Purine nucleoside phosphorylase deficiency induces p53-mediated intrinsic apoptosis in human induced pluripotent stem cell-derived neurons.

Purine nucleoside phosphorylase (PNP) is an important enzyme in the purine degradation and salvage pathway. PNP deficiency results in marked T lineage lymphopenia and severe immunodeficiency. Additionally, PNP-deficient patients and mice suffer from diverse non-infectious neurological abnormalities of unknown etiology. To further investigate the cause for these neurologic abnormalities, induced pluripotent stem cells (iPSC) from two PNP-deficient patients were differentiated into neurons. The iPSC-derived PNP-deficient neurons had significantly reduced soma and nuclei volumes. The PNP-deficient neurons demonstrated increased spontaneous and staurosporine-induced apoptosis, measured by cleaved caspase-3 expression, together with decreased mitochondrial membrane potential and increased cleaved caspase-9 expression, indicative of enhanced intrinsic apoptosis. Greater expression of tumor protein p53 was also observed in these neurons, and inhibition of p53 using pifithrin-α prevented the apoptosis. Importantly, treatment of the iPSC-derived PNP-deficient neurons with exogenous PNP enzyme alleviated the apoptosis. Inhibition of ribonucleotide reductase (RNR) in iPSC derived from PNP-proficient neurons with hydroxyurea or with nicotinamide and trichostatin A increased the intrinsic neuronal apoptosis, implicating RNR dysfunction as the potential mechanism for the damage caused by PNP deficiency. The findings presented here establish a potential mechanism for the neurological defects observed in PNP-deficient patients and reinforce the critical role that PNP has for neuronal viability.

The Use of Induced Pluripotent Stem Cells to Study the Effects of Adenosine Deaminase Deficiency on Human Neutrophil Development.

Inherited defects that abrogate the function of the adenosine deaminase (ADA) enzyme and consequently lead to the accumulation of toxic purine metabolites cause profound lymphopenia and severe combined immune deficiency. Additionally, neutropenia and impaired neutrophil function have been reported among ADA-deficient patients. However, due to the rarity of the disorder, the neutrophil developmental abnormalities and the mechanisms contributing to them have not been characterized. Induced pluripotent stem cells (iPSC) generated from two unrelated ADA-deficient patients and from healthy controls were differentiated through embryoid bodies into neutrophils. ADA deficiency led to a significant reduction in the number of all early multipotent hematopoietic progenitors. At later stages of differentiation, ADA deficiency impeded the formation of granulocyte colonies in methylcellulose cultures, leading to a significant decrease in the number of neutrophils generated from ADA-deficient iPSCs. The viability and apoptosis of ADA-deficient neutrophils isolated from methylcellulose cultures were unaffected, suggesting that the abnormal purine homeostasis in this condition interferes with differentiation or proliferation. Additionally, there was a significant increase in the percentage of hyperlobular ADA-deficient neutrophils, and these neutrophils demonstrated significantly reduced ability to phagocytize fluorescent microspheres. Supplementing iPSCs and methylcellulose cultures with exogenous ADA, which can correct adenosine metabolism, reversed all abnormalities, cementing the critical role of ADA in neutrophil development. Moreover, chemical inhibition of the ribonucleotide reductase (RNR) enzyme, using hydroxyurea or a combination of nicotinamide and trichostatin A in iPSCs from healthy controls, led to abnormal neutrophil differentiation similar to that observed in ADA deficiency, implicating RNR inhibition as a potential mechanism for the neutrophil abnormalities. In conclusion, the findings presented here demonstrate the important role of ADA in the development and function of neutrophils while clarifying the mechanisms responsible for the neutrophil abnormalities in ADA-deficient patients.

Early Enzyme Replacement Therapy Improves Hearing and Immune Defects in Adenosine Deaminase Deficient-Mice.

Background: Inherited defects in adenosine deaminase (ADA) cause severe immune deficiency, which can be corrected by ADA enzyme replacement therapy (ERT). Additionally, ADA-deficient patients suffer from hearing impairment. We hypothesized that ADA-deficient (-/-) mice also exhibit hearing abnormalities and that ERT from an early age will improve the hearing and immune defects in these mice. Methods: Auditory brainstem evoked responses, organ weights, thymocytes numbers, and subpopulations, lymphocytes in peripheral blood as well as T lymphocytes in spleen were analyzed in ADA-/- and ADA-proficient littermate post-partum (pp). The cochlea was visualized by scanning electron microscopy (SEM). The effects of polyethylene glycol conjugated ADA (PEG-ADA) ERT or 40% oxygen initiated at 7 days pp on the hearing and immune abnormalities were assessed. Results: Markedly abnormal hearing thresholds responses were found in ADA-/- mice at low and medium tone frequencies. SEM demonstrated extensive damage to the cochlear hair cells of ADA-/- mice, which were splayed, short or missing, correlating with the hearing deficits. The hearing defects were not reversed when hypoxia in ADA-/- mice was corrected. Progressive immune abnormalities were detected in ADA-/- mice from 4 days pp, initially affecting the thymus followed by peripheral lymphocytes and T cells in the spleen. ERT initiated at 7 days pp significantly improved the hearing of ADA-/- mice as well as the number of thymocytes and T lymphocytes, although not all normalized. Conclusions: ADA deficiency is associated with hearing deficits and damage to cochlear hair cells. Early initiation of ERT improves the hearing and immune abnormalities.

Intracellular Delivery of Human Purine Nucleoside Phosphorylase by Engineered Diphtheria Toxin Rescues Function in Target Cells.

Despite a wealth of potential applications inside target cells, protein-based therapeutics are largely limited to extracellular targets due to the inability of proteins to readily cross biological membranes and enter the cytosol. Bacterial toxins, which deliver a cytotoxic enzyme into cells as part of their intoxication mechanism, hold great potential as platforms for delivering therapeutic protein cargo into cells. Diphtheria toxin (DT) has been shown to be capable of delivering an array of model proteins of varying sizes, structures, and stabilities into mammalian cells as amino-terminal fusions. Here, seeking to expand the utility of DT as a delivery vector, we asked whether an active human enzyme, purine nucleoside phosphorylase (PNP), could be delivered by DT into cells to rescue PNP deficiency. Using a series of biochemical and cellular readouts, we demonstrate that PNP is efficiently delivered into target cells in a receptor- and translocation-dependent manner. In patient-derived PNP-deficient lymphocytes and pluripotent stem cell-differentiated neurons, we show that human PNP is efficiently translocated into target cells by DT, where it is able to restore intracellular hypoxanthine levels. Further, through replacement of the native receptor-binding moiety of DT with single-chain variable fragments that were selected to bind mouse HBEGF, we show that PNP can be retargeted into mouse splenocytes from PNP-deficient mice, resulting in restoration of the proliferative capacity of T-cells. These findings highlight the versatility of the DT delivery platform and provide an attractive approach for the delivery of PNP as well as other cytosolic enzymes implicated in disease.

Cerebellar abnormalities in purine nucleoside phosphorylase deficient mice.

Inherited defects in purine nucleoside phosphorylase (PNP) cause severe T cell immunodeficiency and progressive neurological dysfunction, yet little is known about the effects of PNP deficiency on the brain. PNP-KO mice display metabolic and immune anomalies similar to those observed in patients. Our objectives were to characterize brain abnormalities in PNP-KO mice and determine whether restoring PNP activity prevents these abnormalities. We analyzed structural brain defects in PNP-KO mice by magnetic resonance imaging, while assessing motor deficits using the accelerating rotarod and stationary balance beam tests. We detected morphological abnormalities and apoptosis in the cerebellum of PNP-KO mice by hematoxylin and eosin, electron microscopy, TUNEL and activated caspase 3 staining. We treated PNP-KO mice with PNP fused to the HIV-TAT protein transduction domain (TAT-PNP) from birth or from 4 weeks of age. Magnetic resonance imaging revealed a smaller than normal cerebellum in PNP-KO mice. PNP-KO mice displayed motor abnormalities including rapid fall from the rotating rod and frequent slips from the balance beam. The cerebellum of PNP-KO mice contained reduced purkinje cells (PC), which were irregular in shape and had degenerated dendrites. PC from the cerebellum of PNP-KO mice, expanded ex vivo, demonstrated increased apoptosis, which could be corrected by supplementing cultures with TAT-PNP. TAT-PNP injections restored PNP activity in the cerebellum of PNP-KO mice. TAT-PNP from birth, but not treatment initiated at 4 weeks of age, prevented the cerebellar PC damage and motor deficits. We conclude that PNP deficiency cause cerebellar abnormalities, including PC damage and progressive motor deficits. TAT-PNP treatment from birth can prevent the neurological abnormalities in PNP-KO mice.

Hyperbaric oxygen therapy as a sole agent is not immunosuppressant in a highly immunogenic mouse model.

Background. Hyperbaric oxygen (HBO) therapy, which is used for many conditions, may also have immunosuppressive effects and could be used for prevention or treatment of graft-versus-host disease (GvHD). If HBO is immunosuppressant, then we hypothesize that HBO therapy will delay the T-cell mediated skin graft rejection. Methods. C57/BL6 black-coated (H2B) mice received skin graft from CBA (H2D) white-coated mice. Mice were treated with either 19 session of 240 kpa oxygen or 29 session of 300 kpa oxygen, for 90 minutes. Mice were housed either 4 per cage or separately, to prevent friction and mechanical factors that may affect graft survival. Skin grafts were assessed daily. Results. There was no difference in length of graft survival between mice that received either regimens of HBO therapy and mice that did not receive HBO therapy. Conclusions. HBO therapy, as a sole agent, did not delay skin graft rejection in a highly immunogenic mouse model.

EdU incorporation is an alternative non-radioactive assay to [(3)H]thymidine uptake for in vitro measurement of mice T-cell proliferations.

RATIONALE: T lymphocyte proliferations can be measured by [(3)H]thymidine incorporation. However, many labs avoid this technique because of the need to use radioactive substrates. In addition, [(3)H]thymidine incorporation method does not permit simultaneous characterization of the proliferating cells. We developed the 5-ethynyl-2'-deoxyuridine (EdU) and Cu(I)-catalyzed cycloaddition "click" reaction assay to measure T-cell responses by flow cytometry. METHODS: Spleen cells from normal, immune-deficient purine nucleoside phosphorylase (PNP) defective (PNP-/-) mice or PNP-/- mice with partial immune reconstitution were stimulated with anti-CD3 antibodies. The correlation (r) between [(3)H]thymidine and EdU incorporations into stimulated T cells was measured and the stimulation index (SI), the ratio between stimulated and non-stimulated cells, was calculated. Flow cytometry was used to characterize the proliferating cells. RESULTS: EdU and [(3)H]thymidine incorporation into normal spleen cells were strongly correlated (r=0.89). Following stimulation, EdU incorporation into spleen cells from normal and immune-reconstituted PNP-/- mice was significantly increased compared to PNP-/- immune-deficient mice. Immune-deficient PNP-/- mice had increased [(3)H]thymidine and EdU incorporation into non-stimulated spleen cells, indicative of spontaneous proliferation. Analysis of EdU incorporation showed that the increased proliferation was due primarily to cells expressing CD3, CD4 and IgM. CONCLUSION: EdU-Click technology accurately measures proliferation of murine T lymphocyte and can be used as an alternative to [(3)H]thymidine assays. The EdU-Click technology also allows identification of proliferating cells.

Lentivirus gene therapy for purine nucleoside phosphorylase deficiency.

BACKGROUND: Purine nucleoside phosphorylase (PNP) deficiency causes the accumulation of toxic purine metabolites and lethal T cell immune defects, which might be corrected by expressing PNP by transplanting bone marrow (BM) cells transduced with lentiviral vectors containing the human PNP gene (lentiPNP). METHODS: Lymphocytes from a single PNP-deficient patient as well as lymphocytes, fibroblasts and BM from PNP-deficient (PNP (-/-)) mice were transduced with lentiPNP. Female PNP (-/-) mice were transplanted with lentiPNP transduced BM cells from male PNP (-/-) mice or normal BM. RESULTS: LentiPNP transduction significantly increased PNP expression in PNP-deficient human lymphocytes, murine lymphocytes, fibroblasts and BM cells. LentiPNP transduction also significantly improved the proliferation of PNP (-/-) murine lymphocyte and survival of irradiated PNP (-/-) fibroblasts. Polymerase chain reaction analysis demonstrated efficient transduction of lentiPNP into total and lineage-depleted BM cells grown ex vivo. LentiPNP transduced PNP (-/-) BM cells transplanted into PNP (-/-) mice expressed PNP in vivo, partially restored urinary uric acid secretion, improved thymocytes maturation, increased weight gain and extended survival of the mice. However, 12 weeks after transplant, the benefit of lentiPNP transduced cells and normal BM diminished and the percentage of engrafted donor cells decreased. CONCLUSIONS: This short-term observational study provides the first in vivo proof that gene therapy may correct some of the abnormalities associated with PNP deficiency. Better gene transduction and expression, as well as improved cell engraftment, are required to further advance PNP gene therapy.

Two types of detergent-insoluble, glycosphingolipid/cholesterol-rich membrane domains from isolated myelin.

Two different types of low-density detergent-insoluble glycosphingolipid-enriched membrane domain (DIG) fractions were isolated from myelin by extraction with Triton X-100 (TX-100) in 50 mM sodium phosphate buffer at room temperature (20 degrees C) (procedure 1), in contrast to a single low-density fraction obtained by extraction with TX-100 in Tris buffer containing 150 mM NaCl and 5 mM EDTA at 4 degrees C (procedure 2). Procedure 1 has been used in the past by others for myelin extraction to preserve the cytoskeleton and/or radial component of oligodendrocytes and myelin, whereas procedure 2 is now more commonly used to isolate myelin DIG fractions. The two DIG fractions obtained by procedure 1 gave opaque bands, B1 and B2, at somewhat lower and higher sucrose density respectively than myelin itself. The single DIG fraction obtained by procedure 2 gave a single opaque band at a similar sucrose density to B1. Both B1 and B2 had characteristics of lipid rafts, i.e. high galactosylceramide and cholesterol content and enrichment in GPI-linked 120-kDa neural cell adhesion molecule (NCAM)120, as found by others for the single low-density DIG fraction obtained by procedure 2. However, B2 had most of the myelin GM1 and more of the sulfatide than B1, and they differed significantly in their protein composition. B2 contained 41% of the actin, 100% of the tubulin, and most of the flotillin-1 and caveolin in myelin, whereas B1 contained more NCAM120 and other proteins than B2. The single low-density DIG fraction obtained by procedure 2 contained only low amounts of actin and tubulin. B1 and B2 also had size-isoform selectivity for some proteins, suggesting specific interactions and different functions of the two membrane domains. We propose that B1 may come from non-caveolar raft domains whereas B2 may derive from caveolin-containing raft domains associated with cytoskeletal proteins. Some kinases present were active on myelin basic protein suggesting that the DIGs may come from signaling domains.

A glycosynapse in myelin?

Myelin, the multilayered membrane which surrounds nerve axons, is the only example of a membranous structure where contact between extracellular surfaces of membrane from the same cell occurs. The two major glycosphingolipids (GSLs) of myelin, galactosylceramide (GalC) and its sulfated form, galactosylceramide I(3)-sulfate (SGC), can interact with each other by trans carbohydrate-carbohydrate interactions across apposed membranes. They occur in detergent-insoluble lipid rafts containing kinases and thus may be located in membrane signaling domains. These signaling domains may contact each other across apposed extracellular membranes, thus forming glycosynapses in myelin. Multivalent forms of these carbohydrates, GalC/SGC-containing liposomes, or galactose conjugated to albumin, have been added to cultured oligodendrocytes (OLs) to mimic interactions which might occur between these signaling domains when OL membranes or the extracellular surfaces of myelin come into contact. These interactions between multivalent carbohydrate and the OL membrane cause co-clustering or redistribution of myelin GSLs, GPI-linked proteins, several transmembrane proteins, and signaling proteins to the same membrane domains. They also cause depolymerization of the cytoskeleton, indicating that they cause transmission of a signal across the membrane. Their effects have similarities to those of anti-GSL antibodies on OLs, shown by others, suggesting that the multivalent carbohydrate interacts with GalC/SGC in the OL membrane. Communication between the myelin sheath and the axon regulates both axonal and myelin function and is necessary to prevent neurodegeneration. Participation of transient GalC and SGC interactions in glycosynapses between the apposed extracellular surfaces of mature compact internodal myelin might allow transmission of signals throughout the myelin sheath and thus facilitate myelin-axonal communication.

Attenuation of experimental autoimmune encephalomyelitis and nonimmune demyelination by IFN-beta plus vitamin B12: treatment to modify notch-1/sonic hedgehog balance.

Interferon-beta is a mainstay therapy of demyelinating diseases, but its effects are incomplete in human multiple sclerosis and several of its animal models. In this study, we demonstrate dramatic improvements of clinical, histological, and laboratory parameters in in vivo mouse models of demyelinating disease through combination therapy with IFN-beta plus vitamin B(12) cyanocobalamin (B(12)CN) in nonautoimmune primary demyelinating ND4 (DM20) transgenics, and in acute and chronic experimental autoimmune encephalomyelitis in SJL mice. Clinical improvement (p values <0.0001) was paralleled by near normal motor function, reduced astrocytosis, and reduced demyelination. IFN-beta plus B(12)CN enhanced in vivo and in vitro oligodendrocyte maturation. In vivo and in vitro altered expression patterns of reduced Notch-1 and enhanced expression of sonic hedgehog and its receptor were consistent with oligodendrocyte maturation and remyelination. IFN-beta-B(12)CN combination therapy may be promising for the treatment of multiple sclerosis.

Methotrexate loaded poly(L-lactic acid) microspheres for intra-articular delivery of methotrexate to the joint.

A controlled release delivery system that localizes methotrexate (MTX) in the synovial joint is needed to treat inflammation in rheumatoid arthritis (RA). The purpose of this work was to develop and characterize MTX loaded poly(l-lactic acid) (PLLA) microspheres and evaluate in vivo tolerability and MTX plasma concentrations following intra-articular injection into healthy rabbits. MTX loaded PLLA (2 kg/mole) microspheres were prepared using the solvent evaporation method and characterized in terms of size, molecular weight, thermal properties, and release rates into phosphate buffered saline (PBS) (pH 7.4) at 37 degrees C. Biocompatibility was evaluated by observing the swelling of the joints of the rabbits and histological analysis following the injection of the microspheres. MTX concentrations in the plasma and urine samples of rabbits were evaluated by high-performance liquid chromatography (HPLC). MTX loaded microspheres showed a rapid burst phase followed by a slow release phase. MTX loaded and control microspheres were biocompatible and plasma concentrations of MTX were tenfold higher in rabbits injected intra-articularly with free MTX than MTX microspheres. MTX microspheres may retain the drug in the joint by reducing clearance from the joint into the blood.