Hatem E. Sabaawy

Human Heme Oxygenase-1 Gene Transfer Lowers Blood Pressure and Promotes Growth in Spontaneously Hypertensive Rats

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin, with release of free iron and carbon monoxide. Both heme and carbon monoxide have been implicated in the regulation of vascular tone. A retroviral vector containing human HO-1 cDNA (LSN-HHO-1) was constructed and subjected to purification and concentration of the viral particles to achieve 5×109 to 1×1010 colony-forming units per milliliter. The ability of concentrated infectious viral particles to express human HO-1 (HHO-1) in vivo was tested. A single intracardiac injection of the concentrated infectious viral particles (expressing HHO-1) to 5-day-old spontaneously hypertensive rats resulted in functional expression of the HHO-1 gene and attenuation of the development of hypertension. Rats expressing HHO-1 showed a significant decrease in urinary excretion of a vasoconstrictor arachidonic acid metabolite and a reduction in myogenic responses to increased intraluminal pressure in isolated arterioles. Unexpectedly, HHO-1 chimeric rats showed a simultaneous significant proportionate increase in somatic growth. Thus, delivery of HHO-1 gene by retroviral vector attenuates the development of hypertension and promotes body growth in spontaneously hypertensive rats.

MicroRNA miR-133b is essential for functional recovery after spinal cord injury in adult zebrafish.

MicroRNAs (miRNAs) play important roles during development and also in adult organisms by regulating the expression of multiple target genes. Here, we studied the function of miR‐133b during zebrafish spinal cord regeneration and show upregulation of miR‐133b expression in regenerating neurons of the brainstem after transection of the spinal cord. miR‐133b has been shown to promote tissue regeneration in other tissue, but its ability to do so in the nervous system has yet to be tested. Inhibition of miR‐133b expression by antisense morpholino (MO) application resulted in impaired locomotor recovery and reduced regeneration of axons from neurons in the nucleus of the medial longitudinal fascicle, superior reticular formation and intermediate reticular formation. miR‐133b targets the small GTPase RhoA, which is an inhibitor of axonal growth, as well as other neurite outgrowth‐related molecules. Our results indicate that miR‐133b is an important determinant in spinal cord regeneration of adult zebrafish through reduction in RhoA protein levels by direct interaction with its mRNA. While RhoA has been studied as a therapeutic target in spinal cord injury, this is the first demonstration of endogenous regulation of RhoA by a microRNA that is required for spinal cord regeneration in zebrafish. The ability of miR‐133b to suppress molecules that inhibit axon regrowth may underlie the capacity for adult zebrafish to recover locomotor function after spinal cord injury.

Enrichment of human prostate cancer cells with tumor initiating properties in mouse and zebrafish xenografts by differential adhesion

Prostate tumor‐initiating cells (TICs) have intrinsic resistance to current therapies. TICs are commonly isolated by cell sorting or dye exclusion, however, isolating TICs from limited primary prostate cancer (PCa) tissues is inherently inefficient. We adapted the collagen adherence feature to develop a combined immunophenotypic and time‐of‐adherence assay to identify human prostate TICs.

Autologous Bone Marrow-Derived Cell Therapy Combined With Physical Therapy Induces Functional Improvement in Chronic Spinal Cord Injury Patients

Spinal cord injuries (SCI) cause sensory loss and motor paralysis. They are normally treated with physical therapy, but most patients fail to recover due to limited neural regeneration. Here we describe a strategy in which treatment with autologous adherent bone marrow cells is combined with physical therapy to improve motor and sensory functions in early stage chronic SCI patients. In a phase I/II controlled single-blind clinical trial (clinicaltrials.gov identifier: NCT00816803), 70 chronic cervical and thoracic SCI patients with injury durations of at least 12 months were treated with either intrathecal injection(s) of autologous adherent bone marrow cells combined with physical therapy or with physical therapy alone. Patients were evaluated with clinical and neurological examinations using the American Spinal Injury Association (ASIA) Impairment Scale (AIS), electrophysiological somatosensory-evoked potential, magnetic resonance imaging (MRI), and functional independence measurements. Chronic cervical and thoracic SCI patients (15 AIS A and 35 AIS B) treated with autologous adherent bone marrow cells combined with physical therapy showed functional improvements over patients in the control group (10 AIS A and 10 AIS B) treated with physical therapy alone, and there were no long-term cell therapy-related side effects. At 18 months posttreatment, 23 of the 50 cell therapy-treated cases (46%) showed sustained functional improvement. Compared to those patients with cervical injuries, a higher rate of functional improvement was achieved in thoracic SCI patients with shorter durations of injury and smaller cord lesions. Therefore, when combined with physical therapy, autologous adherent bone marrow cell therapy appears to be a safe and promising therapy for patients with chronic SCI of traumatic origin. Randomized controlled multicenter trials are warranted.

Knockdown of fbxl10/kdm2bb rescues chd7 morphant phenotype in a zebrafish model of CHARGE syndrome

CHARGE syndrome is a sporadic autosomal-dominant genetic disorder characterized by a complex array of birth defects so named for its cardinal features of ocular coloboma, heart defects, choanal atresia, growth retardation, genital abnormalities, and ear abnormalities. Approximately two-thirds of individuals clinically diagnosed with CHARGE syndrome have heterozygous loss-of-function mutations in the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7), an ATP-dependent chromatin remodeler. To examine the role of Chd7 in development, a zebrafish model was generated through morpholino (MO)-mediated targeting of the zebrafish chd7 transcript. High doses of chd7 MO induce lethality early in embryonic development. However, low dose-injected embryos are viable, and by 4 days post-fertilization, morphant fish display multiple defects in organ systems analogous to those affected in humans with CHARGE syndrome. The chd7 morphants show elevated expression of several potent cell-cycle inhibitors including ink4ab (p16/p15), p21 and p27, accompanied by reduced cell proliferation. We also show that Chd7 is required for proper organization of neural crest-derived craniofacial cartilage structures. Strikingly, MO-mediated knockdown of the jumonji domain-containing histone demethylase fbxl10/kdm2bb, a repressor of ribosomal RNA (rRNA) genes, rescues cell proliferation and cartilage defects in chd7 morphant embryos and can lead to complete rescue of the CHARGE syndrome phenotype. These results indicate that CHARGE-like phenotypes in zebrafish can be mitigated through modulation of fbxl10 levels and implicate FBXL10 as a possible therapeutic target in CHARGE syndrome.

Radiosensitization of Primary Human Glioblastoma Stem-like Cells with Low-Dose AKT Inhibition

Glioblastoma (GBM) is the most frequent and lethal brain cancer. The lack of early detection methods, the presence of rapidly growing tumor cells, and the high levels of recurrence due to chemo- and radioresistance make this cancer an extremely difficult disease to treat. Emerging studies have focused on inhibiting AKT activation; here, we demonstrate that in primary GBM tumor samples, full-dose inhibition of AKT activity leads to differential responses among samples in the context of cell death and self-renewal, reinforcing the notion that GBM is a heterogeneous disease. In contrast, low-dose AKT inhibition when combined with fractionation of radiation doses leads to a significant apoptosis-mediated cell death of primary patient–derived GBM cells. Therefore, low-dose–targeted therapies might be better for radiosensitization of primary GBM cells and further allow for reducing the clinical toxicities often associated with targeting the AKT/PI3K/mTOR pathway. This work emphasizes the discrepancies between cell lines and primary tumors in drug testing, and indicates that there are salient differences between patients, highlighting the need for personalized medicine in treating high-grade glioma. Mol Cancer Ther; 14(5); 1171–80. ©2015 AACR.

Detection of central nervous system leukemia in children with acute lymphoblastic leukemia by real-time polymerase chain reaction.

Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. We examined various cerebrospinal fluid (CSF) preservation methods to yield adequate DNA stability for polymerase chain reaction (PCR) analysis and developed a quantitative real-time PCR assay to detect occult CNS leukemia. Sixty CSF specimens were maintained in several storage conditions for varying amounts of time, and we found that preserving CSF in 1:1 serum-free RPMI tissue culture medium offers the best stability of DNA for PCR analysis. Sixty CSF samples (30 at diagnosis and 30 at the end of induction therapy) from 30 children with ALL were tested for CNS leukemic involvement by real-time PCR using patient-specific antigen receptor gene rearrangement primers. Six of thirty patient diagnosis samples were PCR-positive at levels ranging from 0.5 to 66% leukemic blasts in the CSF. Four of these patients had no clinical or cytomorphological evidence of CNS leukemia involvement at that time. All 30 CSF samples drawn at the end of induction therapy were PCR-negative. The data indicate that real-time PCR analysis of CSF is an excellent tool to assess occult CNS leukemia involvement in patients with ALL and can possibly be used to refine CNS status classification.

PDCD2 Controls Hematopoietic Stem Cell Differentiation During Development

Programmed cell death 2 (Pdcd2) is a highly conserved protein of undefined function, and is widely expressed in embryonic and adult tissues. The observations that knockout of Pdcd2 in the mouse is embryonic lethal at preimplantation stages, and that in Drosophila, Zfrp8, the ortholog of Pdcd2, is required for normal lymph gland development suggest that Pdcd2 is important for regulating hematopoietic development. Through genetic and functional studies, we investigated pdcd2 function during the zebrafish ontogeny. Knockdown of pdcd2 expression in zebrafish embryos resulted in defects in embryonic hematopoietic development. Loss of pdcd2 function caused increased expression of progenitor markers, and accumulation of erythroid progenitors during primitive hematopoiesis. Additionally, hematopoietic stem cells (HSCs) failed to appear in the aorta-gonad mesonephros, and were not able to terminally differentiate or reconstitute hematopoiesis. Pdcd2 effects on HSC emergence were cell autonomous and P53-independent, and loss of pdcd2 function was associated with mitotic defects and apoptosis. Restoration of runx1 function(s) and modulation of apoptosis through the inhibition of Jak/Stat signaling rescued the hematopoietic and erythroid defects resulting from pdcd2 knockdown. Our studies suggest that pdcd2 plays a critical role in regulating the transcriptional hierarchy controlling hematopoietic lineage determination. Furthermore, the effects of pdcd2 in regulating mitotic cell death may contribute to its role(s) in directing hematopoietic differentiation during development.

Personalized Medicine Approaches in Prostate Cancer Employing Patient Derived 3D Organoids and Humanized Mice

Prostate cancer (PCa) is the most common malignancy and the second most common cause of cancer death in Western men. Despite its prevalence, PCa has proven very difficult to propagate in vitro. PCa represents a complex organ-like multicellular structure maintained by the dynamic interaction of tumoral cells with parenchymal stroma, endothelial and immune cells, and components of the extracellular matrix (ECM). The lack of PCa models that recapitulate this intricate system has hampered progress toward understanding disease progression and lackluster therapeutic responses. Tissue slices, monolayer cultures and genetically engineered mouse models (GEMM) fail to mimic the complexities of the PCa microenvironment or reproduce the diverse mechanisms of therapy resistance. Moreover, patient derived xenografts (PDXs) are expensive, time consuming, difficult to establish for prostate cancer, lack immune cell-tumor regulation, and often tumors undergo selective engraftments. Here, we describe an interdisciplinary approach using primary PCa and tumor initiating cells (TICs), three-dimensional (3D) tissue engineering, genetic and morphometric profiling, and humanized mice to generate patient-derived organoids for examining personalized therapeutic responses in vitro and in mice co-engrafted with a human immune system (HIS), employing adaptive T-cell- and chimeric antigen receptor- (CAR) immunotherapy. The development of patient specific therapies targeting the vulnerabilities of cancer, when combined with antiproliferative and immunotherapy approaches could help to achieve the full transformative power of cancer precision medicine.

Cartilage regeneration for treatment of osteoarthritis: a paradigm for nonsurgical intervention.

Osteoarthritis (OA) is associated with articular cartilage abnormalities and affects people of older age: preventative or therapeutic treatment measures for OA and related articular cartilage disorders remain challenging. In this perspective review, we have integrated multiple biological, morphological, developmental, stem cell and homeostasis concepts of articular cartilage to develop a paradigm for cartilage regeneration. OA is conceptually defined as an injury of cartilage that initiates chondrocyte activation, expression of proteases and growth factor release from the matrix. This regenerative process results in the local activation of inflammatory response genes in cartilage without migration of inflammatory cells or angiogenesis. The end results are catabolic and anabolic responses, and it is the balance between these two outcomes that controls remodelling of the matrix and regeneration. A tantalizing clinical clue for cartilage regrowth in OA joints has been observed in surgically created joint distraction. We hypothesize that cartilage growth in these distracted joints may have a biological connection with the size of organs and regeneration. Therefore we propose a novel, practical and nonsurgical intervention to validate the role of distraction in cartilage regeneration in OA. The approach permits normal wake-up activity while during sleep; the index knee is subjected to distraction with a pull traction device. Comparison of follow-up magnetic resonance imaging (MRI) at 3 and 6 months of therapy to those taken before therapy will provide much-needed objective evidence for the use of this mode of therapy for OA. We suggest that the paradigm presented here merits investigation for treatment of OA in knee joints.