have protein-calorie malnutrition as well as various vitamins and mineral deficiencies that may limit optimal health and healing. Conclusions: It is essential that those plastic surgeons who treat post-bariatric surgery patients are aware of these nutritional deficiencies, which can be minimized by adhering to eating guidelines and supplemental prescriptions. Although there are many studies documenting relationships between malnutrition and poor wound healing, the optimal nutrient intake in the post-bariatric surgery state to promote wound healing is unknown. It is, however, clear that proteins, vitamin A, vitamin C, arginine, glutamine, zinc, and selenium have significant beneficial effects on wound healing and optimizing the immune system.

Immunomodulation of cancer: potential use of selectively replicating agents.

Cancer immunotherapy relies on the ability of the immune system to identify and destroy tumor cells and to elicit a long-lasting memory of this interaction. Under ordinary circumstances, however, the ability of tumor cells to trigger an effective immune response is limited. The nominal poor immunogenicity of tumor cells results in part from their weak expression of MHC antigens, adhesion molecules, and costimulatory signals that could allow complete T-cell activation. Tumors may also secrete immunosuppressive molecules, such as IL-10, TGF-β, and PGE2, and they often fail to express cytokines that activate local immune responses (1). These evasive strategies can be overcome by introducing immunomodulatory molecules or genes into the tumor milieu. Over the last 2 decades, researchers have sought to identify cytokines and chemokines that induce the maturation, activation, and migration of inflammatory cells and have used these factors to activate the immune system against tumor cells. Most of this work has been conducted in animals, but results have been encouraging enough that several human trials have been initiated.

A Clinical Review of Total Body Lift Surgery

BACKGROUND Until 2001, body contouring surgery after massive weight loss was uncoordinated, with few patients achieving compete rehabilitation. OBJECTIVE The authors report a 5-year, retrospective, 75-case clinical review to determine the effectiveness, reliability, and safety of single and multistage total body lift (TBL) surgery. METHODS Between January 2001 and June 2006, 59 single-stage, 15 two-stage, and 1 three-stage TBL surgeries were performed, involving a total of 605 separate procedures. Outcomes and complications were compared among all TBL patients and a contemporaneous published series. RESULTS Three representative cases are described. Overall, in patients under 55 years of age with a body mass index of less than 30, there was no significant difference in the choice of procedure (ie, single-stage TBL [95% confidence interval, 1.236-2.302] or multiple-stage TBL [95% confidence interval, 1.687-4.892]; P = .1882). Although there was no significant association between major complications and the number of procedures performed in this cohort of patients, there were increased wound healing problems following multiple-stage TBL (P > .5). Single-stage TBL surgery averaged 8.4 hours. Two-stage surgery took 7.4 hours for the first stage and 4.6 hours for the second stage, for a total of 11 hours. Banked blood transfusions for single-stage surgery were 1.5 per single-stage case and 0.78 per multi-stage case. Seventy-six percent of the patients experienced complications, mostly related to wound healing. All preoperative and postoperative Pittsburgh rating grades improved. CONCLUSIONS TBL is customized for individuals who desire a comprehensive approach to improvement of their loose skin. The rate of complications was high and comparable to other published series. There was no difference between the complications of the single-and two-stage patients. While there was an observable reduction in deformity and a high rate of satisfactory aesthetic outcomes, this high number of complications indicates a need to improve clinical performance.

Adjusting transgene expression levels in lymphocytes with a set of inducible promoters.

Inducible gene expression systems are powerful research tools and could be of clinical value in the future, with lymphocytes being likely prime application targets. However, currently available regulatable promoters exhibit variation in their efficiency in a cell line‐dependent‐manner and are notorious for basal leakiness or poor inducibility. Data concerning the regulatory properties of different inducible promoters are scarce for lymphocytes. In the present study, we report a comprehensive analysis of how various inducible promoters perform and how their combination with a transsilencer and a reverse transactivator can result in optimally controlled gene expression in T‐cells.

A doxycycline-inducible urokinase receptor (uPAR) upregulates uPAR activities including resistance to anoikis in human prostate cancer cell lines

BackgroundThe urokinase receptor (uPAR) mediates a diverse array of cellular processes including several events involved in prostate cancer metastasis. Many of these activities are initiated or enhanced by uPAR binding to its proteolytic ligand, urokinase (uPA). Our objective in this study was to generate and test an inducible lentiviral system capable of expressing uPAR and DsRed fluorescent protein in human prostate cancer cell lines.ResultsA DsRed-uPAR fusion construct was inserted into a lentiviral vector. Transduction of human prostate cancer cell lines with this virus and with a virus containing a reverse-tetracycline transactivator (rt-TA) resulted in a stable transgene which induced both uPAR and DsRed proteins in a dose-responsive fashion upon stimulation with doxycycline. Immunoblots and immunofluorescence studies indicated no detectable uPAR expression in non-induced prostate cancer cell lines. Cells with induced-uPAR demonstrated increased cellular adhesion to the matrix substrate vitronectin and increased net cell proliferation compared to uninduced cells. Finally, induced uPAR-expressing prostate cancer cells were resistant to anoikis over an extended time period when grown in suspension.ConclusionThis doxycycline-inducible lentivirus system produces titerable levels of biologically active uPAR in vitro. This tool can be used to dissect cellular events following induction of uPAR in prostate cancer cells.

Management of Upper Abdominal Laxity After Massive Weight Loss: Reverse Abdominoplasty and Inframammary Fold Reconstruction

BackgroundCentral to body contouring after weight loss surgery is treatment of the abdominal region, often through a circumferential abdominoplasty. This procedure, however, neglects the laxity of the lower thoracic/upper abdominal region. A reverse abdominoplasty with reconstruction of a new inframammary fold (IMF) corrects this deformity through removal of excess skin along the IMF. Since 2002, we have performed 88 reverse abdominoplasty procedures within the context of a single or staged total-body lift (TBL).MethodsA retrospective chart review of 129 TBL cases indicated that 88 patients had a combined or staged reverse abdominoplasty and circumferential abdominoplasty. Complication rates were noted as localized or generalized.ResultsFifty-three of our patients had combined reverse abdominoplasty and circumferential abdominoplasty and 35 had the reverse abdominoplasty during a second stage. The complication rates for both groups were about 5% per patient per procedure with differences that were not statistically significant. Also, the revision rates for reverse abdominoplasty and circumferential abdominoplasty were similar for both groups, indicating patient satisfaction with the procedures.ConclusionIn selected patients, effective treatment of the abdominal region demands correction of both the upper and lower abdominal laxity and contour. This can be performed safely, effectively, and reliably by a reverse abdominoplasty with IMF reconstruction independently or simultaneously with circumferential abdominoplasty.