The DNA Polymerase Gamma R953C Mutant Is Associated with Antiretroviral Therapy-Induced Mitochondrial Toxicity.

We found a heterozygous C2857T mutation (R953C) in polymerase gamma (Pol-γ) in an HIV-infected patient with mitochondrial toxicity. The R953C Pol-γ mutant binding affinity for dCTP is 8-fold less than that of the wild type. The R953C mutant shows a 4-fold decrease in discrimination of analog nucleotides relative to the wild type. R953 is located on the "O-helix" that forms the substrate deoxynucleoside triphosphate (dNTP) binding site; the interactions of R953 with E1056 and Y986 may stabilize the O-helix and affect polymerase activity.

Gaucher iPSC-derived macrophages produce elevated levels of inflammatory mediators and serve as a new platform for therapeutic development.

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid ß-glucocerebrosidase (GCase; GBA) gene. The hallmark of GD is the presence of lipid-laden Gaucher macrophages, which infiltrate bone marrow and other organs. These pathological macrophages are believed to be the sources of elevated levels of inflammatory mediators present in the serum of GD patients. The alteration in the immune environment caused by GD is believed to play a role in the increased risk of developing multiple myeloma and other malignancies in GD patients. To determine directly whether Gaucher macrophages are abnormally activated and whether their functional defects can be reversed by pharmacological intervention, we generated GD macrophages by directed differentiation of human induced pluripotent stem cells (hiPSC) derived from patients with types 1, 2, and 3 GD. GD hiPSC-derived macrophages expressed higher levels of tumor necrosis factor α, IL-6, and IL-1ß than control cells, and this phenotype was exacerbated by treatment with lipopolysaccharide. In addition, GD hiPSC macrophages exhibited a striking delay in clearance of phagocytosed red blood cells, recapitulating the presence of red blood cell remnants in Gaucher macrophages from bone marrow aspirates. Incubation of GD hiPSC macrophages with recombinant GCase, or with the chaperones isofagomine and ambroxol, corrected the abnormal phenotypes of GD macrophages to an extent that reflected their known clinical efficacies. We conclude that Gaucher macrophages are the likely source of the elevated levels of inflammatory mediators in the serum of GD patients and that GD hiPSC are valuable new tools for studying disease mechanisms and drug discovery.

Use of a clinicoradiological score to determine the presurgical diagnosis of autoimmune hypophysitis in a teenage girl.

The distinction between autoimmune hypophysitis and other non-hormone secreting pituitary masses is often difficult to determine with certainty without pituitary biopsy and pathological examination. To aid in this distinction, the authors recently published a clinicoradiological scoring system, which they used in the case of a 15-year-old girl presented here. The patient presented with headache, visual field defects, polydipsia, and polyuria, and she was found to have secondary hypogonadism and hypoadrenalism. Magnetic resonance imaging showed a pituitary mass of approximately 2 cm in diameter. Application of the clinicoradiological parameters gave a score of -6, which favored a diagnosis of hypophysitis over that of adenoma. The presence of pituitary autoantibodies substantiated the diagnosis of hypophysitis. The patient was treated conservatively with high-dose prednisolone, and her symptoms improved markedly. This case illustrates the utility of using a clinicoradiological score when autoimmune hypophysitis is suspected since it can identify patients who can be treated without the need for pituitary surgery.

Controlled activation of morphogenesis to generate a functional human microvasculature in a synthetic matrix.

Understanding the role of the extracellular matrix (ECM) in vascular morphogenesis has been possible using natural ECMs as in vitro models to study the underlying molecular mechanisms. However, little is known about vascular morphogenesis in synthetic matrices where properties can be tuned toward both the basic understanding of tubulogenesis in modular environments and as a clinically relevant alternative to natural materials for regenerative medicine. We investigated synthetic, tunable hyaluronic acid (HA) hydrogels and determined both the adhesion and degradation parameters that enable human endothelial colony-forming cells (ECFCs) to form efficient vascular networks. Entrapped ECFCs underwent tubulogenesis dependent on the cellular interactions with the HA hydrogel during each stage of vascular morphogenesis. Vacuole and lumen formed through integrins α(5)ß(1) and α(V)ß(3), while branching and sprouting were enabled by HA hydrogel degradation. Vascular networks formed within HA hydrogels containing ECFCs anastomosed with the host's circulation and supported blood flow in the hydrogel after transplantation. Collectively, we show that the signaling pathways of vascular morphogenesis of ECFCs can be precisely regulated in a synthetic matrix, resulting in a functional microvasculature useful for the study of 3-dimensional vascular biology and toward a range of vascular disorders and approaches in tissue regeneration.

Hyaluronic Acid hydrogels support cord-like structures from endothelial colony-forming cells.

The generation of functional vascular networks has the potential to improve treatment for vascular diseases and to facilitate successful organ transplantation. Endothelial colony-forming cells (ECFCs) have robust proliferative potential and can form vascular networks in vivo. ECFCs are recruited from a bone marrow niche to the site of vascularization, where cues from the extracellular matrix instigate vascular morphogenesis. Although this process has been elucidated using natural matrix, little is known about vascular morphogenesis by ECFCs in synthetic matrix, a xeno-free scaffold that can provide a more controllable and clinically relevant alternative for regenerative medicine. We sought to study hyaluronic acid (HA) hydrogels as three-dimensional scaffolds for capillary-like structure formation from ECFCs, and to determine the crucial parameters needed to design such synthetic scaffolds. We found that ECFCs express HA-specific receptors and that vascular endothelial growth factor stimulates hyaluronidase expression in ECFCs. Using a well-defined and controllable three-dimensional HA culture system, we were able to decouple the effect of matrix viscoelasticity from changes in adhesion peptide density. We determined that decreasing matrix viscoelasticity, which corresponds to a loose ultrastructure, significantly increases ECFC vascular tube length and area, and that the effect of local delivery of vascular endothelial growth factor within the hydrogel depends on the makeup of the synthetic environment. Collectively, these results set forth initial design criteria that need to be considered in developing vascularized tissue constructs.