Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase.

Palmitoyl-protein thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome.

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice.

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.

Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector.

Fabry disease is an X-linked metabolic disorder due to a deficiency of alpha-galactosidase A (alpha-gal A; EC 3.2.1.22). Patients accumulate glycosphingolipids with terminal alpha-galactosyl residues that come from intracellular synthesis, circulating metabolites, or from the biodegradation Of senescent cells. Patients eventually succumb to renal, cardio-, or cerebrovascular disease. No specific therapy exists. One possible approach to ameliorating this disorder is to target corrective gene transfer therapy to circulating hematopoietic cells. Toward this end, an amphotropic virus-producer cell line has been developed that produces a high titer (>10(6) i.p. per ml) recombinant retrovirus constructed to transduce and correct target cells. Virus-producer cells also demonstrate expression of large amounts of both intracellular and secreted alpha-gal A. To examine the utility of this therapeutic vector, skin fibroblasts from Fabry patients were corrected for the metabolic defect by infection with this recombinant virus and secreted enzyme was observed. Furthermore, the secreted enzyme was found to be taken up by uncorrected cells in a mannose-6-phosphate receptor-dependent manner. In related experiments, immortalized B cell lines from Fabry patients, created as a hematologic delivery test system, were transduced. As with the fibroblasts, transduced patient B cell lines demonstrated both endogenous enzyme correction and a small amount of secretion together with uptake by uncorrected cells. These studies demonstrate that endogenous metabolic correction in transduced cells, combined with secretion, may provide a continuous source of corrective material in trans to unmodified patient bystander cells (metabolic cooperativity).

Correction of diabetic alterations by glucokinase.

Hyperglycemia is a common feature of diabetes mellitus. It results from a decrease in glucose utilization by the liver and peripheral tissues and an increase in hepatic glucose production. Glucose phosphorylation by glucokinase is an initial event in glucose metabolism by the liver. However, glucokinase gene expression is very low in diabetic animals. Transgenic mice expressing the P-enolpyruvate carboxykinase/glucokinase chimeric gene were generated to study whether the return of the expression of glucokinase in the liver of diabetic mice might prevent metabolic alterations. In contrast to nontransgenic mice treated with streptozotocin, mice with the transgene previously treated with streptozotocin showed high levels of both glucokinase mRNA and its enzyme activity in the liver, which were associated with an increase in intracellular levels of glucose 6-phosphate and glycogen. The liver of these mice also showed an increase in pyruvate kinase activity and lactate production. Furthermore, normalization of both the expression of genes involved in gluconeogenesis and ketogenesis in the liver and the production of glucose and ketone body by hepatocytes in primary culture were observed in streptozotocin-treated transgenic mice. Thus, glycolysis was induced while gluconeogenesis and ketogenesis were blocked in the liver of diabetic mice expressing glucokinase. This was associated with normalization of blood glucose, ketone bodies, triglycerides, and free fatty acids even in the absence of insulin. These results suggest that the expression of glucokinase during diabetes might be a new approach to the normalization of hyperglycemia.

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.

Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide.

An experimental strategy to facilitate correction of single-base mutations of episomal targets in mammalian cells has been developed. The method utilizes a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. The RNA/DNA sequence is designed to align with the sequence of the mutant locus and to contain the desired nucleotide change. Activity of the chimeric molecule in targeted correction was tested in a model system in which the aim was to correct a point mutation in the gene encoding the human liver/bone/kidney alkaline phosphatase. When the chimeric molecule was introduced into cells containing the mutant gene on an extrachromosomal plasmid, correction of the point mutation was accomplished with a frequency approaching 30%. These results extend the usefulness of the oligonucleotide-based gene targeting approaches by increasing specific targeting frequency. This strategy should enable the design of antiviral agents.

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected.

Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.

Transgenic mice and sheep secrete only low levels of human factor IX in their milk because of an aberrant splicing of the transgene RNA in the mammary gland. Removal of the cryptic 3' splice site prevents this splicing and leads to the production of relatively high levels of factor IX. The purified protein is fully active showing that the mammary gland is capable of the efficient post-translational modification of this protein and that transgenic animals are a suitable means of its production.

Phenotype correction in retinal pigment epithelium in murine mucopolysaccharidosis VII by adenovirus-mediated gene transfer.

We have studied the use of adenovirus-mediated gene transfer to reverse the pathologic changes of lysosomal storage disease caused by beta-glucuronidase deficiency in the eyes of mice with mucopolysaccharidosis VII. A recombinant adenovirus carrying the human beta-glucuronidase cDNA coding region under the control of a non-tissue-specific promoter was injected intravitreally or subretinally into the eyes of mice with mucopolysaccharidosis VII. At 1-3 weeks after injection, the treated and control eyes were examined histochemically for beta-glucuronidase expression and histologically for phenotypic correction of the lysosomal storage defect. Enzymatic expression was detected 1-3 weeks after injection. Storage vacuoles in the retinal pigment epithelium (RPE) were still present 1 week after gene transfer but were reduced to undetectable levels by 3 weeks in both intravitreally and subretinally injected eyes. There was minimal evidence of ocular pathology associated with the viral injection. These data indicate that adenovirus-mediated gene transfer to the eye may provide for adjunctive therapy for lysosomal storage diseases affecting the RPE in conjunction with enzyme replacement and/or gene therapies for correction of systemic disease manifestations. The data also support the view that recombinant adenovirus may be useful as a gene therapy vector for retinal degenerations that result from a primary genetic defect in the RPE cells.

Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene.

The mutagen-sensitive CHO line irs1SF was previously isolated on the basis of hypersensitivity to ionizing radiation and was found to be chromosomally unstable as well as cross-sensitive to diverse kinds of DNA-damaging agents. The analysis of somatic cell hybrids formed between irs1SF and human lymphocytes implicated a human gene (defined as XRCC3; x-ray repair cross-complementing), which partially restored mitomycin C resistance to the mutant. A functional cDNA that confers mitomycin C resistance was transferred to irs1SF cells by transforming them with an expression cDNA library and obtaining primary and secondary transformants. Functional cDNA clones were recovered from a cosmid library prepared from a secondary transformant. Transformants also showed partial correction of sensitivity to cisplatin and gamma-rays, efficient correction of chromosomal instability, and substantially improved plating efficiency and growth rate. The XRCC3 cDNA insert is approximately 2.5 kb and detects an approximately 3.0-kb mRNA on Northern blots. The cDNA was mapped by fluorescence in situ hybridization to human chromosome 14q32.3, which was consistent with the chromosome concordance data of two independent hybrid clone panels.

Impact of artifact correction methods on R-R interbeat signals to quantifying heart rate variability (HRV) according to linear and nonlinear methods.

In the analysis of heart rate variability (HRV) are used temporal series that contains the distances between successive heartbeats in order to assess autonomic regulation of the cardiovascular system. These series are obtained from the electrocardiogram (ECG) signal analysis, which can be affected by different types of artifacts leading to incorrect interpretations in the analysis of the HRV signals. Classic approach to deal with these artifacts implies the use of correction methods, some of them based on interpolation, substitution or statistical techniques. However, there are few studies that shows the accuracy and performance of these correction methods on real HRV signals. This study aims to determine the performance of some linear and non-linear correction methods on HRV signals with induced artefacts by quantification of its linear and nonlinear HRV parameters. As part of the methodology, ECG signals of rats measured using the technique of telemetry were used to generate real heart rate variability signals without any error. In these series were simulated missing points (beats) in different quantities in order to emulate a real experimental situation as accurately as possible. In order to compare recovering efficiency, deletion (DEL), linear interpolation (LI), cubic spline interpolation (CI), moving average window (MAW) and nonlinear predictive interpolation (NPI) were used as correction methods for the series with induced artifacts. The accuracy of each correction method was known through the results obtained after the measurement of the mean value of the series (AVNN), standard deviation (SDNN), root mean square error of the differences between successive heartbeats (RMSSD), Lomb\'s periodogram (LSP), Detrended Fluctuation Analysis (DFA), multiscale entropy (MSE) and symbolic dynamics (SD) on each HRV signal with and without artifacts. The results show that, at low levels of missing points the performance of all correction techniques are very similar with very close values for each HRV parameter. However, at higher levels of losses only the NPI method allows to obtain HRV parameters with low error values and low quantity of significant differences in comparison to the values calculated for the same signals without the presence of missing points.

Ocular residual astigmatism and topographic disparity vector indexes in normal healthy eyes

Purpose: To define a range of normality for the vectorial parameters Ocular Residual Astigmatism (ORA) and topography disparity (TD) and to evaluate their relationship with visual, refractive, anterior and posterior corneal curvature, pachymetric and corneal volume data in normal healthy eyes. Methods: This study comprised a total of 101 consecutive normal healthy eyes of 101 patients ranging in age from 15 to 64 years old. In all cases, a complete corneal analysis was performed using a Scheimpflug photography-based topography system (Pentacam system Oculus Optikgeräte GmbH). Anterior corneal topographic data were imported from the Pentacam system to the iASSORT software (ASSORT Pty. Ltd.), which allowed the calculation of the ocular residual astigmatism (ORA) and topography disparity (TD). Linear regression analysis was used for obtaining a linear expression relating ORA and posterior corneal astigmatism (PCA). Results: Mean magnitude of ORA was 0.79 D (SD: 0.43), with a normality range from 0 to 1.63 D. 90 eyes (89.1%) showed against-the-rule ORA. A weak although statistically significant correlation was found between the magnitudes of posterior corneal astigmatism and ORA (r = 0.34, p < 0.01). Regression analysis showed the presence of a linear relationship between these two variables, although with a very limited predictability (R2: 0.08). Mean magnitude of TD was 0.89 D (SD: 0.50), with a normality range from 0 to 1.87 D. Conclusion: The magnitude of the vector parameters ORA and TD is lower than 1.9 D in the healthy human eye.

New Approach for Correction of Error Associated With Keratometric Estimation of Corneal Power in Keratoconus

The aim of this study was to obtain the exact value of the keratometric index (nkexact) and to clinically validate a variable keratometric index (nkadj) that minimizes this error. Methods: The nkexact value was determined by obtaining differences (DPc) between keratometric corneal power (Pk) and Gaussian corneal power (PGauss c ) equal to 0. The nkexact was defined as the value associated with an equivalent difference in the magnitude of DPc for extreme values of posterior corneal radius (r2c) for each anterior corneal radius value (r1c). This nkadj was considered for the calculation of the adjusted corneal power (Pkadj). Values of r1c ∈ (4.2, 8.5) mm and r2c ∈ (3.1, 8.2) mm were considered. Differences of True Net Power with PGauss c , Pkadj, and Pk(1.3375) were calculated in a clinical sample of 44 eyes with keratoconus. Results: nkexact ranged from 1.3153 to 1.3396 and nkadj from 1.3190 to 1.3339 depending on the eye model analyzed. All the nkadj values adjusted perfectly to 8 linear algorithms. Differences between Pkadj and PGauss c did not exceed 60.7 D (Diopter). Clinically, nk = 1.3375 was not valid in any case. Pkadj and True Net Power and Pk(1.3375) and Pkadj were statistically different (P , 0.01), whereas no differences were found between PGauss c and Pkadj (P . 0.01). Conclusions: The use of a single value of nk for the calculation of the total corneal power in keratoconus has been shown to be imprecise, leading to inaccuracies in the detection and classification of this corneal condition. Furthermore, our study shows the relevance of corneal thickness in corneal power calculations in keratoconus.

Comparison of the predictability of refractive cylinder correction by laser in situ keratomileusis in eyes with low or high ocular residual astigmatism

Purpose: To compare the manifest refractive cylinder (MRC) predictability of myopic astigmatism laser in situ keratomileusis (LASIK) between eyes with low and high ocular residual astigmatism (ORA). Setting: London Vision Clinic, London, United Kingdom. Design: Retrospective case study. Methods: The ORA was considered the vector difference between the MRC and the corneal astigmatism. The index of success (IoS), difference vector ÷ MRC, was analyzed for different groups as follows: stage 1, low ORA (ORA ÷ MRC <1), high ORA (ORA ÷ MRC ≥1); stage 2, low ORA group reduced to match the high ORA group for MRC; stage 3, grouped by ORA magnitude with low ORA (<0.50 diopters [D]), mid ORA (0.50 to 1.24 D), and high ORA (≥1.25 D); stage 4, high ORA group subdivided into low (<0.75 D) and high (≥0.75 D) corneal astigmatism. Results: For stage 1, the mean preoperative MRC and mean IoS were −1.32 D ± 0.65 (SD) (range −0.55 to −3.77 D) and 0.27, respectively, for low ORA and −0.79 ± 0.20 D (range −0.56 to −2.05 D) and 0.37, respectively, for high ORA. For stage 2, the mean IoS increased to 0.32 for low ORA. For stage 3, the mean IoS was 0.28, 0.29, and 0.31 for low ORA, mid ORA, and high ORA, respectively. For stage 4, the mean IoS was 0.20 for high ORA/low corneal astigmatism and 0.35 for high ORA/high corneal astigmatism. Conclusions: The MRC predictability was slightly worse in eyes with high ORA when grouped by the ORA ÷ MRC. Matching for the MRC and grouping by ORA magnitude resulted in similar predictability; however, eyes with high ORA and high corneal astigmatism were less predictable.

Clinical utility of ocular residual astigmatism and topographic disparity vector indexes in subclinical and clinical keratoconus

Purpose. We aimed to characterize the distribution of the vector parameters ocular residual astigmatism (ORA) and topography disparity (TD) in a sample of clinical and subclinical keratoconus eyes, and to evaluate their diagnostic value to discriminate between these conditions and healthy corneas. Methods. This study comprised a total of 43 keratoconic eyes (27 patients, 17–73 years) (keratoconus group), 11 subclinical keratoconus eyes (eight patients, 11–54 years) (subclinical keratoconus group) and 101 healthy eyes (101 patients, 15–64 years) (control group). In all cases, a complete corneal analysis was performed using a Scheimpflug photography-based topography system. Anterior corneal topographic data was imported from it to the iASSORT software (ASSORT Pty. Ltd), which allowed the calculation of ORA and TD. Results. Mean magnitude of the ORA was 3.23 ± 2.38, 1.16 ± 0.50 and 0.79 ± 0.43 D in the keratoconus, subclinical keratoconus and control groups, respectively (p < 0.001). Mean magnitude of the TD was 9.04 ± 8.08, 2.69 ± 2.42 and 0.89 ± 0.50 D in the keratoconus, subclinical keratoconus and control groups, respectively (p < 0.001). Good diagnostic performance of ORA (cutoff point: 1.21 D, sensitivity 83.7 %, specificity 87.1 %) and TD (cutoff point: 1.64 D, sensitivity 93.3 %, specificity 92.1 %) was found for the detection of keratoconus. The diagnostic ability of these parameters for the detection of subclinical keratoconus was more limited (ORA: cutoff 1.17 D, sensitivity 60.0 %, specificity 84.2 %; TD: cutoff 1.29 D, sensitivity 80.0 %, specificity 80.2 %). Conclusion. The vector parameters ORA and TD are able to discriminate with good levels of precision between keratoconus and healthy corneas. For the detection of subclinical keratoconus, only TD seems to be valid.