Patient-reported outcomes in multiple sclerosis: a systematic comparison of available measures.

Multiple patient-reported outcomes (PROs) are currently being used in multiple sclerosis (MS) but their application is inconsistent and guidance on the appropriateness of each tool is lacking. The objective of our study was to identify MS-specific PROs and systematically to assess the development process and the reliability and validity of various instruments. A systematic literature search was conducted on multiple data sources, including MEDLINE, Embase (using the Ovid platform) and Google Scholar, from 1996 to March 2015. Search terms included combinations of MS, PROs and quality of life. Randomized controlled trials or observational studies conducted on patients with MS and published in English were included. In addition, the PROQOLID database was explored. The MS-specific PROs were systematically assessed using the Evaluating the Measurement of Patient-Reported Outcomes tool. In total, 8094 articles were screened and 405 PROs were identified from 1102 relevant articles. PROs were classified into MS-specific (n = 82) and non-MS-specific (n = 323). The results for the eight PROs that are most commonly used in MS clinical trials are presented here. For these eight PROs, the overall summary scores ranged between 50.1 and 68.7. The Multiple Sclerosis Impact Scale-29 had the best overall mean score (68.7), followed by the Leeds Multiple Sclerosis Quality of Life (67.0). This is the first study to provide a standardized assessment of all PROs for MS. There is a lack of data on content validity for PROs used in MS research, which indicates the need for a robust instrument in MS developed according to the US Food and Drug Administration guidelines.

Engineered human Tmpk fused with truncated cell-surface markers: versatile cell-fate control safety cassettes.

Cell-fate control gene therapy (CFCGT)-based strategies can augment existing gene therapy and cell transplantation approaches by providing a safety element in the event of deleterious outcomes. Previously, we described a novel enzyme/prodrug combination for CFCGT. Here, we present results employing novel lentiviral constructs harboring sequences for truncated surface molecules (CD19 or low-affinity nerve growth factor receptor) directly fused to that CFCGT cDNA (TmpkF105Y). This confers an enforced one-to-one correlation between cell marking and eradication functions. In-vitro analysis demonstrated the full functionality of the fusion product. Next, low-dose 3'-azido-3'-deoxythymidine (AZT) administration to non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with transduced clonal K562 cells suppressed tumor growth; furthermore, one integrated vector on average was sufficient to mediate cytotoxicity. Further, in a murine xenogeneic leukemia-lymphoma model we also demonstrated in-vivo control over transduced Raji cells. Finally, in a proof-of-principle study to examine the utility of this cassette in combination with a therapeutic cDNA, we integrated this novel CFCGT fusion construct into a lentivector designed for treatment of Fabry disease. Transduction with this vector restored enzyme activity in Fabry cells and retained AZT sensitivity. In addition, human Fabry patient CD34(+) cells showed high transduction efficiencies and retained normal colony-generating capacity when compared with the non-transduced controls. These collective results demonstrated that this novel and broadly applicable fusion system may enhance general safety in gene- and cell-based therapies.

Lentivirus IL-10 gene therapy down-regulates IL-17 and attenuates mouse orthotopic lung allograft rejection.

The purpose of the study was to examine the effect of lentivirus-mediated IL-10 gene therapy to target lung allograft rejection in a mouse orthotopic left lung transplantation model. IL-10 may regulate posttransplant immunity mediated by IL-17. Lentivirus-mediated trans-airway luciferase gene transfer to the donor lung resulted in persistent luciferase activity up to 6 months posttransplant in the isograft (B6 to B6); luciferase activity decreased in minor-mismatched allograft lungs (B10 to B6) in association with moderate rejection. Fully MHC-mismatched allograft transplantation (BALB/c to B6) resulted in severe rejection and complete loss of luciferase activity. In minor-mismatched allografts, IL-10-encoding lentivirus gene therapy reduced the acute rejection score compared with the lentivirus-luciferase control at posttransplant day 28 (3.0 ± 0.6 vs. 2.0 ± 0.6 (mean ± SD); p = 0.025; n = 6/group). IL-10 gene therapy also significantly reduced gene expression of IL-17, IL-23, and retinoic acid-related orphan receptor (ROR)-γt without affecting levels of IL-12 and interferon-γ (IFN-γ). Cells expressing IL-17 were dramatically reduced in the allograft lung. In conclusion, lentivirus-mediated IL-10 gene therapy significantly reduced expression of IL-17 and other associated genes in the transplanted allograft lung and attenuated posttransplant immune responses after orthotopic lung transplantation.

Differential immune responses mediated by adenovirus- and lentivirus-transduced DCs in a HER-2/neu overexpressing tumor model.

Recent investigations have demonstrated that adenoviral and lentiviral vectors encoding HER-2 can be utilized in cancer immunotherapy. However, it is not known whether both viral systems elicit a similar immune response. Here, we compare the immune response in mice induced by dendritic cells (DCs) infected with either recombinant adenovirus or lentivirus encoding rat HER-2 (rHER-2). Both vaccine types yielded similar control of tumor growth, but we found clear differences in their immune responses 10 days after DC immunization. Adenovirus rHER-2-transduced DCs elicited locally and systemically high frequencies of CD4+ and CD8+ T cells, while lentivirus rHER-2-transduced DCs predominantly led to CD4+ T-cell infiltration at the tumor site. Splenocytes from mice immunized with lentivirus rHER-2-transduced DCs secreted higher levels of interferon (IFN)-γ, mainly by CD4+ T cells, following stimulation by RM-1-mHER-2 tumors. In contrast, the adenovirus vaccinated group exhibited CD4+ and CD8+ T cells that both contributed to IFN-γ production. Besides an established cellular immune response, the rHER-2/DC vaccine elicited a significant humoral response that was highest in the adenovirus group. DC subsets and regulatory T cells in the spleen were also differentially modulated in the two vaccine systems. Finally, adoptive transfer of splenocytes from both groups of immunized mice strongly inhibited in vivo tumor growth. Our results suggest that not only the target antigen but also the virus system may determine the nature and magnitude of antitumor immunity by DC vaccination.

Transduction and transplantation of spermatogonia into the testis of ram lambs through the extra-testicular rete.

Spermatogonial transplantation will provide a new way to study spermatogenesis in domestic animals, disseminate male genetics and produce transgenic animals, if efficiency can be improved. We evaluated a 'surgical' method for transplanting donor cells into testes of ram lambs, where the head of the epididymis is reflected, and a catheter introduced into the extra-testicular rete testis. We also tested transduction of ram spermatogonia with a lentiviral (LV) vector as a means to identify permanent colonization, and introduce genes into donor cells. Eight ram lambs, 11- to 13-week olds, were the recipients: in five, spermatogonia were injected into one testis, and the contralateral testis was an un-manipulated control: in two, spermatogonia were injected into one testis and the contralateral was sham-injected: in one, both testes were injected. Six lambs received spermatogonia labelled with a cell-tracking dye and these were collected 1 or 2 weeks after transplantation; three lambs received spermatogonia transduced with a LV vector driving the expression of enhanced Green Fluorescence Protein and these were collected after 2 months. Donor cells were detected by immunohistochemistry in tubules of seven of nine recipient testes. Approximately 22% of tubule cross-sections contained donor cells immediately after transplantation, and 0.2% contained virally transduced cells 2 months after transplantation. The onset of spermatogenesis was delayed, and there were lesions in both injected and sham-injected testes. Despite the effects of the surgery, elongated spermatids were present in one recipient testis 2 months after surgery. The results suggest that, after modifying the surgical and transduction techniques, this approach will be a means to produce good colonization by donor spermatogonia in sheep testes.

Feasibility of Brain Atrophy Measurement in Clinical Routine without Prior Standardization of the MRI Protocol: Results from MS-MRIUS, a Longitudinal Observational, Multicenter Real-World Outcome Study in Patients with Relapsing-Remitting MS.

BACKGROUND AND PURPOSE: Feasibility of brain atrophy measurement in patients with MS in clinical routine, without prior standardization of the MRI protocol, is unknown. Our aim was to investigate the feasibility of brain atrophy measurement in patients with MS in clinical routine. MATERIALS AND METHODS: Multiple Sclerosis and Clinical Outcome and MR Imaging in the United States (MS-MRIUS) is a multicenter (33 sites), retrospective study that included patients with relapsing-remitting MS who began treatment with fingolimod. Brain MR imaging examinations previously acquired at the baseline and follow-up periods on 1.5T or 3T scanners with no prior standardization were used, to resemble a real-world situation. Brain atrophy outcomes included the percentage brain volume change measured by structural image evaluation with normalization of atrophy on 2D-T1-weighted imaging and 3D-T1WI and the percentage lateral ventricle volume change, measured by VIENA on 2D-T1WI and 3D-T1WI and NeuroSTREAM on T2-fluid-attenuated inversion recovery examinations. RESULTS: A total of 590 patients, followed for 16 months, were included. There were 585 (99.2%) T2-FLAIR, 425 (72%) 2D-T1WI, and 166 (28.2%) 3D-T1WI longitudinal pairs of examinations available. Excluding MR imaging examinations with scanner changes, the analyses were available on 388 (65.8%) patients on T2-FLAIR for the percentage lateral ventricle volume change, 259 and 257 (43.9% and 43.6%, respectively) on 2D-T1WI for the percentage brain volume change and the percentage lateral ventricle volume change, and 110 (18.6%) on 3D-T1WI for the percentage brain volume change and percentage lateral ventricle volume change. The median annualized percentage brain volume change was -0.31% on 2D-T1WI and -0.38% on 3D-T1WI. The median annualized percentage lateral ventricle volume change was 0.95% on 2D-T1WI, 1.47% on 3D-T1WI, and 0.90% on T2-FLAIR. CONCLUSIONS: Brain atrophy was more readily assessed by estimating the percentage lateral ventricle volume change on T2-FLAIR compared with the percentage brain volume change or percentage lateral ventricle volume change using 2D- or 3D-T1WI in this observational retrospective study. Although measurement of the percentage brain volume change on 3D-T1WI remains the criterion standard and should be encouraged in future prospective studies, T2-FLAIR-derived percentage lateral ventricle volume change may be a more feasible surrogate when historical or other practical constraints limit the availability of percentage brain volume change on 3D-T1WI.

Retinoic acid induction of major histocompatibility complex class I genes in NTera-2 embryonal carcinoma cells involves induction of NF-kappa B (p50-p65) and retinoic acid receptor beta-retinoid X receptor beta heterodimers.

Retinoic acid (RA) treatment of human embryonal carcinoma (EC) NTera-2 (NT2) cells induces expression of major histocompatibility complex (MHC) class I and beta-2 microglobulin surface molecules. We found that this induction was accompanied by increased levels of MHC class I mRNA, which was attributable to the activation of the two conserved upstream enhancers, region I (NF-kappa B like) and region II. This activation coincided with the induction of nuclear factor binding activities specific for the two enhancers. Region I binding activity was not present in undifferentiated NT2 cells, but binding of an NF-kappa B heterodimer, p50-p65, was induced following RA treatment. The p50-p65 heterodimer was produced as a result of de novo induction of p50 and p65 mRNAs. Region II binding activity was present in undifferentiated cells at low levels but was greatly augmented by RA treatment because of activation of a nuclear hormone receptor heterodimer composed of the retinoid X receptor (RXR beta) and the RA receptor (RAR beta). The RXR beta-RAR beta heterodimer also bound RA responsive elements present in other genes which are likely to be involved in RA triggering of EC cell differentiation. Furthermore, transfection of p50 and p65 into undifferentiated NT2 cells synergistically activated region I-dependent MHC class I reporter activity. A similar increase in MHC class I reporter activity was demonstrated by cotransfection of RXR beta and RAR beta. These data show that following RA treatment, heterodimers of two transcription factor families are induced to bind to the MHC enhancers, which at least partly accounts for RA induction of MHC class I expression in NT2 EC cells.

Nanodosimetry in a clinical neutron therapy beam using the variance-covariance method and Monte Carlo simulations.

Nanodosimetric single-event distributions or their mean values may contribute to a better understanding of how radiation induced biological damages are produced. They may also provide means for radiation quality characterization in therapy beams. Experimental nanodosimetry is however technically challenging and Monte Carlo simulations are valuable as a complementary tool for such investigations. The dose-mean lineal energy was determined in a therapeutic p(65)+Be neutron beam and in a (60)Co gamma beam using low-pressure gas detectors and the variance-covariance method. The neutron beam was simulated using the condensed history Monte Carlo codes MCNPX and SHIELD-HIT. The dose-mean lineal energy was calculated using the simulated dose and fluence spectra together with published data from track-structure simulations. A comparison between simulated and measured results revealed some systematic differences and different dependencies on the simulated object size. The results show that both experimental and theoretical approaches are needed for an accurate dosimetry in the nanometer region. In line with previously reported results, the dose-mean lineal energy determined at 10 nm was shown to be related to clinical RBE values in the neutron beam and in a simulated 175 MeV proton beam as well.

Sphingolipid pathway enzymes modulate cell fate and immune responses.

Sphingolipids (SLs) are a class of essential, bioactive lipids. The SL family includes over 4000 distinct molecules, characterized by their sphingoid base (long-chain aliphatic amine) backbone. SLs are key components of cell membranes, yet their roles go well beyond structure. SLs are involved in many cellular processes including cell differentiation, apoptosis, growth arrest and senescence. As cancer cells routinely display increased growth properties and escape from cell death, it has been suggested that enzymes involved in SL synthesis or catabolism may be altered in cancer cells. In this review, we discuss the role of SL pathway enzymes in cancer, and in acquired resistance to therapy. The use of inhibitors and gene silencing approaches targeting these SL pathways is also explored. Finally, we elaborate on the role of SL pathway enzymes in the tumor microenvironment and their effect on immune cell function.

An algorithm for real-time dosimetry in intensity-modulated radiation therapy using the radioluminescence signal from Al2O3:C.

Although the radioluminescence (RL) signal from optical fibre Al(2)O(3):C dosemeters used in medical applications is essentially proportional to dose rate, the crystals used so far are imperfect in the sense that their RL sensitivity changes with accumulated dose. A computational algorithm has been developed that corrects for these sensitivity changes. We further report on a new system that effectively separates the RL signal generated in the crystal from fluorescence and Cerenkov emission generated in the optical fibre cable using a gating technique in connection with pulsed linear accelerator radiation beams. The dosimetry system has been used for dose measurements in a phantom during an intensity-modulated radiation therapy (IMRT) treatment with 6 MV photons. The RL measurement results are in excellent agreement (i.e. within 1%) with both the OSL results and the dose delivered according to the treatment planning system. RL signals from Al(2)O(3):C can be used for real-time dose rate measurements with a time resolution of approximately 0.1 s and a spatial resolution only limited by the size of the detector (<0.5 mm).

Characterization of the chimeric retinoic acid receptor RARalpha/VDR.

The chimeric receptor, RARalpha/VDR, contains the DNA-binding domain of the retinoic acid receptor (RARalpha) and the ligand-binding domain of the vitamin D receptor (VDR). The ligand-binding properties of RARalpha/VDR are equivalent to that of VDR, with an observed Kd for 1alpha,25 dihydroxy-vitamin D3 (D3) of 0.5 nM. In CV-1 cells, both RARalpha and RARalpha/VDR induce comparable levels of ligand-mediated transcriptional activity from the retinoic acid responsive reporter gene, beta(RARE)3-TK-luciferase, in the presence of the ligand predicted from the receptor ligand-binding domain. Two chimeric RAR receptors were constructed which contained the ligand-binding domain of the estrogen receptor (ER): RARalpha/ER and ER/RARalpha/ER. Both RARalpha/ER and ER/RARalpha/ER bind beta-estradiol with high affinity, and are transcriptionally active only from palindromic RAREs (TREpal and/or (TRE3)3). Only RARalpha/VDR matched in kind and degree the functional characteristics of RARalpha: (1) maximally active from the beta(RARE); (2) moderately active from the TREs; (3) inactive from the retinoic X receptor response elements (RXREs) ApoA1 and CRBP II; (4) forms heterodimers with RXRalpha; and (5) binds to the betaRARE. F9 embryonal carcinoma cell lines were generated which express RARalpha/VDR mRNA (F9RARalpha/VDR cells) and compared with F9 wild-type (F9-Wt) cells, which do not express VDR mRNA. Treatment with all-trans retinoic acid (tRA) inhibits cell growth and induces the differentiation morphology in both F9-Wt and F9-RARalpha/VDR cells; whereas, treatment with D3 is similarly effective only for F9-RARalpha/VDR cells. It is concluded RARalpha/VDR is an useful 'tool' to pinpoint, or to augment transcription from RAREs in gene pathways controlled by RAR without inhibiting the retinoid responsiveness of endogenous RARs.

The influence of RBE variations in a clinical proton treatment plan for a hypopharynx cancer.

Currently, most clinical range-modulated proton beams are assumed to have a fixed overall relative biological effectiveness (RBE) of 1.1. However, it is well known that the RBE increases with depth in the spread-out Bragg peak (SOBP) and becomes about 10% higher than mid-SOBP RBE at 2 mm from the distal edge (Paganetti 2003 Technol. Cancer Res. Treat. 2 413-26) and can reach values of 1.3-1.4 in vitro at the distal edge (Robertson et al 1975 Cancer 35 1664-77, Courdi et al 1994 Br. J. Radiol. 67 800-4). We present a fast method for applying a variable RBE correction with linear energy transfer (LET) dependent tissue-specific parameters based on the alpharef/betaref ratios suitable for implementation in a treatment planning system. The influence of applying this variable RBE correction on a clinical multiple beam proton dose plan is presented here. The treatment plan is evaluated by RBE weighted dose volume histograms (DVHs) and the calculation of tumour control probability (TCP) and normal tissue complication probability (NTCP) values. The variable RBE correction yields DVHs for the clinical target volumes (CTVs), a primary advanced hypopharynx cancer and subclinical disease in the lymph nodes, that are slightly higher than those achieved by multiplying the absorbed dose with RBE=1.1. Although, more importantly, the RBE weighted DVH for an organ at risk, the spinal cord is considerably increased for the variable RBE. As the spinal cord in this particular case is located 8 mm behind the planning target volume (PTV) and hence receives only low total doses, the NTCP values are zero in spite of the significant increase in the RBE weighted DVHs for the variable RBE. However, high NTCP values for the non-target normal tissue were obtained when applying the variable RBE correction. As RBE variations tend to be smaller for in vivo systems, this study-based on in vitro data since human tissue RBE values are scarce and have large uncertainties-can be interpreted as showing the upper limits of the possible effects of utilizing a variable RBE correction. In conclusion, the results obtained here still indicate a significant difference in introducing a variable RBE compared to applying a generic RBE of 1.1, suggesting it is worth considering such a correction in clinical proton therapy planning, especially when risk organs are located immediately behind the target volume.

In vivo absorbed dose measurements in mammography using a new real-time luminescence technique.

A dosimetry system based on radioluminescence (RL) and optically stimulated luminescence (OSL) from carbon doped aluminium oxide (Al2O3:C) crystals was developed for in vivo absorbed dose measurements in mammography. A small cylindrical crystal of Al2O3:C (diameter 0.48 mm and length 2 mm) was coupled to the end of a 1 mm diameter optical fibre cable. Owing to their small size and characteristic shape, these probes can be placed on the body surface in the field of view during the examination, without compromising the reading of the mammogram. Our new technique was tested with a mammography unit (Siemens Mammomat 3000) and screen-film technique over a range of clinically relevant X-ray energies. The results were compared with those obtained from an ionization chamber usually used for the determination of absorbed dose in mammography. The reproducibility of measurements was around 3% (1 standard deviation) at 4.5 mGy for both RL and OSL data. The dose response was found to be linear between 4.5 mGy and 30 mGy. The energy dependence of the system is around 18% between 23 kV and 35 kV. In vivo measurements were performed during three patient examinations. It was shown that entrance and exit doses could be measured. The presence of the small probes did not significantly interfere with the diagnostic quality of the images. Entrance doses estimated by RL/OSL results agreed within 3% with entrance surface dose values calculated from the ionization chamber measurements. These results indicate a considerable potential for use in routine control and in vivo dose measurements in mammography.

A Monte Carlo study of the energy dependence of Al2O3:C crystals for real-time in vivo dosimetry in mammography.

In a previous experimental study, a novel method for in vivo dosimetry has been investigated, based on radioluminescence (RL) and optically stimulated luminescence (OSL). However, because of the large difference in atomic composition between the detector material and the breast tissue, relatively large energy dependence in low-energy X-ray beams can be expected. In the present work, the energy dependence of Al2O3:C crystals was modelled with the Monte Carlo code EGSnrc using three types of X-ray spectra. The results obtained (5.6-7.3%) agree with a previously determined experimental result (9%) within the combined standard uncertainty of the two methods. The influence of the size of the crystal on the energy dependence was investigated together with the effect of varying the thickness of the surrounding light-protective material. The results obtained indicate a minor effect owing to the thickness of the light-protective material, and a somewhat larger effect from reducing the diameter of the crystal. The outcome of this study can be used to improve the future design of the RL/OSL dosimetry system for use in mammography.

Can the average glandular dose in routine digital mammography screening be reduced? A pilot study using revised image quality criteria.

There is a need for tools that in a simple way can be used for the evaluation of image quality related to clinical requirements in mammography. The aim of this work was to adjust the present European image quality criteria to be relevant also for digital mammography images, and to use as simple and as few criteria as possible. A pilot evaluation of the new set of criteria was made with mammograms of 28 women from a General Electric Senographe 2000D full-field digital mammography system. One breast was exposed using the standard automatic exposure mode, the other using about half of that absorbed dose. Three experienced radiologists evaluated the images using visual grading analysis technique. The results indicate that the new quality criteria can be used for the evaluation of image quality related to clinical requirements in digital mammography in a simple way. The results also suggest that absorbed doses for the mammography system used may be substantially reduced.

Clinical evaluation of a new set of image quality criteria for mammography.

The European Commission (EC) quality criteria for screen-film mammography are used as a tool to assess image quality. A new set of criteria was developed and initially tested in a previous study. In the present study, these criteria are further evaluated using screen-film mammograms that have been digitised, manipulated to simulate different image quality levels and reprinted on film. Expert radiologists have evaluated these manipulated images using both the original (EC) and the new criteria. A comparison of three different simulated dose levels reveals that the new criteria yield a larger separation of image criteria scores than the old ones. These results indicate that the new set of image quality criteria has a higher discriminative power than the old set and thus seems to be more suitable for evaluation of image quality in mammography.

Enzymatic and functional correction along with long-term enzyme secretion from transduced bone marrow hematopoietic stem/progenitor and stromal cells derived from patients with Fabry disease.

Fabry disease is a lysosomal storage disorder that is due to a deficiency in alpha-galactosidase A (alpha-gal A). Previously we have shown that a recombinant retrovirus synthesized for the transfer of the human alpha-gal A coding sequence was able to engineer enzymatic correction of the hydrolase deficiency in fibroblasts and lymphoblasts from Fabry patients. The corrected cells secreted alpha-gal A that was taken up and utilized by uncorrected bystander cells, thus demonstrating metabolic cooperativity. In separate experiments we used transduced murine bone marrow cells and successfully tested and quantitated this phenomenon in vivo. In the present studies, which were designed to bring this therapeutic approach closer to clinical utility, we establish that cells originating from the bone marrow of numerous Fabry patients and normal volunteers can be effectively transduced and that these target cells demonstrate metabolic cooperativity. Both isolated CD34+-enriched cells and long-term bone marrow culture cells, including nonadherent hematopoietic cells and adherent stromal cells, were transduced. The transferred gene generates increased intracellular alpha-gal A enzyme activity in these cells. Further, it causes functional correction of lipid accumulation and provides for long-term alpha-gal A secretion. Collectively, these results indicate that a multifaceted gene transfer approach to bone marrow cells may be of therapeutic benefit for patients with Fabry disease.

Circulating alpha-galactosidase A derived from transduced bone marrow cells: relevance for corrective gene transfer for Fabry disease.

Fabry disease is caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). We previously engineered a retrovirus encoding human alpha-gal A and demonstrated enzymatic correction of patient cells. Further, we demonstrated metabolic cooperativity, in that corrected cells secrete alpha-gal A that can be taken up and utilized by bystander cells in vitro. In the present study, we created a system to examine and quantitate this phenomenon in vivo. To differentiate from endogenous alpha-gal A, we constructed a retroviral vector (pUMFG/alpha-gal A/FLAG) containing a fusion form of alpha-gal A with a specific tag sequence at the carboxy terminus. The catalytic activity of the fusion protein was identical to wild-type alpha-gal A. The fusion protein was overexpressed in and secreted by transduced patient cells. In uptake studies, the fusion protein was detected in the lysosome-enriched fraction of recipient cells. We then examined the effectiveness of the pUMFG/alpha-g A/FLAG retroviral vector in vivo. Murine bone marrow (BM) cells were transduced and transplanted into irradiated hosts. After 9 weeks, proviral DNA was detected by PCR in peripheral blood and BM mononuclear cells. More importantly, specific fusion protein enzymatic activity could be demonstrated in those cells and in plasma. Thus, we have demonstrated that overexpressed alpha-gal A enters the circulation from transduced BM cells and is stable over a significant period of time.

Retrovirus-mediated correction of the metabolic defect in cultured Farber disease cells.

Farber disease is a rare severe lysosomal storage disorder due to a deficient activity of the enzyme acid ceramidase (AC). Patients have granulomas along with lipid-laden macrophages that accumulate in a number of tissues, leading to multiple diverse clinical symptoms. There is no therapy for the disorder and most patients succumb to the disease in early childhood. The severity of the disease progression seems to correlate with the amount of the accumulated ceramide substrate. Since the cDNA for human AC has been elucidated we sought to establish if genetic transfer of this sequence would lead to enzymatic and, especially, functional correction of the catabolic defect in Farber patient cells. To do this, a novel amphotropic recombinant retrovirus was constructed that engineers transfer of the human AC cDNA. On infection of patient fibroblasts, AC enzyme activity in cell extracts was completely restored. Further, substrate-loading assays of intact living cells showed a fully normalized catabolism of lysosomal ceramide. Lastly, as reported for some other corrected enzymatic defects of lysosomes, metabolic cooperativity was seen, in that functionally corrected patient fibroblasts secreted AC that was taken up through the mannose 6-phosphate receptor and used by uncorrected fibroblasts as well as recipient Farber lymphoblastoid cells. This overall transduction and uptake scenario for Farber disease allows future treatment of this severe disorder to be envisioned using gene transfer approaches.

Increasing stroke incidence in Sweden between 1989 and 2000 among persons aged 30 to 65 years: evidence from the Swedish Hospital Discharge Register.

BACKGROUND AND PURPOSE: Stroke mortality is decreasing in Sweden, as is the case in other Western European countries. However, both decreases and increases have been reported in Sweden for persons younger than age 65 years. The aim of this study was to compare the incidence of stroke in Sweden between the periods 1989 and 1991 and 1998 and 2000 in persons aged 30 to 65 years. METHODS: All first-ever stroke patients aged 30 to 65 years in the Swedish Hospital Discharge Register between 1989 and 2000 were included. RESULTS: The age-standardized, 3-year average incidence increased by 19%, from 98.9 to 118.0 per 100 000 among men, and by 33%, from 48.4 to 64.4 among women, between 1989 and 1991 and 1998 and 2000. The largest increase was seen among those younger than 60 years. On a county level, the change in age-standardized stroke incidence varied from small decreases (-3%) to large increases (82%). CONCLUSIONS: Stroke incidence increased in Sweden for both men and women between 1989 and 2000. The increase was larger among women. This calls for action when it comes to studying risk factors and planning for prevention and health promotion and indicates the need for gender-specific studies.