The use of cell fusion to study exchange of information at the molecular level between the nucleus and the cytoplasm of cells during regulation of gene expression was pioneered by Harris and Ringertz more than three decades ago. The ability to make heterokaryons with cells from different species or genetic strains is especially useful because genetic differences in gene products allow the origin of trans-acting regulatory factors to be determined. Heterokaryons between adult nucleated erythroid cells of one species and embryonic/larval nucleated erythroid cells of another species, for example, show cross-induction between the two types of nuclei, resulting in reprogramming of the adult nucleus to embryonic/larval globin gene expression and/or reprogramming of the embryonic/larval cell nucleus to adult globin expression. These experiments provided definitive evidence that developmental program switching is mediated by trans-acting factors. Other possible uses of this cell fusion protocol in stem cell biology and transplantation of genetically engineered cells for tissue regeneration are briefly discussed.

Gene regulation of developmental hemoglobin switching holds the potential for therapeutic relief from all symptoms associated with Sickle Cell Disease (SCD). Reactivation of fetal gamma-globin expression (HbF) can replace mutant betaS-globin (HbS) to produce functional hemoglobin tetramers and eliminate the hemoglobin polymerization that is characteristic of sickled red blood cells. We have discovered a protein that regulates this developmental switch, and have identified a compound that stimulates expression of this protein. EdX-17 promotes expression of the anti-stress factor ferritin heavy chain (FtH), which enters the nucleus of erythroid precursor cells and activates expression of fetal gamma-globin, producing HbF (PNAS 98:9145-50, 2001; Blood 108:790a, 2006). Mononuclear cells were isolated from SCD patient blood and matured in vitro to the advanced erythroblast stage using a 28-day, 2-phase culture system (Methods in Molecular Biology 482:127-40, 2009; Blood 119:6296-306, 2012). Treatment with EdX-17 for 24h resulted in a dose-responsive induction of gamma-globin gene expression and a concomitant dose-responsive increase in HbF was observed after 28 days in culture. These studies demonstrate that EdX-17 doses in the picomolar range are sufficient to significantly enhance HbF. Furthermore, EdX-17 treatment reconstitutes fetal hemoglobin (HbF) in transgenic betaYAC mice to levels above 25-30% - the range thought to be sufficient to ameliorate symptoms of SCD – with no detectable ill effects. In fact, mice treated with EdX-17 tend to have shinier coats, are more alert and stronger than age-matched, untreated mice. Development of this novel therapeutic is expected to ameliorate SCD symptoms, decrease pain and morbidity, increase life-span, greatly improve patient quality of life, and significantly reduce treatment costs. Supported in part by The Sickle Cell Cure Foundation, Inc., the Bill & Melinda Gates Foundation, and EpimedX, LLC. Disclosures Broyles: EpimedX, LLC: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Curtis:EpimedX, LLC: Employment, Equity Ownership, Research Funding. Roth:EpimedX, LLC: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Floyd:EpimedX, LLC: Employment, Equity Ownership, Research Funding. Belegu:EpimedX, LLC: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Floyd:EpimedX, LLC: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Tissue-mimicking materials (TMM) are often used as surrogates for human tissue when developing prospective treatments such as thermal ablation of tumors. Localized heating or ablation may be applied by methods including high-intensity focused ultrasound (HIFU), radio frequency (RF), microwave, and laser treatment. In such methods, confining the heated region to a narrow target is an important concern for minimizing collateral damage to surrounding healthy tissue. Mechanical compression can potentially assist in confining heat near a target region by constricting microvascular blood flow. However, characterization of the effects of compression on thermal properties of the tissue itself (apart from microvasculature) is needed for accurate modeling of heat transfer. Accordingly this study presents a method and material characterization results that quantify the extent to which mechanical compression alters thermal conductivity, specific heat capacity, and thermal diffusivity of a polyacrylamide-based TMM. Cylindrical test specimens were cast from polyacrylamide material with diameter of 50 mm and height of 45 mm. Compression was applied using custom apparatus for applying prescribed uniaxial displacement, with a modular configuration for testing under ambient temperature as well as on a hot plate. Compression force at room temperature was measured with a load cell that was positioned in-line between compression plates. Prescribed heat flux was delivered based on power input, as quantified with the use of a reference sample in a thermal resistance network. Temperature was measured by an array of thermocouples. Software simulations were performed using finite element analysis (FEA) for structural deformation and computational fluid dynamics (CFD) for heat transfer under the combined effects of conduction and convection. The simulations provided estimates of deformed shape and thermal losses that were compared to experimental measurements. Mechanical stress-strain tests using three TMM replicate specimens at room temperature showed a linear stress-strain relationship from approximately 2% to 14% strain and a compressive modulus of elasticity ranging from 7.56 kPa to 12.7 kPa. Distributed temperature measurements under an imposed heat flux resulted in thermal conductivity between 0.89 W/(m·K) and 1.04 W/(m·K), specific heat capacity between 5590 J/(kg·K) and 6720 J/(kg·K) and thermal diffusivity between 1.29 × 10−7 m 2 /s to 1.71 × 10−7 m2/ s. Viscoelastic effects were observed to reach steady state after approximately 20 seconds, with full elastic recovery upon unloading. Thermal conductivity and thermal diffusivity were observed to decrease under mechanical compression, while specific heat capacity was observed to increase. The results affirm that thermal properties of tissue-mimicking material can be altered by mechanical compression. These findings can be applied to future investigation of temperature distribution during localized ablation by methods such as HIFU, and can be extended to refined material modeling of perfused tissue under compression.

We have found that ferritin heavy chain (FtH), an antioxidant/stress response/iron-storage protein, localizes to the nucleus in K562 cells and represses the human adult beta-globin promoter in transient assays in primate cells (Broyles et al., PNAS98: 9145, 2001). Since other work indicates FtH is also a gene activator of fetal-globin genes, we hypothesize that FtH is a long-sought developmental hemoglobin (Hb) switching factor and that delivery of FtH to human adult erythroid cell precursors will reverse the phenotype to HbF, offering a phenotypic cure for sickle cell disease (SCD). Chromatin immunoprecipitation (ChIP) assays, antisense treatments, and an FtH transgenic mouse have confirmed that FtH is a globin gene regulatory protein in vivo. With erythroid precursor cells from pediatric SCD patients, under an IRB-approved protocol, we have used a two-phase culture system for in vitro maturation of erythroid cells in the presence of FtH, delivered to the cells as pure protein, as an expression plasmid, or as a priority inducer compound that activates the endogenous FtH gene. HPLC with a PolyCAT A column was used to separate and quantify human Hbs. With each mode of delivery, FtH stimulated a complete switch from HbS to HbF. This result was repeatable in multiple experiments using erythroid precursor cells from three different SCD donors. Fluorescently-labeled recombinant human FtH protein was taken into red cell precursors in culture, suggesting that the purified protein can be directly delivered without gene therapy. This method of producing a phenotypic cure in SCD patients should be easy and inexpensive to deliver in vivo.

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