Publications by authors named "Nishat Sultana"

32 Publications

Direct Reprogramming Induces Vascular Regeneration Post Muscle Ischemic Injury.

Mol Ther 2021 Jul 28. Epub 2021 Jul 28.

Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029. Electronic address:

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail of four cardiac-reprogramming genes (Gata4 (G), Mef2c (M), Tbx5 (T) and Hand2 (H)) together with three reprogramming-helper genes (Dominant Negative (DN)-TGFβ, DN-Wnt8a and Acid ceramidase (AC)), termed 7G-modRNA, to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival and capillary density. Mechanistically, we determined that while 7G-modRNA cannot create de-novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.
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http://dx.doi.org/10.1016/j.ymthe.2021.07.014DOI Listing
July 2021

Therapeutic Delivery of Pip4k2c-Modified mRNA Attenuates Cardiac Hypertrophy and Fibrosis in the Failing Heart.

Adv Sci (Weinh) 2021 05 12;8(10):2004661. Epub 2021 Mar 12.

Cardiovascular Research Center Icahn School of Medicine at Mount Sinai New York NY 10029 USA.

Heart failure (HF) remains a major cause of morbidity and mortality worldwide. One of the risk factors for HF is cardiac hypertrophy (CH), which is frequently accompanied by cardiac fibrosis (CF). CH and CF are controlled by master regulators mTORC1 and TGF-, respectively. Type-2-phosphatidylinositol-5-phosphate-4-kinase-gamma (Pip4k2c) is a known mTORC1 regulator. It is shown that Pip4k2c is significantly downregulated in the hearts of CH and HF patients as compared to non-injured hearts. The role of Pip4k2c in the heart during development and disease is unknown. It is shown that deleting Pip4k2c does not affect normal embryonic cardiac development; however, three weeks after TAC, adult Pip4k2c mice has higher rates of CH, CF, and sudden death than wild-type mice. In a gain-of-function study using a TAC mouse model, Pip4k2c is transiently upregulated using a modified mRNA (modRNA) gene delivery platform, which significantly improve heart function, reverse CH and CF, and lead to increased survival. Mechanistically, it is shown that Pip4k2c inhibits TGF1 via its N-terminal motif, Pip5k1, phospho-AKT 1/2/3, and phospho-Smad3. In sum, loss-and-gain-of-function studies in a TAC mouse model are used to identify Pip4k2c as a potential therapeutic target for CF, CH, and HF, for which modRNA is a highly translatable gene therapy approach.
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http://dx.doi.org/10.1002/advs.202004661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132051PMC
May 2021

In Vitro Synthesis of Modified RNA for Cardiac Gene Therapy.

Methods Mol Biol 2021 ;2158:281-294

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Modified mRNA (modRNA) is a promising new gene therapy approach that has safely and effectively delivered genes into different tissues, including the heart. Current efforts to use DNA-based or viral gene therapy to induce cardiac regeneration postmyocardial infarction (MI) or in heart failure (HF) have encountered key challenges, e.g., genome integration and delayed and noncontrolled expression. By contrast, modRNA is a transient, safe, non-immunogenic, and controlled gene delivery method that is not integrated into the genome. For most therapeutic applications, especially in regenerative medicine, the ability to deliver genes to the heart transiently and with control is vital for achieving therapeutic effect. Additionally, modRNA synthesis is comparatively simple and inexpensive compared to other gene delivery methods (e.g., protein), though a simple, clear in vitro transcription (IVT) protocol for synthesizing modRNA is needed for it to be more widely used. Here, we describe a simple and improved step-by-step IVT protocol to synthesize modRNA for in vitro or in vivo applications.
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http://dx.doi.org/10.1007/978-1-0716-0668-1_21DOI Listing
March 2021

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model.

J Vis Exp 2020 06 11(160). Epub 2020 Jun 11.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai;

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising treatment option for heart disease, owing to its efficiency and exceptional therapeutic effects. In an effort to repair the damaged tissue post-MI, various studies have employed DNA-based or viral gene therapy but have faced considerable hurdles due to the poor and uncontrolled expression of the delivered genes, edema, arrhythmia, and cardiac hypertrophy. Synthetic modified mRNA (modRNA) presents a novel gene therapy approach that offers high, transient, safe, nonimmunogenic, and controlled mRNA delivery to the heart tissue without any risk of genomic integration. Due to these remarkable characteristics combined with its bell-shaped pharmacokinetics in the heart, modRNA has become an attractive approach for the treatment of heart disease. However, to increase its effectiveness in vivo, a consistent and reliable delivery method needs to be followed. Hence, to maximize modRNA delivery efficiency and yield consistency in modRNA use for in vivo applications, an optimized method of preparation and delivery of modRNA intracardiac injection in a mouse MI model is presented. This protocol will make modRNA delivery more accessible for basic and translational research.
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http://dx.doi.org/10.3791/60832DOI Listing
June 2020

Optimization of 5' Untranslated Region of Modified mRNA for Use in Cardiac or Hepatic Ischemic Injury.

Mol Ther Methods Clin Dev 2020 Jun 31;17:622-633. Epub 2020 Mar 31.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Modified mRNA (modRNA) is a gene-delivery platform for transiently introducing a single gene or several genes of interest to different cell types and tissues. modRNA is considered to be a safe vector for gene transfer, as it negligibly activates the innate immune system and does not compromise the genome integrity. The use of modRNA in basic and translational science is rising, due to the clinical potential of modRNA. We are currently using modRNA to induce cardiac regeneration post-ischemic injury. Major obstacles in using modRNA for cardiac ischemic disease include the need for the direct and single administration of modRNA to the heart and the inefficient translation of modRNA due to its short half-life. Modulation of the 5' untranslated region (5' UTR) to enhance translation efficiency in ischemic cardiac disease has great value, as it can reduce the amount of modRNA needed per delivery and will achieve higher and longer protein production post-single delivery. Here, we identified that 5' UTR, from the fatty acid metabolism gene carboxylesterase 1D (Ces1d), enhanced the translation of firefly luciferase (Luc) modRNA by 2-fold in the heart post-myocardial infarction (MI). Moreover, we identified, in the Ces1d, a specific RNA element (element D) that is responsible for the improvement of modRNA translation and leads to a 2.5-fold translation increment over Luc modRNA carrying artificial 5' UTR, post-MI. Importantly, we were able to show that 5' UTR Ces1d also enhances modRNA translation in the liver, but not in the kidney, post-ischemic injury, indicating that Ces1d 5' UTR and element D may play a wider role in translation of protein under an ischemic condition.
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http://dx.doi.org/10.1016/j.omtm.2020.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150433PMC
June 2020

Altering Sphingolipid Metabolism Attenuates Cell Death and Inflammatory Response After Myocardial Infarction.

Circulation 2020 03 29;141(11):916-930. Epub 2020 Jan 29.

Cardiovascular Research Center (Y.H., E.Y., M.M.Ż., E.C., N.S., M.T.K.S., R.K., A.A.K., K.K., A.M., N.H., L.Z., A.F, M.G.K.), Icahn School of Medicine at Mount Sinai, New York.

Background: Sphingolipids have recently emerged as a biomarker of recurrence and mortality after myocardial infarction (MI). The increased ceramide levels in mammalian heart tissues during acute MI, as demonstrated by several groups, is associated with higher cell death rates in the left ventricle and deteriorated cardiac function. Ceramidase, the only enzyme known to hydrolyze proapoptotic ceramide, generates sphingosine, which is then phosphorylated by sphingosine kinase to produce the prosurvival molecule sphingosine-1-phosphate. We hypothesized that Acid Ceramidase (AC) overexpression would counteract the negative effects of elevated ceramide and promote cell survival, thereby providing cardioprotection after MI.

Methods: We performed transcriptomic, sphingolipid, and protein analyses to evaluate sphingolipid metabolism and signaling post-MI. We investigated the effect of altering ceramide metabolism through a loss (chemical inhibitors) or gain (modified mRNA [modRNA]) of AC function post hypoxia or MI.

Results: We found that several genes involved in de novo ceramide synthesis were upregulated and that ceramide (C16, C20, C20:1, and C24) levels had significantly increased 24 hours after MI. AC inhibition after hypoxia or MI resulted in reduced AC activity and increased cell death. By contrast, enhancing AC activity via AC modRNA treatment increased cell survival after hypoxia or MI. AC modRNA-treated mice had significantly better heart function, longer survival, and smaller scar size than control mice 28 days post-MI. We attributed the improvement in heart function post-MI after AC modRNA delivery to decreased ceramide levels, lower cell death rates, and changes in the composition of the immune cell population in the left ventricle manifested by lowered abundance of proinflammatory detrimental neutrophils.

Conclusions: Our findings suggest that transiently altering sphingolipid metabolism through AC overexpression is sufficient and necessary to induce cardioprotection post-MI, thereby highlighting the therapeutic potential of AC modRNA in ischemic heart disease.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.041882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135928PMC
March 2020

Optimizing Modified mRNA Synthesis Protocol for Heart Gene Therapy.

Mol Ther Methods Clin Dev 2019 Sep 30;14:300-305. Epub 2019 Jul 30.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Synthetic modified RNA (modRNA) is a novel vector for gene transfer to the heart and other organs. modRNA can mediate strong, transient protein expression with minimal induction of the innate immune response and risk for genome integration. modRNA is already being used in several human clinical trials, and its use in basic and translational science is growing. Due to the complexity of preparing modRNA and the high cost of its reagents, there is a need for an improved, cost-efficient protocol to make modRNA. Here we show that changing the ratio between anti-reverse cap analog (ARCA) and N1-methyl-pseudouridine (N1mΨ), favoring ARCA over N1mΨ, significantly increases the yield per reaction, improves modRNA translation, and reduces its immunogenicity . This protocol will make modRNA preparation more accessible and financially affordable for basic and translational research.
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http://dx.doi.org/10.1016/j.omtm.2019.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722299PMC
September 2019

Field evaluation of a locally produced rapid diagnostic test for early detection of cholera in Bangladesh.

PLoS Negl Trop Dis 2019 01 31;13(1):e0007124. Epub 2019 Jan 31.

International Centre for Diarrheal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh.

Background: Cholera remains a substantial health burden in Asia and Africa particularly in resource poor settings. The standard procedures to identify the etiological organism V. cholerae are isolation from microbiological culture from stool as well as Polymerase Chain Reaction (PCR). Both the processes are highly lab oriented, labor extensive, time consuming, and expensive. In an effort to control for outbreaks and epidemics; an effective, convenient, quick and relatively less expensive detection method is imperative, without compromising the sensitivity and specificity that exists at present. The objective of this component of the study was to evaluate the effectiveness of a locally produced rapid diagnostic test (RDT) for cholera diagnosis.

Methods: In Bangladesh, nationwide cholera surveillance is ongoing in 22 hospitals covering all 8 divisions of the country since June, 2016. In the surveillance, stool samples have been collected from patients presenting to hospitals with acute watery diarrhea. Crystal VCTM (Span diagnostics, India) and Cholkit (locally produced RDT) have been used to detect V. cholerae from stool samples. Samples have also been sent to the main laboratory at icddr,b where the culture based isolation is routinely performed. All the tests were carried out for both direct and enriched stool samples. RDT sensitivity and specificity were calculated using stool culture as the gold standard.

Results: A total of 7720 samples were tested. Among these, 5865 samples were solely tested with Crystal VC and 1355 samples with Cholkit whereas 381 samples were tested with both the RDTs. In comparison with culture, direct testing with Crystal VC showed a sensitivity of 72% (95% CI: 50.6% to 87.9%) and specificity of 86.8% (95% CI: 82.8% to 90.1%). After enrichment the sensitivity and specificity was 68% (95% CI: 46.5% to 85.1%) and 97.5% (95% CI: 95.3% to 98.8%) respectively. The direct Cholkit test showed sensitivity of 76% (95% CI: 54.9% to 90.6%) and specificity of 90.2% (95% CI: 86.6% to 93.1%).

Conclusion: This evaluation has demonstrated that the sensitivity and specificity of Cholkit is similar to the commercially available test, Crystal VC when used in field settings for detecting V. cholerae from stool specimens. The findings from this study suggest that the Cholkit could be a possible alternative for cholera endemic regions where V. cholerae O1 is the major causative organism causing cholera.
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http://dx.doi.org/10.1371/journal.pntd.0007124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372204PMC
January 2019

Cardiac Sca-1 Cells Are Not Intrinsic Stem Cells for Myocardial Development, Renewal, and Repair.

Circulation 2018 12;138(25):2919-2930

Riley Heart Research Center and Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis (L. Zhang, F.Y., C.-L.C.).

Background: For more than a decade, Sca-1 cells within the mouse heart have been widely recognized as a stem cell population with multipotency that can give rise to cardiomyocytes, endothelial cells, and smooth muscle cells in vitro and after cardiac grafting. However, the developmental origin and authentic nature of these cells remain elusive.

Methods: Here, we used a series of high-fidelity genetic mouse models to characterize the identity and regenerative potential of cardiac resident Sca-1 cells.

Results: With these novel genetic tools, we found that Sca-1 does not label cardiac precursor cells during early embryonic heart formation. Postnatal cardiac resident Sca-1 cells are in fact a pure endothelial cell population. They retain endothelial properties and exhibit minimal cardiomyogenic potential during development, normal aging and upon ischemic injury.

Conclusions: Our study provides definitive insights into the nature of cardiac resident Sca-1 cells. The observations challenge the current dogma that cardiac resident Sca-1 cells are intrinsic stem cells for myocardial development, renewal, and repair, and suggest that the mechanisms of transplanted Sca-1 cells in heart repair need to be reassessed.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.035200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366943PMC
December 2018

Smad4 deficiency impairs chondrocyte hypertrophy via the Runx2 transcription factor in mouse skeletal development.

J Biol Chem 2018 06 7;293(24):9162-9175. Epub 2018 May 7.

From the Department of Developmental and Regenerative Biology, The Mindich Child Health and Development Institute, and The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029,

Chondrocyte hypertrophy is the terminal step in chondrocyte differentiation and is crucial for endochondral bone formation. How signaling pathways regulate chondrocyte hypertrophic differentiation remains incompletely understood. In this study, using a () gene-deletion approach, we selectively deleted the gene for the signaling protein SMAD family member 4 ( ) in the limbs of mice. We found that the -deficient mice develop a prominent shortened limb, with decreased expression of chondrocyte differentiation markers, including and , in the humerus at mid-to-late gestation. The most striking defects in these mice were the absence of stylopod elements and failure of chondrocyte hypertrophy in the humerus. Moreover, expression levels of the chondrocyte hypertrophy-related markers and were significantly decreased. Of note, we also observed that the expression of runt-related transcription factor 2 (), a critical mediator of chondrocyte hypertrophy, was also down-regulated in -deficient limbs. To determine how the skeletal defects arose in the mouse mutants, we performed RNA-Seq with ChIP-Seq analyses and found that Smad4 directly binds to regulatory elements in the promoter. Our results suggest a new mechanism whereby Smad4 controls chondrocyte hypertrophy by up-regulating expression during skeletal development. The regulatory mechanism involving Smad4-mediated activation uncovered here provides critical insights into bone development and pathogenesis of chondrodysplasia.
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http://dx.doi.org/10.1074/jbc.RA118.001825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6005452PMC
June 2018

Development of a new dipstick (Cholkit) for rapid detection of Vibrio cholerae O1 in acute watery diarrheal stools.

PLoS Negl Trop Dis 2018 03 14;12(3):e0006286. Epub 2018 Mar 14.

Infectious Diseases Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh.

Recognizing cholera cases early, especially in the initial phase of an outbreak and in areas where cholera has not previously circulated, is a high public health priority. Laboratory capacity in such settings is often limited. To address this, we have developed a rapid diagnostic test (RDT) termed Cholkit that is based on an immunochromatographic lateral flow assay for the diagnosis of cholera cases using stool. Cholkit contains a monoclonal antibody (ICL-33) to the O-specific polysaccharide (OSP) component of V. cholerae O1 lipopolysaccharide, and recognizes both Inaba and Ogawa serotypes. We tested the Cholkit dipstick using fresh stool specimens of 76 adults and children presenting with acute watery diarrhea at the icddr,b hospital in Dhaka, Bangladesh. We compared Cholkit's performance with those of microbial culture, PCR (targeting the rfb and ctxA genes of V. cholerae) and the commercially available RDT, Crystal VC (Span Diagnostics; Surat, India). We found that all stool specimens with a positive culture for V. cholerae O1 (n = 19) were positive by Cholkit as well as Crystal VC. We then used Bayesian latent class modeling to estimate the sensitivity and specificity of each diagnostic assay. The sensitivity of Cholkit, microbiological culture, PCR and Crystal VC was 98% (95% CI: 88-100), 71% (95% CI: 59-81), 74% (95% CI: 59-86) and 98% (95% CI: 88-100), respectively. The specificity for V. cholerae O1 was 97% (95% CI: 89-100), 100%, 97% (95% CI: 93-99) and 98% (95% CI: 92-100), respectively. Of note, two Crystal VC dipsticks were positive for V. cholerae O139 but negative by culture and PCR in this area without known circulating epidemic V. cholerae O139. In conclusion, the Cholkit dipstick is simple to use, requires no dedicated laboratory capacity, and has a sensitivity and specificity for V. cholerae O1 of 98% and 97%, respectively. Cholkit warrants further evaluation in other settings.
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http://dx.doi.org/10.1371/journal.pntd.0006286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862499PMC
March 2018

Study of lip prints in different ethno-racial groups in India.

Indian J Dent Res 2017 Sep-Oct;28(5):545-548

MDS, Bangalore, Karnataka, India.

Context (Background): Lips prints are unique and are a tool for personal identification.

Aims: Indian population can be divided into different ethno-racial groups. In this study, we aimed at finding the most and the least prevalent lip print patterns in these groups and also to observe any similarities or differences that may exist in these groups in terms of lip print patterns.

Settings And Design: Lip prints in 755 individuals categorized into different ethno-racial groups were studied.

Materials And Methods: Brown- and pink-colored lipsticks, cellophane tape, and magnifying lens were used to record and study the lip prints.

Results: Among all the three ethno-racial groups, Type I was the most prevalent lip print pattern observed. The least prevalent lip print pattern in all the three groups was Type IV. Inference/Conclusion: Lip prints hold potential as supplementary tools for identification where they can be recorded with ease. The observation and classification of lip print patterns in different ethno-racial groups not only provide some useful data but also open a new window to a field that can contribute extensively to criminal investigation and identification.
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http://dx.doi.org/10.4103/ijdr.IJDR_352_14DOI Listing
July 2018

Optimizing Cardiac Delivery of Modified mRNA.

Mol Ther 2017 06 4;25(6):1306-1315. Epub 2017 Apr 4.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

Modified mRNA (modRNA) is a new technology in the field of somatic gene transfer that has been used for the delivery of genes into different tissues, including the heart. Our group and others have shown that modRNAs injected into the heart are robustly translated into the encoded protein and can potentially improve outcome in heart injury models. However, the optimal compositions of the modRNA and the reagents necessary to achieve optimal expression in the heart have not been characterized yet. In this study, our aim was to elucidate those parameters by testing different nucleotide modifications, modRNA doses, and transfection reagents both in vitro and in vivo in cardiac cells and tissue. Our results indicate that optimal cardiac delivery of modRNA is with N1-Methylpseudouridine-5'-Triphosphate nucleotide modification and achieved using 0.013 μg modRNA/mm/500 cardiomyocytes (CMs) transfected with positively charged transfection reagent in vitro and 100 μg/mouse heart (1.6 μg modRNA/μL in 60 μL total) sucrose-citrate buffer in vivo. We have optimized the conditions for cardiac delivery of modRNA in vitro and in vivo. Using the described methods and conditions may allow for successful gene delivery using modRNA in various models of cardiovascular disease.
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http://dx.doi.org/10.1016/j.ymthe.2017.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474881PMC
June 2017

Synthesis of Modified mRNA for Myocardial Delivery.

Methods Mol Biol 2017 ;1521:127-138

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Cardiac gene therapy shows tremendous promise in combating the growing problem of heart disease. Modified mRNA (modRNA) is a novel gene delivery system used in vitro or in vivo to achieve transient expression of therapeutic proteins in a heterogeneous population of cells. Incorporation of specific modified nucleosides enables modRNA to be translated efficiently without triggering antiviral and innate immune responses. ModRNA has been shown to be effective at delivering short-term robust gene expression to the heart and its use in the field of cardiac gene therapy is expanding. Here, we describe a stepwise protocol for the synthesis of modRNA for in vivo myocardial delivery.
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http://dx.doi.org/10.1007/978-1-4939-6588-5_8DOI Listing
January 2018

Insulin-Like Growth Factor 1 Receptor-Dependent Pathway Drives Epicardial Adipose Tissue Formation After Myocardial Injury.

Circulation 2017 Jan 1;135(1):59-72. Epub 2016 Nov 1.

From Cardiovascular Research Center, Department of Genetics and Genomic Sciences, and Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York (L.Z., N.S., Y.H.); Department of Cardiology, Boston Children's Hospital, MA (L.Z., M.S.O., L.Y.Y., Q.M., W.T.P.); Cardiovascular and Metabolic Diseases Innovative Medicine Biotech Unit, AstraZeneca, Möllndal, Sweden (D.S., Q.-D.W.); The State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (B.Z.); Department of Genetics (W.L.C.), Harvard Stem Cell Institute (W.E., W.T.P.), Harvard Medical School, and Cardiovascular Research Center, Massachusetts General Hospital (M.A.), Harvard Medical School, Boston, MA; and Department of Cell and Molecular Biology and Medicine, Karolinska Institutet, Stockholm, Sweden (K.R.C.).

Background: Epicardial adipose tissue volume and coronary artery disease are strongly associated, even after accounting for overall body mass. Despite its pathophysiological significance, the origin and paracrine signaling pathways that regulate epicardial adipose tissue's formation and expansion are unclear.

Methods: We used a novel modified mRNA-based screening approach to probe the effect of individual paracrine factors on epicardial progenitors in the adult heart.

Results: Using 2 independent lineage-tracing strategies in murine models, we show that cells originating from the Wt1 mesothelial lineage, which includes epicardial cells, differentiate into epicardial adipose tissue after myocardial infarction. This differentiation process required Wt1 expression in this lineage and was stimulated by insulin-like growth factor 1 receptor (IGF1R) activation. IGF1R inhibition within this lineage significantly reduced its adipogenic differentiation in the context of exogenous, IGF1-modified mRNA stimulation. Moreover, IGF1R inhibition significantly reduced Wt1 lineage cell differentiation into adipocytes after myocardial infarction.

Conclusions: Our results establish IGF1R signaling as a key pathway that governs epicardial adipose tissue formation in the context of myocardial injury by redirecting the fate of Wt1 lineage cells. Our study also demonstrates the power of modified mRNA -based paracrine factor library screening to dissect signaling pathways that govern progenitor cell activity in homeostasis and disease.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.116.022064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5195872PMC
January 2017

A series of robust genetic indicators for definitive identification of cardiomyocytes.

J Mol Cell Cardiol 2016 08 3;97:278-85. Epub 2016 Jun 3.

Department of Developmental and Regenerative Biology, The Black Family Stem Cell Institute, and The Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

Definitively identifying the cell type of newly generated cells in the heart and defining their origins are central questions in cardiac regenerative medicine. Currently, it is challenging to ascertain the myocardial identity and to track myocardial progeny during heart development and disease due to lack of proper genetic tools. This may lead to many misinterpretations of the findings in cardiac regenerative biology. In this study, we developed a set of novel mouse models by inserting double reporter genes nlacZ/H2B-GFP, mGFP/H2B-mCherry into the start codon of Tnnt2 and Myh6. nlacZ (nuclear lacZ) and mGFP (membrane GFP) are flanked by two LoxP sites in these animals. We found that the reporter genes faithfully recapitulated Tnnt2 and Myh6 cardiac expression from embryonic stage and adulthood. The reporter mice provide unprecedented robustness and fidelity for visualizing and tracing cardiomyocytes with nuclear or cell membrane localization signals. These animal models offer superior genetic tools to meet a critical need in studies of heart development, cardiac stem cell biology and cardiac regenerative medicine.
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http://dx.doi.org/10.1016/j.yjmcc.2016.06.003DOI Listing
August 2016

Development of a Simple, Peripheral-Blood-Based Lateral-Flow Dipstick Assay for Accurate Detection of Patients with Enteric Fever.

Clin Vaccine Immunol 2016 05 6;23(5):403-409. Epub 2016 May 6.

International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh

Enteric fever is a systemic infection caused by typhoidal strains of Salmonella enterica and is a significant cause of mortality and morbidity in many parts of the world, especially in resource-limited areas. Unfortunately, currently available diagnostic tests for enteric fever lack sensitivity and/or specificity. No true clinically practical gold standard for diagnosing patients with enteric fever exists. Unfortunately, microbiologic culturing of blood is only 30 to 70% sensitive although 100% specific. Here, we report the development of a lateral-flow immunochromatographic dipstick assay based on the detection of Salmonella enterica serovar Typhi (S Typhi) lipopolysaccharide (LPS)-specific IgG in lymphocyte culture secretion. We tested the assay using samples from 142 clinically suspected enteric fever patients, 28 healthy individuals residing in a zone where enteric fever is endemic, and 35 patients with other febrile illnesses. In our analysis, the dipstick detected all blood culture-confirmed S Typhi cases (48/48) and 5 of 6 Salmonella enterica serovar Paratyphi A blood cultured-confirmed cases. The test was negative in all 35 individuals febrile with other illnesses and all 28 healthy controls from the zone of endemicity. The test was positive in 19 of 88 individuals with suspected enteric fever but with negative blood cultures. Thus, the dipstick had a sensitivity of 98% compared to blood culture results and a specificity that ranged from 78 to 100% (95% confidence interval [CI], 70 to 100%), depending on the definition of a true negative. These results suggest that this dipstick assay can be very useful for the detection of enteric fever patients especially in regions of endemicity.
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http://dx.doi.org/10.1128/CVI.00690-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860473PMC
May 2016

Resident c-kit(+) cells in the heart are not cardiac stem cells.

Nat Commun 2015 Oct 30;6:8701. Epub 2015 Oct 30.

Department of Developmental and Regenerative Biology, The Black Family Stem Cell Institute, and The Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA.

Identifying a bona fide population of cardiac stem cells (CSCs) is a critical step for developing cell-based therapies for heart failure patients. Previously, cardiac c-kit(+) cells were reported to be CSCs with a potential to become myocardial, endothelial and smooth muscle cells in vitro and after cardiac injury. Here we provide further insights into the nature of cardiac c-kit(+) cells. By targeting the c-kit locus with multiple reporter genes in mice, we find that c-kit expression rarely co-localizes with the expression of the cardiac progenitor and myogenic marker Nkx2.5, or that of the myocardial marker, cardiac troponin T (cTnT). Instead, c-kit predominantly labels a cardiac endothelial cell population in developing and adult hearts. After acute cardiac injury, c-kit(+) cells retain their endothelial identity and do not become myogenic progenitors or cardiomyocytes. Thus, our work strongly suggests that c-kit(+) cells in the murine heart are endothelial cells and not CSCs.
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http://dx.doi.org/10.1038/ncomms9701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846318PMC
October 2015

A Murine Myh6MerCreMer Knock-In Allele Specifically Mediates Temporal Genetic Deletion in Cardiomyocytes after Tamoxifen Induction.

PLoS One 2015 23;10(7):e0133472. Epub 2015 Jul 23.

Department of Developmental and Regenerative Biology, The Black Family Stem Cell Institute, and The Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States of America.

A mouse model that mediates temporal, specific, and efficient myocardial deletion with Cre-LoxP technology will be a valuable tool to determine the function of genes during heart formation. Mhy6 encodes a cardiac muscle specific protein: alpha-myosin heavy chain. Here, we generated a new Myh6-MerCreMer (Myh6(MerCreMer/+)) inducible Cre knock-in mouse by inserting a MerCreMer cassette into the Myh6 start codon. By crossing knock-in mice with Rosa26 reporter lines, we found the Myh6(MerCreMer/+) mice mediate complete Cre-LoxP recombination in cardiomyocytes after tamoxifen induction. X-gal staining and immunohistochemistry analysis revealed that Myh6-driven Cre recombinase was specifically activated in cardiomyocytes at embryonic and adult stages. Furthermore, echocardiography showed that Myh6(MerCreMer/+) mice maintained normal cardiac structure and function before and after tamoxifen administration. These results suggest that the new Myh6(MerCreMer/+) mouse can serve as a robust tool to dissect the roles of genes in heart development and function. Additionally, myocardial progeny during heart development and after cardiac injury can be traced using this mouse line.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133472PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4512710PMC
May 2016

Generation of a tamoxifen inducible Tnnt2MerCreMer knock-in mouse model for cardiac studies.

Genesis 2015 Jun 13;53(6):377-86. Epub 2015 Jun 13.

Department of Developmental and Regenerative Biology, The Black Family Stem Cell Institute, and the Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, New York.

Tnnt2, encoding thin-filament sarcomeric protein cardiac troponin T, plays critical roles in heart development and function in mammals. To develop an inducible genetic deletion strategy in myocardial cells, we generated a new Tnnt2:MerCreMer (Tnnt2(MerCreMer/+)) knock-in mouse. Rosa26 reporter lines were used to examine the specificity and efficiency of the inducible Cre recombinase. We found that Cre was specifically and robustly expressed in the cardiomyocytes at embryonic and adult stages following tamoxifen induction. The knock-in allele on Tnnt2 locus does not impact cardiac function. These results suggest that this new Tnnt2(MerCreMer/+) mouse could be applied towards the temporal genetic deletion of genes of interests in cardiomyocytes with Cre-LoxP technology. The Tnnt2(MerCreMer/+) mouse model also provides a useful tool to trace myocardial lineage during development and repair after cardiac injury.
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http://dx.doi.org/10.1002/dvg.22861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480198PMC
June 2015

Cheiloscopic patterns in Indian population and their efficacy in sex determination: A randomized cross-sectional study.

J Forensic Dent Sci 2015 May-Aug;7(2):101-6

Department of Oral Medicine and Radiology, Vydehi Institute of Dental Sciences and Research Centre, Bangalore, Karnataka, India.

Background: Cheiloscopy or the study of lip prints is an important tool in forensic dentistry which is gaining popularity in current times. Not only has it proven effective in identification of individuals, but its role in sex determination has also been investigated. There are six different types of cheiloscopic/lip print patterns according to Tsuchihashi.

Aims: The present study was conducted to find the prevalence of cheiloscopic/lip print pattern in 755 individuals and also to assess their efficacy in sex determination.

Materials And Methods: According to classification by Tsuchihashi, the lip prints were analysed. Sex determination using lip print was done by method given by Vahanwala et al.

Results: It was found that Type I was the most prevalent cheiloscopic pattern. Based on the method of sex determination using lip prints by Vahanwala et al., the percentage of individuals correctly identified was less.

Conclusion: Although cheiloscopy holds promise as a supplementary tool along with other modes to recognize the sex of an individual, there is need for further studies using standardized methods to evaluate the same in larger population sizes.
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http://dx.doi.org/10.4103/0975-1475.156192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4430566PMC
May 2015

Smad4 regulates ureteral smooth muscle cell differentiation during mouse embryogenesis.

PLoS One 2014 15;9(8):e104503. Epub 2014 Aug 15.

Department of Developmental and Regenerative Biology, The Mindich Child Health and Development Institute, and The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.

Proper formation of ureteral smooth muscle cells (SMCs) during embryogenesis is essential for ureter peristalsis that propels urine from the kidney to the bladder in mammals. Currently the molecular factors that regulate differentiation of ureteral mesenchymal cells into SMCs are incompletely understood. A recent study has reported that Smad4 deficiency reduces the number of ureteral SMCs. However, its precise role in the ureteral smooth muscle development remains largely unknown. Here, we used Tbx18:Cre knock-in mouse line to delete Smad4 to examine its requirement in the development of ureteral mesenchyme and SMC differentiation. We found that mice with specific deletion of Smad4 in Tbx18-expressing ureteral mesenchyme exhibited hydroureter and hydronephrosis at embryonic day (E) 16.5, and the mutant mesenchymal cells failed to differentiate into SMCs with increased apoptosis and decreased proliferation. Molecular markers for SMCs including alpha smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) were absent in the mutant ureters. Moreover, disruption of Smad4 significantly reduced the expression of genes, including Sox9, Tbx18 and Myocardin associated with SMC differentiation. These findings suggest that Smad4 is essential for initiating the SMC differentiation program during ureter development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104503PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134214PMC
February 2016

Topical tacrolimus in the management of oral lichen planus: literature review.

J Calif Dent Assoc 2014 Mar;42(3):165-70

Tacrolimus is an immunosuppressive property approved for the treatment of atopic dermatitis. Studies have shown that topical tacrolimus is effective for a broad spectrum of mucocutaneous diseases, including oral lichen planus. The objective of this article is to review the pharmacology of tacrolimus, its usage in oral lichen planus, adverse effects and advantages of tacrolimus over other conventional drugs, thus making it a popular and alternative drug for the treatment of lichen planus.
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March 2014

Botulinum toxin: The Midas touch.

J Nat Sci Biol Med 2014 Jan;5(1):8-14

Department of Oral Medicine and Radiology, Vydehi Institute of Dental Sciences, No. 82, EPIP Area, White Field, Bangalore, India.

Botulinum Toxin (BT) is a natural molecule produced during growth and autolysis of bacterium called Clostridium botulinum. Use of BT for cosmetic purposes has gained popularity over past two decades, and recently, other therapeutic uses of BT has been extensively studied. BT is considered as a minimally invasive agent that can be used in the treatment of various orofacial disorders and improving the quality of life in such patients. The objective of this article is to review the nature, mechanism of action of BT, and its application in various head and neck diseases.
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http://dx.doi.org/10.4103/0976-9668.127274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3961958PMC
January 2014

Mesodermal Nkx2.5 is necessary and sufficient for early second heart field development.

Dev Biol 2014 Jun 5;390(1):68-79. Epub 2014 Mar 5.

Department of Developmental and Regenerative Biology, The Mindich Child Health and Development Institute, and The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA. Electronic address:

The vertebrate heart develops from mesoderm and requires inductive signals secreted from early endoderm. During embryogenesis, Nkx2.5 acts as a key transcription factor and plays essential roles for heart formation from Drosophila to human. In mice, Nkx2.5 is expressed in the early first heart field, second heart field pharyngeal mesoderm, as well as pharyngeal endodermal cells underlying the second heart field. Currently, the specific requirements for Nkx2.5 in the endoderm versus mesoderm with regard to early heart formation are incompletely understood. Here, we performed tissue-specific deletion in mice to dissect the roles of Nkx2.5 in the pharyngeal endoderm and mesoderm. We found that heart development appeared normal after endodermal deletion of Nkx2.5 whereas mesodermal deletion engendered cardiac defects almost identical to those observed on Nkx2.5 null embryos (Nkx2.5(-/-)). Furthermore, re-expression of Nkx2.5 in the mesoderm rescued Nkx2.5(-/-) heart defects. Our findings reveal that Nkx2.5 in the mesoderm is essential while endodermal expression is dispensable for early heart formation in mammals.
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http://dx.doi.org/10.1016/j.ydbio.2014.02.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461860PMC
June 2014

Stem cells: Boon to dentistry and medicine.

Dent Res J (Isfahan) 2013 Mar;10(2):149-54

Department of Oral Medicine and Radiology, Vydehi Institute of Dental Sciences and Research Center, Bangalore, India.

Stem cell research has received considerable attention since the discovery that adult stem cells have the capacity to form many different tissue types. Stem cells are a booming field for the research and have been extensively studied in the field of medicine, as well as dentistry. Their application in oncology has been a boon to many of the patients. Dental stem cells have been novel approach to treat diseases like periodontitis, dental caries and many more. Their potential uses in dentistry have provided a new generation of treatments for dental diseases and stem cells have become the focus in dental research. This review highlights about the biology, sources and potential applications of stem cells in dentistry with emphasis on a dentist's role in enabling both medical and dental applications using stem cells from teeth.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731952PMC
http://dx.doi.org/10.4103/1735-3327.113321DOI Listing
March 2013

Tbx20 acts upstream of Wnt signaling to regulate endocardial cushion formation and valve remodeling during mouse cardiogenesis.

Development 2013 Aug 3;140(15):3176-87. Epub 2013 Jul 3.

Department of Developmental and Regenerative Biology, The Mindich Child Health and Development Institute, and The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Cardiac valves are essential to direct forward blood flow through the cardiac chambers efficiently. Congenital valvular defects are prevalent among newborns and can cause an immediate threat to survival as well as long-term morbidity. Valve leaflet formation is a rigorously programmed process consisting of endocardial epithelial-mesenchymal transformation (EMT), mesenchymal cell proliferation, valve elongation and remodeling. Currently, little is known about the coordination of the diverse signals that regulate endocardial cushion development and valve elongation. Here, we report that the T-box transcription factor Tbx20 is expressed in the developing endocardial cushions and valves throughout heart development. Ablation of Tbx20 in endocardial cells causes severe valve elongation defects and impaired cardiac function in mice. Our study reveals that endocardial Tbx20 is crucial for valve endocardial cell proliferation and extracellular matrix development, but is not required for initiation of EMT. Elimination of Tbx20 also causes aberrant Wnt/β-catenin signaling in the endocardial cushions. In addition, Tbx20 regulates Lef1, a key transcriptional mediator for Wnt/β-catenin signaling, in this developmental process. Our study suggests a model in which Tbx20 regulates the Wnt pathway to direct endocardial cushion maturation and valve elongation, and provides new insights into the etiology of valve defects in humans.
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http://dx.doi.org/10.1242/dev.092502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931733PMC
August 2013

Generation and characterization of Tbx1-AmCyan1 transgenic reporter mouse line that selectively labels developing thymus primordium.

Transgenic Res 2013 Jun 2;22(3):659-66. Epub 2012 Nov 2.

Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192, Shizuoka, Japan.

Thymus development is a complicated process that includes highly dynamic morphological changes and reciprocal tissue interactions between endoderm-derived epithelial cells of the anterior foregut and neural crest-derived mesenchymal cells. We generated and characterized a Tbx1-AmCyan1 reporter transgenic mouse to visualize thymus precursor cells during early embryonic development. In transgenic embryos, AmCyan1 fluorescence was specifically detected in the endoderm of the developing 3rd and 4th pharyngeal pouches and later in thymus epithelium until E14.5. Cells expressing AmCyan1 that were isolated based on AmCyan1 fluorescence expressed endodermal, thymic, and parathyroid markers, but they did not express neural crest or endothelial markers; these findings indicated that this transgenic mouse strain could be used to collect thymic or parathyroid precursor cells or both. We also showed that in nude mice, which exhibit defects in thymus development, the thymus precursors were clearly labeled with AmCyan1. In summary, these AmCyan1-fluorescent transgenic mice are useful for investigating early thymus development.
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http://dx.doi.org/10.1007/s11248-012-9664-5DOI Listing
June 2013

Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes.

Biochem Biophys Res Commun 2012 Jan 11;417(1):601-6. Epub 2011 Dec 11.

Department of Biochemistry, Hamamatsu University School of Medicine, Higashi-ku, Hamamatsu, Japan.

The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, we generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with ∼50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age.
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http://dx.doi.org/10.1016/j.bbrc.2011.12.014DOI Listing
January 2012

Expression of human factors CD81, claudin-1, scavenger receptor, and occludin in mouse hepatocytes does not confer susceptibility to HCV entry.

Biomed Res 2011 Apr;32(2):143-50

Department of Biochemistry, Hamamatsu University School of Medicine, Handa-yama, Higashi-ku, Hamamatsu, Shizuoka, Japan.

No suitable mouse model is available for studying chronic liver disease caused by hepatitis C virus (HCV). CD81, claudin-1, scavenger receptor class B type I, and occludin were recently reported to be the important factors in HCV entry into hepatocytes. We made transgenic mice (Alb-CCSO) expressing the four human proteins and examined whether HCV from a patient serum or HCV pseudoparticles (HCVpp) were capable of infecting them. HCV was not detected in the mouse serum after injecting the mice with HCV from a patient serum. We also found no indications of HCVpp entry into primary hepatocytes from Alb-CCSO mice. In addition, HCV-infectible Hep3B cells were fused with HCV-resistant primary mouse hepatocytes and the fused cells showed 35-fold lower infectivity compared to wild-type Hep3B cells, indicating that primary mouse hepatocytes have the inhibitory factor(s) in HCVpp entry. Our results suggest that the expression of the human factors does not confer susceptibility to HCV entry into the liver.
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http://dx.doi.org/10.2220/biomedres.32.143DOI Listing
April 2011
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