Long-term strategies for the treatment of Refsum's disease using therapeutic apheresis.

Refsum's disease is a rare autosomal recessive disorder of fatty acid metabolism. Poorly metabolized phytanic acid accumulates in fatty tissues, including myelin sheaths and internal organs, leading to retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia, and renal, cardiac or liver impairment. Dietary restriction of phytanic acid in some cases is not sufficient to prevent acute attacks and stabilize the progressive course. Phytanic acid bound to large low density lipoproteins (LDL) and very low density lipoproteins (VLDL) molecules offers the possibility of extracorporeal elimination by lipid apheresis. We report on the long-term lipid apheresis treatment of four patients with severe Refsum's disease. Retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia, anosmia, and sensorineural hearing loss were major symptoms exhibiting a progressive course. Lipid apheresis was performed for 5-13 years without severe complications. Maximum levels of phytanic acid before commencing chronic lipid apheresis were >300 mg/l. During steady state with lipid apheresis, mean phytanic acid before treatments was 87 mg/l and was reduced to 36 mg/l. Mean reduction rate was 59% per treatment. In all patients, abnormal motor nerve conduction velocity with signs of chronic denervation improved, morphological and functional stabilization of eye involvement was observed. Lipid apheresis prevented the extension of the disease to previously unaffected organs in three patients. Extracorporeal elimination of lipoprotein-phytanic acid complexes by lipid apheresis represents a pathophysiologically guided therapeutic approach, resulting in long-term improvement or stabilization of overall rehabilitation in patients with progressive Refsum's disease.

[Rheopheresis for recurrent sudden hearing loss : therapeutic options for patients refractory to infusion therapy]. / Rheopherese bei rezidivierendem Hörsturz : Therapieoption für Patienten nach erfolgloser Infusionstherapie.

BACKGROUND AND OBJECTIVE: For patients suffering from recurrent sudden hearing loss (SHL) that is refractory to infusion therapy, new therapeutic options must be established. PATIENTS AND METHODS: Patients suffering from recurrent and progressive SHL refractory to infusion therapy according to German guidelines were analysed retrospectively. After unsuccessful infusion therapy following the last onset of SHL, patients were treated with Rheopheresis twice. Hearing gain and recovery of speech discrimination were analysed. RESULTS: Twenty-five patients with a mean of 2.1+/-0.4 events of SHL within 30.0+/-21.6 months were examined. The patients' mean hearing loss before the first onset of SHL was 34 dB and was reduced by infusion therapy to 20 dB. With the second onset of SHL, hearing loss remained almost unchanged after infusion therapy. Patients showed a mean improvement of 20 dB after two consecutive Rheopheresis treatments. Forty percent showed complete remission of SHL, and a further 28% showed partial remission. CONCLUSION: Rheopheresis can efficiently improve the hearing of patients with recurrent SHL refractory to infusion therapy.

Functional comparison of homologous members of three groups of Kunitz-type enzyme inhibitors from potato tubers (Solanum tuberosum L.).

For functional studies, nine cDNAs encoding Kunitz-type enzyme inhibitors from potato tubers were expressed as GST (glutathione S transferase)-tagged fusion proteins in the fission yeast Schizosaccharomyces pombe. The inhibitors represented the three major homology groups A, B and C found in tubers. Members of the same homology group were at least 90% identical in sequence. The purified GST fusion proteins were tested for their ability to inhibit the proteases trypsin, alpha-chymotrypsin, subtilisin, papain and aspergillopepsin I, and for inhibition of the growth of fungi. Fusion proteins belonging to the same and different homology groups were found to exhibit distinct protease inhibition profiles. Removal of the GST tag by cleavage with enterokinase did not change the inhibition profile but increased the inhibitory activity. Group A and B inhibitors affected the proteases to different extents, whereas group C inhibitors showed only weak or no protease inhibition. One fusion protein completely inhibited aspergillopepsin I. One fusion protein each of groups A and B strongly inhibited mycelial growth of the fungus Fusarium moniliforme. The results suggest functional polymorphism among closely related members of the Kunitz-type inhibitor family.

Structural diversity and organization of three gene families for Kunitz-type enzyme inhibitors from potato tubers (Solanum tuberosum L.).

In the potato, Kunitz-type enzyme inhibitors are abundant and highly polymorphic small proteins found in tubers. DNA sequence analysis of 1596 unselected ESTs (expressed sequence tags) from mature tubers of the cultivars Provita and Saturna resulted in the identification of 55 different DNA sequences with high sequence similarity to Kunitz-type enzyme inhibitors. The frequency of Kunitz-type inhibitor ESTs in Provita was four times higher than in Saturna tubers, and none of the Provita ESTs was identical to any of the Saturna ESTs. A phenogram constructed from the deduced amino acid sequences of the inhibitors revealed three major homology groups-A, B and C. Group A inhibitors were all derived from Provita ESTs. Inhibitor groups A and B were more similar to each other than to group C inhibitors, and for most members within-group similarity was at least 90%. Non-conservative amino acid substitutions and insertion/deletion polymorphisms suggest functional differentiation between members of the gene family. A minimum of 21 genes for Kunitz-type enzyme inhibitors (six for group A, nine for group B and six for group C) was estimated to exist in the potato genome. Genetic mapping and the identification of BAC (bacterial artificial chromosome) clones containing more than one member of the gene family indicated that most inhibitor genes of groups A, B and C are organized in a cluster that maps to a single region on potato chromosome III.

Determination of BOD-values of starch-containing waste water by a BOD-biosensor.

The control of waste water plants is difficult or even impossible using the classical determination method for biological oxygen demand (BOD), because of its high time consumption of five days. A determination within some minutes is possible by microbial BOD-sensors. However, high molecular weight substances cannot be detected, a problem which can be overcome by the use of additional enzymes. For the application in a flow-through system to analyse starch containing waste water, alpha-amylase and amyloglucosidase were immobilized by adsorption to polystyrene or polypropylene carriers followed by crosslinking. Furthermore, covalent coupling to different nylon carriers, derivatives of chitin, silanized glass beads and silanized beads of foamed glass was tried. Chitin and Lewatit were the best suited carriers for the immobilization of alpha-amylase and amyloglucosidase. Two glass columns were filled with the immobilized enzymes and inserted into a commercial BOD-sensor containing the yeast Trichosporon cutaneum as biological component. The system was stable for more than two months under storage and one month under working conditions. A comparison of different starch types resulted in a hydrolysis of more than 80% in case of potato starch whereas grain starch was hydrolized only for 40-50%. Sensor-determined BOD-values of waste water with potato starch were nearly identical with BOD5-values resulting from the classical method.