T-cell function, T-cell phenotype and its role in responsiveness to recombinant human erythropoietin in hemodialysis patients.

Resistance to recombinant human erythropoietin (Epo) occurs in a small proportion of hemodialysis (HD) patients. In this study we investigated the relationship between T-cell phenotype (using flow cytometry), T-cell function (by measuring in vitro cytokine production) and responsiveness to Epo in HD patients and to compare the results with those from healthy controls. T-cell phenotypes were assessed and T-cell function was studied. The study included 24 chronic renal failure (CRF) patients on HD treated with rHuEPO as well as 14 normal control subjects. Dual-colour immunofluorescence and flow cytometry were used to compare the surface antigen expression on freshly isolated CD4+ and CD8+ T-cells from PBMC of the studied groups. Levels of a panel of selected cytokines (IL-4, IFN-gamma, slL-2R and IL-10) were determined in PBMC culture supernatants and in plasma samples (TNF-alpha, IFN-gamma, IL-6, slL-2R) using (ELISA) kits. Patients were followed-up for 24 months and a survival study was carried out. T-cells from poor responders showed increased proportions of CD4+/CD28- cells and CD8+/CD28- cells compared with both good responders and controls. Compared with their CD28+ counterparts, CD4+/CD28- T-cells produced significantly more IFN-gamma, enabling them to function as pro-inflammatory cells. There was no difference in secretion of IFN-gamma, slL-2R or IL-4 in PBMC cultures obtained from HD patients and controls. However, Unstimulated PBMC from poor responders generated increased levels of IL-10 poor compared with both good responders and controls. Plasma slL-2R and IL-6 were significantly elevated in both good and poor responders compared with controls. Plasma levels of IFN-gamma and TNF-alpha were undetectable in both HD patients and controls. In the follow up period, more deaths were occurring among the poor responders than the good responders. Based on the finding of the this study we may suggest that, in the absence of any obvious cause, poor response to Epo may be mediated by generation of cytokines from a subpopulation of activated T-cells, which might promote apoptosis in erythroid progenitor cells in the bone marrow.

Characterization of the D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate.

The murine gene for the glucuronyl C5-epimerase involved in heparan sulfate biosynthesis was cloned, using a previously isolated bovine lung cDNA fragment (Li, J.-P., Hagner-McWhirter, A., Kjellén, L., Palgi, J., Jalkanen, M., and Lindahl, U. (1997) J. Biol. Chem. 272, 28158-28163) as probe. The approximately 11-kilobase pair mouse gene contains 3 exons from the first ATG to stop codon and is localized to chromosome 9. Southern analysis of the genomic DNA and chromosome mapping suggested the occurrence of a single epimerase gene. Based on the genomic sequence, a mouse liver cDNA was isolated that encodes a 618-amino acid residue protein, thus extending by 174 N-terminal residues the sequence deduced from the (incomplete) bovine cDNA. Comparison of murine, bovine, and human epimerase cDNA structures indicated 96-99% identity at the amino acid level. A cDNA identical to the mouse liver species was demonstrated in mouse mast cells committed to heparin biosynthesis. These findings suggest that the iduronic acid residues in heparin and heparan sulfate, despite different structural contexts, are generated by the same C5-epimerase enzyme. The catalytic activity of the recombinant full-length mouse liver epimerase, expressed in insect cells, was found to be >2 orders of magnitude higher than that of the previously cloned, smaller bovine recombinant protein. The approximately 52-kDa, similarly highly active, enzyme originally purified from bovine liver (Campbell, P., Hannesson, H. H., Sandbäck, D., Rodén, L., Lindahl, U., and Li, J.-P. (1994) J. Biol. Chem. 269, 26953-26958) was found to be associated with an approximately 22-kDa peptide generated by a single proteolytic cleavage of the full-sized protein.

Production and characterization of the extracellular domain of recombinant human fibroblast growth factor receptor 4.

Among the members of the fibroblast growth factor receptor family the FGFR4 has demonstrated strong dependence on heparin-like material for its activation by fibroblast growth factors. We have produced and characterized a recombinant human FGFR4 extracellular domain (FGFR4ed), in order to study its biochemical properties in isolated conditions. The FGFR4ed was expressed in an insect cell system and purified from the culture medium by Ni(2+)-affinity and gel filtration chromatography. Pure FGFR4ed was tested for FGF- and heparin-binding by covalent crosslinking experiments and by biosensor analysis. In solution, FGFR4ed formed complexes with acidic FGF (FGF-1) and basic FGF (FGF-2), both in the presence and absence of heparin. Immobilized FGFR4 also bound FGF-8 besides FGF-1 and FGF-2. Furthermore, heparin alone induced receptor oligomerization on the surface of the receptor coupled chip. Thus, the recombinant FGFR4ed revealed properties described for the cellular form of this receptor and can be used for interaction studies.

Extensive small bowel varices as a cause of severe anemia in Klippel-Trenaunay-Weber syndrome.

Klippel-Trenaunay-Weber (KTW) syndrome is a rare syndrome characterized by hemangiomata, varicose veins, and both bony and soft tissue hemihypertrophy. Abdominal viscera affected by ipsilateral hemangiomata include colon, liver, spleen, jejunum, kidney, and liver. We report a case of this syndrome that presented with severe anemia and extensive jeujenal varices without any other significant digestive tract lesions.

Engineering proteins, subcloning and hyperexpressing oxidoreductase genes.

A very efficient system for subcloning and studying protein sequences, combining previously established elements for hyperexpression, replication and screening, was used to hyperproduce and characterize seven different products. It expedited the cloning of genes, in a multipurpose recombinant DNA construct, for all the requirements to study and engineer proteins with a strain of Escherichia coli. Genes encoding six heme proteins and a flavoprotein have been subcloned and expressed to 13-30% of the total cell protein, greatly facilitating purification and analyses. Three of the heme proteins and the flavoprotein incorporated prosthetic groups in E. coli, and exhibited the expected activities. Four of the enzymes have been purified to homogeneity and two of these crystallized for X-ray diffraction analysis. A rapid mutagenesis protocol, based on polymerase chain reactions, was successfully applied to clone derivatives of one of these enzymes, cytochrome c peroxidase. Thus, this system fulfills all criteria for engineering proteins in an efficient and concerted manner.

Characterization of recombinant Bacillus megaterium cytochrome P-450 BM-3 and its two functional domains.

Bacillus megaterium cytochrome P-450BM-3 and its two functional domains, the heme and flavin domains, have been purified and characterized using an Escherichia coli expression system. Recombinant P-450BM-3 behaves both spectrally and enzymatically the same as the enzyme produced from the natural host, B. megaterium, and another E. coli system recently described (Bouddupalli, S. S., Estabrook, R. W., and Peterson, J. A. (1990) J. Biol. Chem. 265, 4233-4239). Reduction of the flavins in P-450BM-3 domain with NADPH appears to be very similar to microsomal P-450 reductases where two reducing equivalents are consumed to fully reduce the FMN while the FAD is converted to the semiquinone in an one electron reduction. NADPH reduction of the heme occurs only in the presence of substrate suggesting, by analogy with the cytochrome P-450CAM system, a possible increase in iron redox potential of the heme upon substrate binding which facilitates electron transfer from the flavins to the heme. The flavin domain retains a high level of cytochrome c reductase activity and also reacts with NADPH to give a 3-electron reduced product. The heme domain retains the ability to bind substrate and generates the characteristic 450-nm absorption band upon reduction in the presence of CO. The heme domain has been crystallized and a preliminary set of x-ray diffraction data obtained.

Novel regulation of heat shock genes during carrot somatic embryo development.

We have determined that somatic embryos of carrot exhibit a number of interesting and unusual properties when exposed to heat shock at different times in their development. Specifically, we have seen that mid-globular embryos can be arrested irreversibly in their development when heat-shocked, whereas all other stages of embryogenesis, both before and after this stage, are fully capable of normal development after the stress. In investigating the molecular basis of this developmental sensitivity to heat shock, using a cloned heat shock gene encoding a small heat shock protein, we have determined that globular embryos both synthesize and accumulate significantly less heat shock mRNA when compared with embryos of any other stage or to callus suspension cells. In fact, there appears to be no transcriptional induction of heat shock gene expression in response to heat shock during this time period; the gene is expressed at the same relatively low level both before and after heat shock. However, in spite of the low level of heat shock mRNA available, globular embryos synthesize the full complement of heat shock proteins in response to heat treatment. The globular embryos appear to accomplish this by translating the existing heat shock mRNAs at an elevated rate, which compensates for the low level of available mRNA. Once the embryos have progressed beyond the globular stage of development, regulation at the transcriptional level resumes, and the embryos again exhibit normal development after heat shock.