Biochemical and behavioural profile of NTBC treated Tyrosinemie type 1 mice.

Background: Tyrosinemia type 1 (HT1) is a rare metabolic disorder caused by a defect in the tyrosine catabolic pathway. Since HT1 patients are treated with NTBC, outcome improved and life expectancy greatly increased. However extensive neurocognitive and behavioural problems have been described, which might be related to treatment with NTBC, the biochemical changes induced by NTBC, or metabolites accumulating due to the enzymatic defect characterizing the disease.

Modeling the cognitive effects of diet discontinuation in adults with phenylketonuria (PKU) using pegvaliase therapy in PAH-deficient mice.

Existing phenylalanine hydroxylase (PAH)-deficient mice strains are useful models of untreated or late-treated human phenylketonuria (PKU), as most contemporary therapies can only be initiated after weaning and the pups have already suffered irreversible consequences of chronic hyperphenylalaninemia (HPA) during early brain development. Therefore, we sought to evaluate whether enzyme substitution therapy with pegvaliase initiated near birth and administered repetitively to C57Bl/6-Pah mice would prevent HPA-related behavioral and cognitive deficits and form a model for early-treated PKU. The main results of three reported experiments are: 1) lifelong weekly pegvaliase treatment prevented the cognitive deficits associated with HPA in contrast to persisting deficits in mice treated with pegvaliase only as adults.

Therapeutic liver repopulation by transient acetaminophen selection of gene-modified hepatocytes.

Gene therapy by integrating vectors is promising for monogenic liver diseases, especially in children where episomal vectors remain transient. However, reaching the therapeutic threshold with genome-integrating vectors is challenging. Therefore, we developed a method to expand hepatocytes bearing therapeutic transgenes.

Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing.

Phenylketonuria (PKU) is a metabolic disorder whereby phenylalanine metabolism is deficient due to allelic variations in the gene for phenylalanine hydroxylase (PAH). There is no cure for PKU other than orthotopic liver transplantation, and the standard of care for patients is limited to dietary restrictions and key amino acid supplementation. Therefore, Pah was edited in pig fibroblasts for the generation of PKU clone piglets that harbor a common and severe human mutation, R408W.

Cardiac tissue citric acid cycle intermediates in exercised very long-chain acyl-CoA dehydrogenase-deficient mice fed triheptanoin or medium-chain triglyceride.

Anaplerotic odd-chain fatty acid supplementation has been suggested as an approach to replenish citric acid cycle intermediate (CACi) pools and facilitate adenosine triphosphate (ATP) production in subjects with long-chain fatty acid oxidation disorders, but the evidence that cellular CACi depletion exists and that repletion occurs following anaplerotic substrate supplementation is limited. We exercised very long-chain acyl-CoA dehydrogenase-deficient (VLCAD-/-) and wild-type (WT) mice to exhaustion and collected cardiac tissue for measurement of CACi by targeted metabolomics. In a second experimental group, VLCAD-/- and WT mice that had been fed chow prepared with either medium-chain triglyceride (MCT) oil or triheptanoin for 4 weeks were exercised for 60 minutes.

A novel Pah-exon1 deleted murine model of phenylalanine hydroxylase (PAH) deficiency.

Phenylalanine hydroxylase (PAH) deficiency, colloquially known as phenylketonuria (PKU), is among the most common inborn errors of metabolism and in the past decade has become a target for the development of novel therapeutics such as gene therapy. PAH deficient mouse models have been key to new treatment development, but all prior existing models natively express liver PAH polypeptide as inactive or partially active PAH monomers, which complicates the experimental assessment of protein expression following therapeutic gene, mRNA, protein, or cell transfer. The mutant PAH monomers are able to form hetero-tetramers with and inhibit the overall holoenzyme activity of wild type PAH monomers produced from a therapeutic vector.

AAV-Mediated CRISPR/Cas9 Gene Editing in Murine Phenylketonuria.

Phenylketonuria (PKU) due to recessively inherited phenylalanine hydroxylase (PAH) deficiency results in hyperphenylalaninemia, which is toxic to the central nervous system. Restriction of dietary phenylalanine intake remains the standard of PKU care and prevents the major neurologic manifestations of the disease, yet shortcomings of dietary therapy remain, including poor adherence to a difficult and unpalatable diet, an increased incidence of neuropsychiatric illness, and imperfect neurocognitive outcomes. Gene therapy for PKU is a promising novel approach to promote lifelong neurological protection while allowing unrestricted dietary phenylalanine intake.

Tetrahydrobiopterin treatment reduces brain L-Phe but only partially improves serotonin in hyperphenylalaninemic ENU1/2 mice.

Hyperphenylalaninemia (HPA) caused by hepatic phenylalanine hydroxylase (PAH) deficiency has severe consequences on brain monoamine neurotransmitter metabolism. We have studied monoamine neurotransmitter status and the effect of tetrahydrobiopterin (BH) treatment in Pah (ENU1/2) mice, a model of partial PAH deficiency. These mice exhibit elevated blood L-phenylalanine (L-Phe) concentrations similar to that of mild hyperphenylalaninemia (HPA), but brain levels of L-Phe are still ~5-fold elevated compared to wild-type.

Blood phenylalanine reduction corrects CNS dopamine and serotonin deficiencies and partially improves behavioral performance in adult phenylketonuric mice.

Central nervous system (CNS) deficiencies of the monoamine neurotransmitters dopamine and serotonin have been implicated in the pathophysiology of neuropsychiatric dysfunction in human phenylketonuria (PKU). In this study, we confirmed the occurrence of brain dopamine and serotonin deficiencies in association with severe behavioral alterations and cognitive impairments in hyperphenylalaninemic C57BL/6-Pah mice, a model of human PKU. Phenylalanine-reducing treatments, including either dietary phenylalanine restriction or liver-directed gene therapy, initiated during adulthood were associated with increased brain monoamine content along with improvements in nesting behavior but without a change in the severe cognitive deficits exhibited by these mice.

High dose sapropterin dihydrochloride therapy improves monoamine neurotransmitter turnover in murine phenylketonuria (PKU).

Central nervous system (CNS) deficiencies of the monoamine neurotransmitters, dopamine and serotonin, have been implicated in the pathophysiology of neuropsychiatric dysfunction in phenylketonuria (PKU). Increased brain phenylalanine concentration likely competitively inhibits the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), the rate limiting steps in dopamine and serotonin synthesis respectively. Tetrahydrobiopterin (BH4) is a required cofactor for TH and TPH activity.

The Influence of Vitamin D Metabolism on Gene Expression, Matrix Production and Mineralization During Osteoprecursor Cell-Based Bone Development.

Background: Multipotential precursor cell lines derived from human bone marrow, capable of differentiating into cartilage or bone, may provide a useful tissue development model for studying the regulation and metabolism of putative growth and differentiation factors necessary for tissue regeneration. In mammals, the process of bone development depends on the proliferation and differentiation of osteoblast lineage cells, and the subsequent synthesis and mineralization of bone extracellular matrix (ECM). Vitamin D metabolites play a pivotal role in bone and mineral homeostasis, and are positive factors on bone development.

Pharmacologic inhibition of L-tyrosine degradation ameliorates cerebral dopamine deficiency in murine phenylketonuria (PKU).

Monoamine neurotransmitter deficiency has been implicated in the etiology of neuropsychiatric symptoms associated with chronic hyperphenylalaninemia in phenylketonuria (PKU). Two proposed explanations for neurotransmitter deficiency in PKU include first, that chronically elevated blood L-phenylalanine (Phe) inhibits the transport of L-tyrosine (Tyr) and L-tryptophan (Trp), the substrates for dopamine and serotonin synthesis respectively, into brain. In the second hypothesis, elevated Phe competitively inhibits brain tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities, the rate limiting steps in dopamine and serotonin synthesis.

Extrahepatic 25-Hydroxylation of Vitamin D in an Engineered Osteoblast Precursor Cell Line Exploring the Influence on Cellular Proliferation and Matrix Maturation during Bone Development.

Osteoblastic precursors experience distinct stages during differentiation and bone development, which include proliferation, extracellular matrix (ECM) maturation, and ECM mineralization. It is well known that vitamin D plays a large role in the regulation of bone mineralization and homeostasis via the endocrine system. The activation of vitamin D requires two sequential hydroxylation steps, first in the kidney and then in the liver, in order to carry out its role in calcium homeostasis.

Hepatocytes from wild-type or heterozygous donors are equally effective in achieving successful therapeutic liver repopulation in murine phenylketonuria (PKU).

Successful restoration of phenylalanine (Phe) clearance following liver-directed gene therapy in murine phenylketonuria (PKU) is likely dependent upon both the number of cells successfully transduced and the amount of phenylalanine hydroxylase (PAH) activity expressed per cell. At low levels of transduction, Phe clearance could be limited by the low absolute number of PAH-expressing cells rather than the total amount of PAH activity produced in the liver. We have evaluated the interrelationship between the number of PAH positive cells, the amount of PAH activity produced and Phe clearance through experiments with hepatocyte-mediated therapeutic liver repopulation in the Pah(enu2) mouse, a model of PKU.

Three-Dimensional Culture of Cells and Matrix Biomolecules for Engineered Tissue Development and Biokinetics Model Validation.

There has been considerable progress in cellular and molecular engineering due to recent advances in multiscale technology. Such technologies allow controlled manipulation of physiochemical interactions among cells in tissue culture. In particular, a novel chemomechanical bioreactor has recently been designed for the study of bone and cartilage tissue development, with particular focus on extracellular matrix formation.

Evaluating revascularization and flap survival using vascular endothelial growth factor in an irradiated rat model.

Objective: To evaluate the role of vascular endothelial growth factor (VEGF) plasmid DNA (pDNA) in improving flap revascularization in a previously developed rat model. Our hypothesis was that the uptake and expression of VEGF pDNA in the wound bed would improve revascularization and flap viability.

Design: Twenty-eight male Sprague-Dawley rats received a total dose of 40 Gy electron beam radiation to the ventral abdominal wall.

Manipulation of Suspended Single Cells by Microfluidics and Optical Tweezers.

Chondrocytes and osteoblasts experience multiple stresses in vivo. The optimum mechanical conditions for cell health are not fully understood. This paper describes the optical and microfluidic mechanical manipulation of single suspended cells enabled by the μPIVOT, an integrated micron resolution particle image velocimeter (μPIV) and dual optical tweezers instrument (OT).

Development of an irradiated rodent model to study flap revascularization.

Objective: To develop a reproducible free-flap animal model to study the effects of irradiation on flap revascularization.

Design: After institutional animal care and use committee review and approval, 16 Sprague-Dawley rats were subjected to either 23- or 40-Gy electron beam irradiation to their ventral abdominal wall. After a recovery period, the animals then underwent a ventral fasciocutaneous flap pedicled on the inferior epigastric vessels with subsequent pedicle ligation at 10 days.

VEGF gene therapy augments localized angiogenesis and promotes anastomotic wound healing: a pilot study in a clinically relevant animal model.

Background: Anastomotic leak related to ischemia is a source of significant morbidity and mortality in gastrointestinal surgery. The aim of this study was to apply growth factor gene transfection for the purpose of up-regulating angiogenesis, increasing anastomotic strength, and ultimately preventing dehiscence.

Methods: An opossum esophagogastrostomy model was employed.

Revascularization of rat fasciocutaneous flap using CROSSEAL with VEGF protein or plasmid DNA expressing VEGF.

Background: Fasciocutaneous tissue transfer is a common reconstructive procedure. Revascularization of flap tissue is an important component of tissue healing. Gene therapy offers an avenue through which the period of pedicle vascular dependency can be reduced.

Reduction of rib fractures with a bioresorbable plating system: preliminary observations.

Background: Operative fixation of rib fractures can reduce morbidity and mortality. Currently, resorbable fixation devices are used in a variety of surgical procedures.

Methods: A standard osteotomy was prepared in 30 New Zealand white rabbits at the 12th rib.

Reduction mammaplasty: a review of managed care medical policy coverage criteria.

Background: Insurance companies evaluate the medical necessity for breast reduction surgery based on internal company medical policies, but the correlation of insurance company criteria to the scientifically established indications for reduction mammaplasty has never been studied. The authors obtained 90 insurance company medical policies for reduction mammaplasty to determine whether the criteria on which coverage determinations are made are consistent with published data regarding the indications for this procedure.

Methods: The authors reviewed the medical literature on reduction mammaplasty and identified what conclusions can reasonably be drawn from this literature on the common insurance criteria used to determine medical necessity for reduction mammaplasty.

rhBMP-4 gene therapy in a juvenile canine alveolar defect model.

Background: Autologous bone grafts have the disadvantages of donor-site pain and morbidity, finite supply, increased costs, and prolonged hospitalizations. Using a juvenile canine model, the authors hypothesized that recombinant human (rh) bone morphogenetic protein (BMP)-4 gene therapy-treated alveolar defects would promote bone healing and canine tooth eruption equal to their autografted counterparts.

Methods: Sixty-five maxillary alveolar defects were created in juvenile foxhound dogs with a mean age of 80.

Osteogenic response of bone marrow stromal cells from normal and ovariectomized rats treated with a low dose of basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a potent mitogen that exhibits stimulatory effects on bone tissue regeneration. To gain further insight into the potential of bFGF for systemic therapy in osteoporosis, we investigated the responsiveness of bone marrow stromal cells (BMSCs) explanted from 7-month-old normal and ovariectomized (OVX) rats that were intravenously treated with a low dose of bFGF (25 microg/kg) for 2 weeks. The BMSCs were obtained using femoral aspiration and maintained in an osteogenic medium.

Bone marrow cells from normal and ovariectomized rats respond differently to basic fibroblast growth factor and bone morphogenetic protein 2 treatment in vitro.

The protein growth factors basic fibroblast growth factor (bFGF) and bone morphogenetic protein 2 (BMP-2) are being actively pursued for bone tissue engineering. Although both proteins are capable of stimulating osteogenic activity of bone marrow cells (BMCs), no studies have addressed the effect of estrogen deficiency on the growth factor responsiveness of BMCs. This study investigated the osteogenic response of BMCs from normal and ovariectomized (OVX) rats to bFGF and BMP- 2.