Sachin David

Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors

The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12–48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6–16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5–2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

Management of Hemophilia in Patients with Inhibitors: The Perspective from Developing Countries

Data are limited on inhibitors in people with hemophilia (PWH) in developing countries. There is a perception that the overall prevalence of inhibitors, ranging from 7 to 19% in different reports, may be lower in these countries as compared with that reported from developed countries. This is possible given the fact that most patients are treated after 2 years of age with plasma-derived clotting factor concentrates. Whether genetic or other environmental factors also contribute to this needs further evaluation. There is a need to develop laboratory infrastructure and establish quality control programs for laboratory tests for inhibitors in developing countries. Management options vary widely given the socioeconomic diversity among these countries. Significant individualization of approach to management is therefore required depending on the available resources, particularly with regard to the use of bypassing agents. The limited data on immune tolerance induction with some low-dose regimens deserve further evaluation. Even in resource-constrained environments, education and a policy of systematic screening of patients associated with judicious use of bypassing agents can significantly improve the care of PWH who develop inhibitors.

Molecular basis of Bernard-Soulier syndrome in 27 patients from India.

To cite this article: Sumitha E, Jayandharan GR, David S, Jacob RR, Sankari Devi G, Bargavi B, Shenbagapriya S, Nair SC, Abraham A, George B, Viswabandya A, Mathews V, Chandy M, Srivastava A. Molecular basis of Bernard–Soulier syndrome in 27 patients from India. J Thromb Haemost 2011; 9: 1590–8.

Molecular basis of Wiskott–Aldrich syndrome in patients from India

To the Editor: Wiskott–Aldrich syndrome (WAS) [OMIM: 301000] is an X-linked immunodeficiency disease characterized by thrombocytopenia and small platelets, eczema, recurrent infections, with an increased risk for autoimmunity and malignancy (1, 2). The gene responsible for this syndrome, WAS, comprises 12 exons and approximately 1.8 kb in length (3). WAS encodes a 502-amino-acid protein (WASp) that is expressed selectively in hematopoietic stem cell– derived lineages and is involved in cell signaling and cytoskeleton reorganization (4). A milder allelic variant caused by WAS gene mutation leads to X-linked thrombocytopenia (XLT), a congenital disorder characterized by thrombocytopenia and small platelets but, in general, without the other complications of WAS (5). So far, ~369mutations have been reported in WAS gene (http://www.hgmd.cf.ac.uk/ ac/gene.php?gene=WAS). Detection of additional mutations in this gene is important for the precise genetic diagnosis in families affected by this disorder as well as for studying the molecular basis of this disease (6). We report here for the first time the WAS gene mutations identified in patients with WAS/XLT from India and their genotype–phenotype correlations. Ten patients from eight families were evaluated at the Department of Haematology, Christian Medical College, Vellore, with clinical features suggestive of WAS on written, informed consent. All patients underwent hematological and biochemical evaluation (Table 1). Blood was collected in citrated buffer and in ethylenediamine tetra-acetic acid (EDTA) from the probands and, whenever available, from family members. Similar samples were also collected from healthy controls. Blood smears were stained using modified Giemsa–Wright stain (Beckman, Duarte, CA, USA) and evaluated for morphology. Platelet count and the mean platelet volume (MPV) were estimated in a cell counter (Coulter LH 755; Beckman Coulter). A mononuclear cell suspension in phosphate-buffered saline (PBS) was isolated from heparinized blood by Ficoll gradient centrifugation. The cells were fixed (Fix and Perm Medium; Invitrogen, Carlsbad, CA, USA) and permeabilized (Fix and Perm medium B; Invitrogen) for intracellular staining and incubated with phycoerythrin (PE)-conjugated antibody against WASP DI (SantaCruz Biotechnology, SantaCruz, CA, USA) and WAS B9 (Santa Cruz Biotechnology) with appropriate IgG1 and IgG2a controls (BD Pharmingen, San Jose, CA, USA). After processing, cells were analyzed for intracellular bound florescence in a flow cytometer (BD FACS Calibur, Manifield, MA, USA). The patient WASP expression was compared to normal controls processed at the same time, and the data are expressed as percentage of normal WASP-positive cells (7). Genomic DNA from EDTA anti-coagulated blood was isolated by standard phenol–chloroform method. The human WAS gene exonic and flanking intronic regions were amplified by twelve pairs of primers as described previously (8). Nucleotide changes in the amplified fragments were screened by a conformation-sensitive gel electrophoresis (CSGE) and DNA sequencing strategy (9). Samples displaying abnormal CSGE patterns were sequenced by the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) on an ABI 3130 genetic analyzer (PE Applied Biosystems, Foster City, CA, USA). All the novel mutations identified were confirmed as unique to the patients by screening 100 normal control alleles. The normal controls were recruited based on a written informed consent. Mutations at or near the splice junction consensus sequences were analyzed by ‘Splice Site Prediction program’ (http://www.fruitfly.org/seq_tools/splice.html) to predict changes in RNA splicing. The clinical features, and hematological and molecular genetic data of all the 10 patients are detailed in Table 1. Diagnosis was based on low platelet count (9000–95 000/ mm), presence of small platelets (mean platelet volume – 6.2 ± 1.5) and reduced levels of WASp by flow cytometry. Of these, two were sibling pairs (WAS-9/WAS-10; WAS15/WAS-35). The median age at first clinical symptoms of these patients was 2.5 yr (range 1–12 yr). Six of them had a family history of bleeding. To differentiate between classical WAS and XLT in these patients, the disease was scored from 1 to 5 based on the presence of thrombocytopenia, small platelets, eczema, immunodeficiency, infections, autoimmunity or malignancy and congenital neutropenia as described previously (10). In patients who were lost to follow-up, we could not comprehensively analyze the biochemical or genetic data, and this was a limitation to our study. Genotypic analysis revealed mutations in eight of ten patients (Table 1). Among them, three had nonsense mutations, two splice site variations, two deletions, and one missense mutation. Two of these eight mutations were novel. These included a single ‘T-nucleotide’ deletion (c.108delT),

Retraction: Association Between 5-HTR2C -759C/T (rs3813929) and -697G/C (rs518147) Gene Polymorphisms and Risperidone-Induced Insulin Resistance Syndrome in an Indian Population

The above article from the Journal of Clinical Pharmacology, first published online on 22 September 2017 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement among the authors, the journal Editor in Chief, Joseph Bertino, and Wiley Periodicals, Inc. The retraction has been agreed upon due to the article having been submitted by the lead author without agreement from all co‐authors. Having been alerted to this irregularity, the journal was also advised by the Senior Author of inaccuracies in the genotyping data.

Mechanisms and management of coagulopathy in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is a subtype of leukemia which is associated with unique and distinctive coagulopathy. In the absence of treatment it is rapidly fatal and even after initiation of therapy the major cause of early mortality is related to hemorrhagic complications. The coagulopathy can be exacerbated with the start of treatment. In the absence of early hemorrhage related deaths the probability of cure exceeds 90% in low and intermediate risk patients and 80% even in high risk patients, highlighting the importance of understanding the pathophysiology of this complication and instituting prompt and appropriate management strategies. The coagulopathy in APL is complex and results from a combination of thrombocytopenia, disseminated intravascular coagulation and hyperfibronlysis. Recently the effect of all-trans retinioc acid (ATRA) induced ETosis on exacerbating coagulopathy in the first few days after starting therapy with this agent raises the potential for potentially novel strategies to reduce the risk of hemorrhage. Currently management is mainly related to rapid initiation of therapy with ATRA along with appropriate and adequate replacement of blood products to correct the coagulopathy. There is limited role for the use of low dose anti-coagulants and anti-fibrinolytic agents in the initial management of this disease. There is limited data on the use of rFVIIa or the use of global tests of hemostasis in the management of this condition.

Arsenic Trioxide Enhances the NK Cell Cytotoxicity Against Acute Promyelocytic Leukemia While Simultaneously Inhibiting Its Bio-Genesis

Natural killer cells (NK) contribute significantly to eradication of cancer cells, and there is increased interest in strategies to enhance it’s efficacy. Therapeutic agents used in the treatment of cancer can impact the immune system in a quantitative and qualitative manner. In this study, we evaluated the impact of arsenic trioxide (ATO) used in the management of acute promyelocytic leukemia (APL) on NK cell reconstitution and function. In patients with APL treated with single agent ATO, there was a significant delay in the reconstitution of circulating NK cells to reach median normal levels from the time of diagnosis (655 days for NK cells vs 145 and 265 days for T cells and B cells, respectively). In vitro experiments demonstrated that ATO significantly reduced the CD34 hematopoietic stem cell (HSC) differentiation to NK cells. Additional experimental data demonstrate that CD34+ sorted cells when exposed to ATO lead to a significant decrease in the expression of IKZF2, ETS1, and TOX transcription factors involved in NK cell differentiation and maturation. In contrast, exposure of NK cells and leukemic cells to low doses of ATO modulates NK cell receptors and malignant cell ligand profile in a direction that enhances NK cell mediated cytolytic activity. We have demonstrated that NK cytolytic activity toward NB4 cell line when exposed to ATO was significantly higher when compared with controls. We also validated this beneficial effect in a mouse model of APL were the median survival with ATO alone and ATO + NK was 44 days (range: 33–46) vs 54 days (range: 52–75). In conclusion, ATO has a differential quantitative and qualitative effect on NK cell activity. This information can potentially be exploited in the management of leukemia.