[Gene transfer into hematopoietic stem cells].
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Animals↗
Biomedical subjects
A Kume
Publications and source records attributed to A Kume.
Hematopoietic stem cell gene therapy: a current overview.
Retrovirus-mediated gene transfer into murine hematopoietic stem cells and reconstitution of syngeneic mice have demonstrated persistence and functioning of the transgenes over extended periods of time. In contrast, clinically relevant levels of gene transfer into large animal and human stem cells have not been widely achieved. Results of current clinical gene transfer studies have raised fundamental questions about the physiology of primitive human hematopoietic cells and gene therapy vectors. Efforts are being undertaken to answer these problems and to develop more efficient gene therapy strategies.
Animals↗
[A case of typical Melkersson-Rosenthal syndrome with possible autosomal dominant inheritance].
Here we presented a case of 40-year-old woman who suffered from bilateral facial palsy and headache. She had allegedly had an episode of facial palsy, and facial edema at her age of 14 years. Physical examination revealed swelling of the lips, upward disturbance of the left eye, hypogeusia, the fissured tongue, and bilateral facial palsy. Oral administration of prednisolone 20 mg/day yielded gradual but complete improvement of the facial palsy and hypogeusia within two weeks. Careful analysis of family history disclosed that four members had oro-facio-cervical edema and three had the fissured tongue. A diagnosis of Melkersson-Rosenthal syndrome with possible autosomal dominant inheritance was made based on the clinical findings and familial aggregation of the incomplete form of this syndrome.
Administration, Oral↗
Caspase-3 cleaves the expanded androgen receptor protein of spinal and bulbar muscular atrophy in a polyglutamine repeat length-dependent manner.
Spinal and bulbar muscular atrophy (SBMA) is one of a group of human inherited neurodegenerative diseases caused by polyglutamine expansion. There is increasing evidence that generation of truncated proteins containing an expanded polyglutamine tract may be an important step in the pathogenesis of these disorders. We have previously demonstrated that the SBMA gene product, the androgen receptor (AR) protein, is toxic when truncated. We now report that in vitro translated full-length AR proteins containing different sized polyglutamine repeats (24, 65 and 97 repeats, respectively) are specifically cleaved by recombinant caspase-3, liberating a polyglutamine containing fragment, and that the susceptibility to cleavage is polyglutamine repeat length-dependent. These findings suggest that AR protein is one of the "death substrates" cleaved by caspase-3 and that caspase-3 might be involved in the pathogenesis of SBMA.
Bulbar Palsy, Progressive↗
Prevention of dopaminergic neuron death by adeno-associated virus vector-mediated GDNF gene transfer in rat mesencephalic cells in vitro.
Glial cell line-derived neurotrophic factor (GDNF) is known as a potent neurotrophic factor for dopaminergic neurons. Since adeno-associated virus (AAV) vector is a suitable vehicle for gene transfer into neurons, rat E14 mesencephalic cells were transduced with an AAV vector expressing GDNF. When compared with mock transduction, a larger number of dopaminergic neurons survived in AAV-GDNF-transduced cultures (234% and 325% of controls at 1 and 2 weeks, respectively; P < 0.01). Furthermore, the dopaminergic neurons in the latter cultures grew more prominent neurites than those in the former. These findings suggest that AAV vector-mediated GDNF gene transfer may prevent dopaminergic neuron death, and is therefore a logical approach for the treatment of Parkinson's disease.
Animals↗
Efficient gene transfer into cardiac myocytes using adeno-associated virus (AAV) vectors.
Adeno-associated virus (AAV) vectors, derived from a non-pathogenic parvovirus, are considered to be an appropriate gene transfer vehicle for both dividing and non-dividing cells. In the present study, we investigated whether the rat heart could be efficiently transduced with AAV vectors. Rat cardiac myocytes (CM) were infected with AAV-lacZ vector containing beta-galactosidase (beta-gal) gene in vitro, and the expression of beta-gal in CM was evaluated by X-gal staining and beta-gal ELISA. With increasing multiplicities of infection (MOI), more than 60% of CM were stained positively with X-gal, and the beta-gal expression increased to 31.1 +/- 4.6 ng/mg protein in a MOI-dependent manner (MOI: 10(4) to 10(6) particles/cell). The beta-gal expression was also increased in an incubation period-dependent manner (1-24 h). beta-gal expression was maximal at day 3 and then gradually decreased with time. However, beta-gal expression at day 14 was almost the same level as that at day 1 (45.5 +/- 5.9 v 55.2 +/- 6.2 ng/mg protein). Excised rat right ventricular papillary muscles were also infected with AAV-lacZ ex vivo. When the beta-gal expression was evaluated by X-gal staining, more than 80% of CM in the papillary muscles were stained positively, indicating efficient gene transfer into CM using AAV vectors. These findings suggest that AAV vectors are promising for cardiac gene therapy.
Animals↗
Apoptosis-mediated regulation of recombinant human granulocyte colony-stimulating factor production by genetically engineered fibroblasts.
We investigated the feasibility of an inducible apoptosis system to regulate cells genetically engineered for ectopic cytokine production. In a previous study, cDNA encoding the ligand-binding domain of the rat estrogen receptor was fused to the sequence for murine Fas transmembrane and cytoplasmic regions, and expression of the fusion protein (MfasER) in L929 fibroblasts resulted in estrogen-dependent apoptosis. We applied this MfasER/estrogen strategy to apoptosis-mediated regulation of cytokine production, using the human granulocyte colony-stimulating factor (G-CSF) as a model. Upon estrogen treatment, the G-CSF producers expressing MfasER showed an apoptotic phenotype and died in several hours, with termination of G-CSF production. This estrogen-induced apoptosis was not influenced by whether the target cells were proliferating or resting, unlike a conventional suicide system involving the herpes simplex virus 1 thymidine kinase (HSVtk). That is, estrogen induced prompt and extensive apoptosis in the resting cells which expressed MfasER, while ganciclovir treatment induced only partial reduction of the resting cells which expressed HSVtk. These results imply the feasibility of apoptosis-mediated regulation of cytokine production by genetically modified cells for supplement gene therapy.
Animals↗
Fas and mutant estrogen receptor chimeric gene: a novel suicide vector for tamoxifen-inducible apoptosis.
Several cancer gene therapy strategies involve suicide genes to kill the neoplasm, or to regulate effector cells such as lymphocytes. We have developed an inducible apoptosis system with a Fas-estrogen receptor fusion protein (MfasER) for rapid elimination of transduced cells. In the present study, we further improved this molecular switch for estrogen-inducible apoptosis to overcome concerns with the wild-type estrogen receptor and its natural ligand, 17beta-estradiol (E2). The ligand-binding domain of MfasER was replaced with that of a mutant estrogen receptor which is unable to bind estrogen yet retains affinity for a synthetic ligand, 4-hydroxytamoxifen (Tm). The resultant fusion protein (MfasTmR) and MfasER were expressed in L929 cells for examination of their ligand specificities. Tm induced apoptosis in MfasTmR-expressing cells (L929MfasTmR) at 10(-8) M or higher concentrations, but induced no apoptosis in MfasER-expressing cells (L929MfasER) at up to 10(-6) M. On the other hand, E2 induced apoptosis in L929MfasER at concentrations as low as 10(-10)-10(-9) M, while it did so partially in L929MfasTmR at concentrations greater than 10(-7) M. Thus, L929MfasTmR cells were highly susceptible to Tm, but refractory to E2, with 100-1,000 times more tolerance than L929MfasER. These results suggest that the MfasTmR/Tm system would induce apoptosis in the target cells more safely in vivo, working independently of endogenous estrogen.
Animals↗
Allergic contact dermatitis due to diclofenac and indomethacin.
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Administration, Topical↗
Focal adhesion kinase upregulated by granulocyte-macrophage colony-stimulating factor but not by interleukin-3 in differentiating myeloid cells.
The involvement of focal adhesion kinase (FAK) in myeloid differentiation was investigated in primary murine bone marrow (BM) cells. In unstimulated BM, FAK mRNA was detected in myeloid and lymphoid cells, but not in erythroid precursors. When the BM cells were incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), FAK expression showed a remarkable difference depending on the cytokine. Although FAK was upregulated in the cells stimulated by GM-CSF (GM-treated cells), the kinase was barely detectable in the cells cultured with IL-3 (IL-3-treated cells). Morphology and flow cytometry analysis showed GM-CSF promoted the growth and differentiation of monocyte/macrophage lineage stronger than IL-3. In addition, motility of the cytokine-differentiated cells showed an overt distinction between the cultures, which was closely correlated with FAK expression. After 7 days of stimulation, GM-treated cells showed active migration and chemoattractant-induced morphologic polarization. In contrast, IL-3-treated cells showed minimal migration and polarization. These results suggest an important role of GM-CSF in the terminal differentiation of monocytes/macrophages, and possible involvement of FAK in functional maturity of this lineage.
Animals↗
Cutaneous infiltration in acute promyelocytic leukemia.
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Aged↗
Equilibrium versus compartmental analysis for assessment of the vesicular monoamine transporter using (+)-alpha-[11C]dihydrotetrabenazine (DTBZ) and positron emission tomography.
This work compares equilibrium to kinetic analysis of positron emission tomography data for the assessment of vesicular monoamine transporter (VMAT2) binding density using (+)-alpha-[11C]dihydrotetrabenazine ((+)-alpha-[11C]DTBZ). Studies were performed for 80 minutes after intravenous administration of 18 +/- 1 mCi (+)-alpha-[11C]DTBZ on 9 young control subjects, 20 to 45 years of age. A 9-mCi bolus was injected over the first minute of the study, whereas the remaining 9 mCi were infused at a constant rate over the following 79 minutes. Steady-state was reached in both blood and brain by approximately 30 minutes after initiation of the study. Nonlinear least-squares analysis using two- and three-compartment models, weighted integral analysis using a two-compartment configuration, and Logan plot analysis all yielded kinetic estimates of the total tissue distribution volume, DVtot(kin). These results were compared with equilibrium distribution volume estimates, DVtot(eq), calculated from the tissue to metabolite corrected arterial plasma concentration ratio after 30 minutes. Kinetic modeling results from this study were in close agreement with prior bolus-injection (+)-alpha-[11C]DTBZ studies. In the current study, coefficients of variation in DVtot(kin) (19% to 23% across regions) and DVtot(eq) (18% to 22%) were nearly identical. Equilibrium estimates of DVtot were slightly lower than kinetic estimates, averaging 5% +/- 9% lower (P = 0.04, paired t test) in regions of high binding density (caudate and putamen), but only 2% +/- 6% (P = 0.09) in lower binding density regions (cortex, thalamus, cerebellum). DVtot(eq) estimates, however, still correlated highly with DVtot(kin) estimates (r = 0.977-0.989). Steady-state conditions can be achieved in both tissue and blood by 30 minutes, and the tissue-to-blood ratios of (+)-alpha-[11C]DTBZ at equilibrium yield DVtot(eq) measures that are in close agreement with DVtot(kin) estimates. Thus, a simple, easily tolerated protocol using a loading bolus followed by continuous infusion can provide excellent measures of VMAT2 binding.
Adult↗
High-level reconstitution of respiratory burst activity in a human X-linked chronic granulomatous disease (X-CGD) cell line and correction of murine X-CGD bone marrow cells by retroviral-mediated gene transfer of human gp91phox.
The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, a 91-kD membrane glycoprotein that is the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a new retroviral vector for expression of human gp91phox, MSCV-h91Neo, based on murine stem cell virus vectors, was evaluated using a human X-CGD myeloid cell line (X-CGD PLB-985 cells) and murine bone marrow cells. Expression of recombinant gp91phox in transduced X-CGD PLB-985 cells was substantially improved compared with levels achieved previously using a different retroviral construct, and respiratory burst oxidase activity was fully reconstituted in the majority of clones analyzed. Expression of gp91phox transcripts was also observed in primary and secondary murine colony-forming unit-spleen derived from transduced bone marrow cells. Furthermore, respiratory burst activity was restored to granulocyte-monocyte progeny of transduced X-CGD mice bone marrow cells cultured in vitro. This observation is the first reported use of gene transfer to correct the enzymatic defect in murine CGD phagocytes and is also consistent with the high conservation of the oxidase complex among different species. Taken together, these data suggest that the MSCV-h91Neo vector may be useful for gene replacement therapy in X-linked CGD, in which high-level reconstitution of phagocyte oxidase activity may be important for full correction of phagocyte microbicidal function.
3T3 Cells↗
Paroxysmal dystonic choreoathetosis: tight linkage to chromosome 2q.
Paroxysmal dystonic choreoathetosis (PDC) is characterized by attacks of involuntary movements that last up to several hours and occur at rest both spontaneously and following caffeine or alcohol consumption. We analyzed a Polish-American kindred with autosomal dominant PDC and identified tight linkage between the disorder and microsatellite markers on chromosome 2q (maximum two-point LOD score 4.77; recombination fraction 0). Our results clearly establish the existence of a locus for autosomal dominant PDC on distal chromosome 2q. The fact that three other paroxysmal neurological disorders (periodic ataxia with myokymia and hypo- and hyperkalemic periodic paralysis) are due to mutation in ion-channel genes raises the possibility that PDC is also due to an ion-channel gene mutation. It is noteworthy that a cluster of sodium-channel genes is located on distal chromosome 2q, near the PDC locus. Identifying the PDC locus on chromosome 2q will facilitate discovery of the PDC gene and enable investigators to determine whether PDC is genetically homogeneous and whether other paroxysmal movement disorders are also genetically linked to the PDC locus.
Athetosis↗
Felbamate inhibits [3H]t-butylbicycloorthobenzoate (TBOB) binding and enhances Cl- current at the gamma-aminobutyric AcidA (GABAA) receptor.
We investigated the interaction of felbamate (FBM) with gamma-aminobutyric acid type A receptors using receptor autoradiography with [3H]t-butylbicycloorthobenzoate (TBOB) and whole-cell patch-clamp recordings of cultured mouse cortical neurons. FBM produced dose-dependent inhibition of [3H]T-BOB binding with IC50 values of approximately 250 microM. Saturation analysis in the presence of FBM revealed increased Kd and decreased Bmax. Dissociation initiated by picrotoxin (PTX) was accelerated by FBM. The regional pattern of [3H]TBOB binding inhibition by FBM was different from the regional modulation of [3H]TBOB binding produced by gamma-aminobutyric acid (GABA) agonists, bicuculline, zinc or neurosteroids. With electrophysiological recordings, FBM enhanced GABA-elicited Cl- currents at GABA concentrations of 10 microM but not 3 microM or 100 microM. FBM enhancement was not blocked by the benzodiazepine antagonist flumazenil, and FBM did not affect pentobarbital potentiation of GABA-elicited currents. FBM also had no effect on PTX inhibition of GABA-elicited Cl- currents. These results suggest that FBM potentiates gamma-aminobutyric acid type A receptor function, at least in part, by acting at a site that interacts with the PTX site but is distinct from the PTX, barbiturate, GABA, benzodiazepine, zinc and neurosteroid sites.
Animals↗
Synthesis of carbon-11-, fluorine-18-, and iodine-125-labeled GABAA-gated chloride ion channel blockers: substituted 5-tert-butyl-2-phenyl-1,3-dithianes and -dithiane oxides.
A series of substituted 5-tert-butyl-2-phenyl-1,3-dithianes and 5-tert-butyl-2-phenyl-1,1,3,3-tetraoxo-1,3-dithianes was synthesized as ligands for the GABAA receptor complex-associated neuronal chloride ion channels. The in vitro binding affinities of these compounds for the GABA-gated chloride ion channel were determined by their ability to compete with [3H]TBOB for binding to rat brain slices. Of the eight compounds tested, trans-5-tert-butyl-2-(4-cyanophenyl)-2-methyl-1,1,3,3-tetraoxo+ ++-1,3-dithiane, 9b, trans-5-tert-butyl-2-(4-fluorophenyl)-1,1,3,3-tetraoxo-1,3-dithian e, 10, and trans-5-tert-butyl-2-(4-iodophenyl)-2-methyl-1,1,3,3-tetraoxo-1,3- dithiane, 11, showed moderately high binding affinities (Ki = 41, 180, and 105 nM, respectively). Four radioligand candidates from this series, 5-tert-butyl-2-(4-cyanophenyl)-2-[11C]methyl-1,3-dithiane, [11C]6, 5-tert-butyl-2-(4-[18F]fluorophenyl)-1,3-dithiane, [18F]7, 5-tert-butyl-2-(4-[18F]-fluorophenyl)-1,1,3,3-tetraoxo-1,3- dithiane, [18F]10, and 5-tert-butyl-2-(4-[125I]iodophenyl)-2-methyl-1,1,3,3- tetraoxo-1,3-dithiane, [125I]11, have been successfully prepared for evaluation as in vivo imaging agents useful for positron emission tomography and single photon emission computed tomography. Preliminary in vivo studies indicate significant uptake into mouse brain for [18F]7, [18F]10, and [125I]11.
Animals↗
Distribution of neuronal cytoplasmic inclusions in multiple system atrophy.
Neuronal cytoplasmic inclusions (NCIs) were demonstrated by means of silver staining (Gallyas staining) in the central nervous systems of 18 deceased patients with multiple system atrophy (MSA) - 6 with olivopontocerebellar atrophy (OPCA), 6 with striatonigral degeneration (SND) and 6 with Shy-Drager syndrome (SDS). We observed NCIs in the cerebral cortex, putamen, pons, medulla oblongata and spinal cord, and especially in the putamen and pons of all cases with MSA. No NCIs were observed in the cerebellum and midbrain. The findings were common to all 3 subtypes of MSA. NCIs were not present in patients with other neurodegenerative disorders and non-neurological disorders. Our findings indicate that NCIs represent a special neuronal alteration characteristic of MSA and support the theory that OPCA, SND and SDS represent manifestations of a single condition i.e. MSA.
Atrophy↗
Retrovirus-mediated reconstitution of respiratory burst activity in X-linked chronic granulomatous disease cells.
X-linked chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a recombinant retrovirus vector was constructed and evaluated for its expression of human gp91phox in a human X-CGD myeloid cell line in which the endogenous gp91phox gene had been disrupted by gene targeting. The retrovirus construct, Zip/PGKgp91, was first introduced into the GP+envAm12 amphotropic packaging line and yielded virus producer clones with estimated titers of up to 1 x 10(5) cfu/mL. Coculture infection of X-CGD myeloid cells with Zip/PGKgp91 resulted in restoration of respiratory burst activity to 15% of the cells. Isolated clonal infectants expressed relatively low levels of recombinant gp91phox (< or = 12% of wild-type), but exhibited considerable superoxide-generating activity (up to nearly 60% of wild-type). These results show the feasibility of phenotypic correction of CGD using gene replacement therapy and suggest that even modest levels of gp91phox expression may lead to considerable functional correction of X-CGD neutrophils.
Blotting, Northern↗