Faculty Bibliography

Human bone marrow contains mesenchymal stem cells (MSCs) that can differentiate into various cells of mesenchymal origin. We developed an efficient method of isolating and culture expanding a homogenous population of MSCs from bone marrow and determined that MSCs express alpha-L-iduronidase, arylsulfatase-A and B, glucocerebrosidase, and adrenoleukodystrophy protein. These findings raised the possibility that MSCs may be useful in the treatment of storage disorders. To determine if donor derived MSCs are transferred to the recipients with lysosomal or peroxisomal storage diseases by allogeneic hematopoietic stem cell (HSC) transplantation, we investigated bone marrow derived MSCs of 13 patients 1-14 years after allogeneic transplantation. Highly purified MSCs were genotyped either by fluorescence in situ hybridization using probes for X and Y-chromosomes in gender mis-matched recipients or by radiolabeled PCR amplification of polymorphic simple sequence repeats. Phenotype was determined by the measurement of disease specific protein/enzyme activity in purified MSCs. We found that MSCs isolated from recipients of allogeneic HSC transplantation are not of donor genotype and have persistent phenotypic defects despite successful donor type hematopoietic engraftment. Whether culture expanded normal MSCs can be successfully transplanted into patients with storage diseases and provide therapeutic benefit needs to be determined

In 225 adults aged 18 to 80 years, normative warm and cold perception thresholds were assessed at the volar distal forearm, thenar eminence, lower medial calf, and lateral dorsal foot using the method of limits and a Thermotest (Somedic, Stockholm, Sweden). A 1.5-cm x 2.5-cm thermode, a 1 degrees C/s stimulus change rate, and a 32 degrees C baseline temperature were applied. Thresholds of five consecutive stimuli were averaged. At the thenar eminence a 3 degrees C/s stimulation was applied in addition to the 1 degree C/s stimulation. Effects of spatial summation were studied at the calf and forearm by additional testing with a 2.5-cm x 5.0-cm thermode. To evaluate the influence of skin temperature, thresholds were correlated with the pretest skin temperature at the tested sites. Reproducibility of stimulus perception was determined by comparing the lowest to the highest response to five consecutive stimuli. Results showed sufficient accuracy of thermal perception thresholds. Thresholds were higher with the 3 degrees C/s stimulation than with the 1 degree C/s stimulation. Thresholds were lower with the large than with the small probe. Skin temperature had only minimal influence on thresholds. The use of a 32 degrees C baseline temperature and a 1 degree C/s stimulus change rate is recommended. The large probe should be used at body sites where the entire thermode surface adjusts planely to the skin. Warming up the tested skin area is not necessary before thermotesting

An adenovirus, AdCDTK, expressing both bacterial cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSVTK) was constructed and introduced into glioma cells. AdCDTK selectively rendered glioma cells sensitive to both 5-fluorocytosine (5-FCyt) and ganciclovir (GCV) (termed AdCDTK/5-FCyt-GCV). AdCDTK/5-FCyt-GCV not only potently mediated apoptosis and the arrest of glioma cell growth in vitro, but also significantly increased the survival time of glioma-bearing rats as compared with controls. The 90-day survival time was observed in 50% of rats. Interferon-alpha (IFN-alpha) further enhanced the tumor cell killing of AdCDTK/5-FCyt-GCV. In the group of AdCDTK/5-FCyt-GCV/IFN-alpha, the average survival time was significantly increased, and the average tumor size was smaller than that in the group of AdCDTK/5-FCyt-GCV. Ninety-day survival increased from 50% in the group of AdCDTK/5-FCyt-GCV to 75% in the group of AdCDTK/5-FCyt-GCV/IFN-alpha. Complete tumor regression was observed in 50% of rats in the group of AdCDTK/5-FCyt-GCV/IFN-alpha. The data indicate that AdCDTK/5-FCyt-GCV induces glioma cell killing greater than that induced by either CD/5-FCyt or HSVTK/GCV alone. IFN-alpha synergistically enhances this effect by increasing apoptosis

To determine whether sympathetic skin response (SSR) testing evaluates afferent small or efferent sympathetic nerve fiber dysfunction, we studied SSR in patients with familial dysautonomia (FD) in whom both afferent small and efferent sympathetic fibers are largely reduced. We analyzed whether the response pattern to a combination of stimuli specific for large or small fiber activation allows differentiation between afferent and efferent small fiber dysfunction. In 52 volunteers and 13 FD patients, SSR was studied at palms and soles after warm, cold and heat as well as electrical, acoustic, and inspiratory gasp stimulation. In addition, thermal thresholds were assessed at four body sites using a Thermotest device (Somedic; Stockholm, Sweden). In volunteers, any stimulus induced reproducible SSRs. Only cold failed to evoke SSR in two volunteers. In all FD patients, electrical SSR was present, but amplitudes were reduced. Five patients had no acoustic SSR, four had no inspiratory SSR. Thermal SSR was absent in 10 patients with abnormal thermal perception and present in one patient with preserved thermal sensation. In two patients, thermal SSR was present only when skin areas with preserved temperature perception were stimulated. In patients with FD, preserved electrical SSR demonstrated the overall integrity of the SSR reflex but amplitude reduction suggested impaired sudomotor activation. SSR responses were dependent on the perception of the stimulus. In the presence of preserved electrical SSR, absent thermal SSR reflects afferent small fiber dysfunction. A combination of SSR stimulus types allows differentiation between afferent small or efferent sympathetic nerve fiber dysfunction

Mucolipidosis IV (ML IV) is an inherited lysosomal disorder for which the primary biochemical defect has not been identified. In order to detect any defect in glycosphingolipid metabolism, we have examined the metabolism of sphingosine-labeled (3H)G(M2) in situ in fibroblasts from patients diagnosed with ML IV. Fibroblasts were exposed for 10 days in medium containing (3H)G(M2) (15 uM; Sp. Act. 35000 cpm/nmole), washed, harvested and analyzed for radioactivity in extracted lipids. Control cells metabolized about half of the internalized ganglioside, mostly to ceramide. In ML IV fibroblasts, 70-80% of the cellular radioactivity was present as G(M2) indicating reduced degradation. This is not as severe as in G(M2) gangliosidosis as a small amount of G(M2) was metabolized in ML IV cells to ceramide. Since there is no defect in the lysosomal enzyme profile in these cells, it is possible that an abnormality in the translocation of membrane constituents to the lysosomes may explain the slower ganglioside metabolism