In vitro study of encapsulation therapy for Fabry disease using genetically engineered CHO cell line.

Fabry disease is an X-linked recessive disorder caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A (alpha-gal). The deficiency of this enzyme leads to the systemic deposition of ceramide trihexoside (CTH) in various tissues and organs. Enzyme replacement using IV doses of recombinant human alpha-gal produced in CHO cells or in human fibroblasts is currently being evaluated in clinical trials as a potential therapy for this disease. However, it requires lifelong therapy involving a large amount of purified alpha-gal. As a novel approach for treatment of Fabry disease we used polymer encapsulated Chinese hamster ovary (CHO) cells genetically modified to express alpha-gal. The secreted high levels of alpha-gal passed through the semipermeable polymeric membrane. Using coculture system with Fabry fibroblasts, the secreted enzyme was taken up in cells, resulting in reduced accumulation of CTH in Fabry fibroblasts. This in vitro study demonstrated that an encapsulated alpha-gal-secreting cell line can be used to treat Fabry mice by transplantation in vivo. Judging from the protection against immune rejection by a semipermeable synthetic membrane, this novel approach may be applied to treat patients with Fabry disease and other lysosomal storage diseases.

A novel approach to ex vivo gene therapy for familial hypercholesterolemia using human amniotic epithelial cells as a transgene carrier.

This study has demonstrated the potential of human amniotic epithelial cells (HAEC) as a transgene carrier to treat patients with familial hypercholesterolemia (FH). One approach to liver-directed gene therapy is represented by transplantation of autologous hepatocytes that have been genetically modified in vitro. However, the hepatocytes must be isolated from surgically resected tissue and it is difficult to expand the hepatocytes in culture. In contrast, the advantages for using HAEC are the higher availability and the nonimmunogenicity after allotransplantation. Our strategy involved isolating HAEC from an amnion, transducing a human low-density lipoprotein receptor (LDLR) gene into these cells with a recombinant adenovirus, and transplanting the genetically modified cells into the liver of an animal model of FH. Each animal, treated with the LDLR-transduced HAEC, exhibited a substantial decrease in serum cholesterol with an eventual return to pretreatment level. Moreover, the transplanted HAEC migrated out of the sinusoids into the hepatic parenchyma and expressed the LDLRs until at least 20 days after transplantation. However, the transplanted HAEC markedly decreased in number after 10 days post-transplant with an increase of inflammatory cells. The temporary nature of the metabolic improvement may be associated with xenograft rejection and transient function of the adenoviral vector.

Enzymatic corrections for cells derived from Fabry disease patients by a recombinant adenovirus vector.

Fabry disease is an X-linked inherited metabolic disorder caused by a deficiency of alpha-galactosidase (alpha-gal), resulting in the accumulation of ceramide trihexoside (CTH) in body fluids and in many organs and tissues. We constructed a recombinant adenovirus with a human alpha-gal cDNA (AxCAG alpha-gal), and transfected this vector to skin fibroblasts from Fabry patients. Transfected cells expressed high amounts of alpha-gal in their cytoplasm, and a high level of alpha-gal activity was detected in the medium. The accumulated CTH in the fibroblasts disappeared 3 days after infection. The secreted alpha-gal also eliminated the accumulated CTH from uninfected patient's cells. The enzyme may be taken up through mannose-6-phosphate receptors, as the addition of mannose-6-phosphate to the medium completely inhibited the uptake of the enzyme. The infected cells continued to express alpha-gal for more than 10 days. These results suggest that AxCAG alpha-gal could be used as enzyme replacement gene therapy for Fabry disease.

Gene expression of oligodendrocyte markers in human amniotic epithelial cells using neural cell-type-specific expression system.

We have previously reported that human amniotic epithelial (HAE) cells expressed neuronal and glial cell markers using immunostaining and western blotting. To study the expression system of these cell markers in HAE cells, we investigated the expression of mRNA for oligodendrocyte markers in HAE cells by reverse-transcriptase-polymerase chain reaction (RT-PCR) and northern blotting. Neural cell-specific expression system was used to examine the transcriptional activity of myelin basic protein (MBP). Oligodendrocyte markers were expressed such as CNPase, MBP and proteolipid protein (PLP and DM-20). PLP gene transcripts in the cells were in a lower level than DM-20, compared with those of human brain. Neural cell-type-specific expression system disclosed HAE cells were about 20% positive for beta-Gal using AdexMBP-NL-LacZ. This strongly indicates that HAE cells have MBP-specific gene expressing cells.

Hox gene expression, AV-1 antigen expression, and cartilage pattern formation in chick recombinant limb buds.

A recombinant limb bud composed of dissociated and reaggregated mesenchyme and an ectodermal jacket develops a limblike structure with bifurcated and segmented cartilage. We compared the cartilage structure formed from recombinants that were composed of mesenchymal cells derived from the limb bud progress zone at various stages. In the case of recombinants containing distal mesenchyme of early-stage limb buds (stage 18 or 20), long and thick cartilage structures were formed in the proximal region, and segmented digitlike structures were formed in the distal region. On the contrary, in the case of recombinants containing distal mesenchyme of late-stage limb buds (stage 25 or 27), only poorly developed cartilage structures were formed. Next, we analyzed expression patterns of the position-specific genes HoxA11, A13 and D12 and the position-specific antigen AV-1 protein in recombinants containing distal mesenchyme of stage 20 limb buds (stage 20 recombinants) or stage 25 limb buds (stage 25 recombinants). In stage 20 recombinants, HoxA11 was expressed throughout the mesenchyme, but HoxA13 was expressed only in the distal half of the mesenchyme. In stage 25 recombinants, HoxA13 was expressed throughout the mesenchyme, but HoxA11 was only faintly expressed. The expression pattern of HoxD12 was similar to that of HoxA13 in both stage 20 and stage 25 recombinants, and no asymmetric expression pattern, which is observed in normal limb buds, was detected. AV-1 antigen was expressed in the core region of stage 20 recombinants, and anteroposterior asymmetry, which is observed in the anterior-ventral-distal region of normal limb buds, was not found. No AV-1 expression was observed in stage 25 recombinants. These results suggest that the mesenchyme in recombinants shows spatially controlled gene/protein expressions along the proximodistal axis, and that these differences in gene/protein expressions may affect cartilage pattern formation in recombinants.

Cell cycle length affects gene expression and pattern formation in limbs.

The relationship between growth and pattern specification during development remains elusive. Some molecules known to function as growth factors are also potent agents of pattern formation. This raises the possibility that growth factors could act in pattern formation via an effect on the cell cycle. We have tested the significance of the length of the cell cycle for gene expression and pattern formation in developing chick limb buds by locally slowing the cell cycle. When anterior cell cycles are lengthened by reversible inhibition of DNA replication or by other means, some genes characteristic of the posterior polarizing region are expressed, and digit duplication is observed. Conversely, when posterior cell cycles are slowed, expression of some posterior-specific genes is inhibited, but the pattern is normal. These results indicate that control of the length of the cell cycle could play a primary role in pattern formation by influencing the complement of genes expressed in a particular region of the embryo.

Evidence for active acetylcholine metabolism in human amniotic epithelial cells: applicable to intracerebral allografting for neurologic disease.

Human amniotic epithelial (HAE) cells have been used for allotransplantation in patients with lysosomal storage disease due to lack of expression of HLA antigens. Previously, we have reported the expression of differentiation markers for both neural stem cells, and neuron and glial cells. In the present study, we investigated the presence of choline acetyltransferase (ChAT) and acetylcholine (ACh) in HAE cells using different experimental approaches. Cultured HAE cells showed strong immunoreactivity against ChAT antibody. ChAT activity in primary cells was 24.9 +/- 8.5 pmol/mg protein/h. Using HPLC with electrochemical detection, ACh was detected in both cell incubation media and cell pellets indicating that these cells synthesize and release ACh in a time-dependent manner. Additional confirmation of this hypothesis was gained from the data obtained from RT-PCR and Western blot analyses which revealed the expression of ChAT mRNA and ChAT protein, respectively, in HAE cells. Results of the present study suggest that HAE cells can possibly be applied for intracerebral allografting to treat neurologic diseases in which cholinergic neurons are damaged.

Microdissection-mediated selection of chromosome region-specific cDNAs.

K562 is a cell line with two acrocentric marker chromosomes containing abnormally banded regions (ABRs), derived from a Ph-positive chronic myelogenous leukemia (CML) patient. Using reverse and forward chromosome painting FISH analysis, we found that 9q34, 13q31, and 22q11 regions co-amplified in the ABRs-bearing acrocentric marker chromosomes of K562. Utilizing the ABRs of the cell line as target DNA for cDNA selection, we established a simple procedure for chromosome region-specific cDNA isolation. After first strand cDNA synthesis from fetal brain mRNAs, short fragment cDNAs (sf-cDNAs) were synthesized with a two-step amplification system by use of our modified Degenerate Oligonucleotide Primed Shuttle Polymerase Chain Reaction (DOP-Shuttle-PCR) method. The sf-cDNAs were hybridized onto RNase A treated metaphases from K562, and the ABRs were microdissected and reamplified with DOP-Shuttle-PCR primer-II. The reamplified sf-cDNAs were cloned into a pBluescript vector. Twenty randomly chosen clones were sequenced and classified into 8 groups. Three out of the 8 grouped clones had been mapped to the long arm of chromosome 22 (22q11), whereas the other 5 were novel cDNAs. Quantitative Southern blot analysis indicated that 7 out of the 8 grouped clones (87.5%) were derived from the co-amplified regions.

Oxidative stress triggers tyrosine phosphorylation in B cells through a redox- and inflammatory cytokine-sensitive mechanism.

Exposure to oxidants such as hydrogen peroxide (H2O2) and gamma-ray irradiation has been recently shown to trigger tyrosine phosphorylation in B cells as does cross-linking surface immunoglobulin (sIg) by antigens or anti-immunoglobulins. We studied the mechanism by which H2O2 induced tyrosine phosphorylation in B cells and compared it with the mechanism utilized by sIg. Both anti-immunoglobulin M (anti-IgM) and H2O2 induced tyrosine phosphorylation through protein tyrosine kinase (PTK) activation. However, the tyrosine phosphorylation caused by H2O2 but not that induced by anti-IgM, was modulated by agents affecting cellular thiols and glutathione contents including dithiothreitol, 2-mercaptoethanol, and buthionine sulphoximine. Moreover, the tyrosine phosphorylation caused by the oxidant but not that induced by anti-IgM was markedly augmented by two inflammatory cytokines, tumour necrosis factor-alpha and interleukin-1 alpha, although these agents by themselves did not stimulate PTK activity nor induce tyrosine phosphorylation. These findings demonstrate that oxidative stress but not surface IgM (sIgM) ligation triggers tyrosine phosphorylation through a mechanism that is sensitive to cellular thiols and these inflammatory cytokines.

Purification of AV-1, a position-specific molecule, and the effects of its antiserum on chick limb pattern formation.

AV-1 protein is a membrane-localized glycoprotein which is expressed in a stage- and position-specific manner during limb development and is a candidate pattern formation molecule. In this study, we have purified the AV-1 protein using an affinity column coupled with the anti-AV-1 monoclonal antibody. Using the purified protein, we produced a rat anti-AV-1 antiserum. Characterization of the antiserum by immunoblotting and immunostaining showed highly specific reactivity which was identical to that of the anti-AV-1 monoclonal antibody. The rat anti-AV-1 antiserum was injected into the vitelline vein of stage 20 chick embryos and these embryos were allowed to develop further to examine the effect of the antibody on limb pattern. The injected embryos developed deformed limbs. Many of the abnormalities in limb cartilage pattern were related to the normal pattern of the AV-1 expression, suggesting an involvement of the AV-1 protein in the process of limb pattern formation.

Digit induction by Hensen's node and notochord involves the expression of shh but not RAR-beta 2.

It is well established that Hensen's nodes can induce the formation of supernumerary digits after grafting into the anterior margin of the developing limb bud. The recent finding that distinct mesodermal cell populations are segregated within the node has made it possible to isolate different prospective cell types in an attempt to correlate digit-inducing ability with cell fate. We find that the prospective notochord cells contained within Hensen's node are able to induce supernumerary digits, whereas presumptive somite cells cannot. This early difference in inducing ability persists into later stages of development: epithelial somites are unable to induce while notochord from all lengths of the neuraxis continues to induce. Using probes to retinoic acid receptor-beta 2 and sonic hedgehog (shh) we find no evidence to support the idea that inducing tissues generate extra digits by releasing retinoic acid into adjacent limb tissue but find that the inducing ability of a tissue correlates with its expression of shh.

MRC-5 cells induce the AER prior to the duplicated pattern formation in chick limb bud.

We have previously shown that MRC-5 cells induce the duplication of the chick limb bud following the implantation into the anterior limb bud only during pre-limb-bud stages. We now report the process of duplicated pattern formation caused by MRC-5 cells. The duplicated patterns are also formed following the implantation into the center of the limb bud and an excess apical ectodermal ridge (AER) with Msx2 expression is induced prior to these duplicated pattern formulations. Only after the implantation into the anterior leg bud, the shh gene is expressed additionally in the anterior leg bud and the mirror-symmetric duplication along the anteroposterior (A-P) axis is formed. The map of the polarizing activity in stage 21 embryo suggests that the high polarizing activity of the normal flank region is responsible for the changes in the A-P polarity when MRC-5 cells are grafted into the anterior leg bud. These results indicate that MRC-5 cells induce the AER and that the excess AER produces the duplicated cartilage pattern of the limb bud.

Formation of distal structures from stumps of chick wing buds at stages 24-25 following the grafting of quail tissue from X-irradiated distal limb buds.

To examine the regulatory activity of the proximal region of chick limb buds, distal halves of chick wing buds at stages 24-25 were removed, and the distal tips of X-irradiated stage 21 quail limb buds with an apical ectodermal ridge (AER) were grafted onto the chick stumps. The host stumps formed only humerus after removal of the donor distal tips or with tip mesoderm alone. However, when distal tips with an AER were grafted onto the stumps, one or two cartilage elements of host origin were formed at the distal end of the humerus. To examine whether the stump cells have changed to cells of a more distal, progress zone (PZ) cells, the chick stumps with quail tips were immunostained with antibody against AV-1, which reacts with the anterior PZ region of chick limb buds and is specific for an antigen that is expressed under the control of the AER. Within two days of grafting, cells positive for the antigen reappeared in the stump tissues. These results suggest that the some of the stump cells may be converted to PZ cells and their positional values may change to those of more distal structures under the influence of the AER.

Expression of AV-1, a position-specific molecule, in response to retinoic acid beads and ZPA grafts in chick limb development.

Local application of retinoic acid (RA) to the anterior-distal margin of chick limb buds causes digit pattern duplications identical to those that result from zone of polarizing activity (ZPA) grafts. The AV-1 antigen is a cell membrane glycoprotein which is expressed during chick limb bud development in a stage- and position-specific manner that correlates with cartilage pattern formation. In order to further understand the limb patterning mechanisms affected by RA and by ZPA grafts, we first examined the effects of RA treatment on AV-1 expression. We then compared the effect of RA with those of ZPA grafts. When beads soaked in varying concentrations of RA were applied to the anterior-distal margin of wing buds, changes in the pattern of AV-1 antigen expression were observed. AV-1 expression appears to correlate with regions that will give rise to a digit 3. These results suggest that the AV-1 protein is one of the molecules which may be involved in the process of cartilage pattern formation during limb development. ZPA grafts produced similar alterations in AV-1 expression, but the initial changes were observed earlier with ZPA grafts than with RA beads. These results support the idea that RA changes anterior limb bud cells into ZPA cells.

EBV increases phosphoinositide kinase activities in human B cells.

To determine whether EBV affects phosphoinositide kinase activities of human B cells, we compared the activities between EBV- and EBV+ human B cell lymphoma lines. The two types of human B cells contained both phosphatidylinositol (PtdIns) 4-kinase and phosphatidylinositol 4-phosphate (PtdIns(4)P) kinase activities irrespective of the presence of EBV. However, both activities were increased in EBV+ cells compared to EBV- cells. The increases were associated with neither altered Km values for substrates nor altered elution profiles in DEAE-cellulose chromatography. Furthermore, expression of a latent EBV protein, EBV nuclear Ag1 (EBNA1) in BHK cells by the transfection of EBNA1 DNA was accompanied by increased PtdIns 4-kinase and PtdIns(4)P kinase activities. These increases also were not associated with altered Km values for substrates. However, phospholipase C activity was altered in neither EBV+ cells nor in EBNA1-expressing cells. These results indicate that EBV selectively increases the two phosphoinositide kinase activities in human B cells, although the viral gene product has no intrinsic phosphoinositide kinase activity. PtdIns 4-kinase and PtdIns(4)P kinase cooperatively synthesize PtdIns 4,5-bisphosphate, the major source of 1,2-diacylglycerol and inositol 1,4,5-triphosphate, the two second messengers in transducing signals for cell activation. Such increase therefore may play a role in EBV-induced human B cell activation.

Chicken homeobox gene Msx-1: structure, expression in limb buds and effect of retinoic acid.

A chicken gene carrying a homeobox highly homologous to the Drosophila muscle segment homeobox (msh) gene was isolated and designated as Msx-1. Conceptual translation from the longest ORF gave a protein of 259 amino acids lacking the conserved hexapeptide. Northern analysis detected a single 2.6 kb transcript. As early as day 2 of incubation, the transcript was detected but was not found in adult tissue. In situ hybridization analysis revealed that Msx-1 expression is closely related to a particular mesenchymal cell lineage during limb bud formation. In early stage embryos, Msx-1 was expressed in the somatopleure. When primordial mesenchyme cells for limb bud were generated from the Wolffian ridge of the somatopleure, Msx-1 expression began to diminish in the posterior half of the limb bud then in the presumptive cartilage-forming mesenchyme. In developing limb buds, remarkable expression was seen in the apical ectodermal ridge (AER), which is responsible for the sustained outgrowth and development of the limb. The Msx-1 transcripts were found in the limb mesenchymal cells in the region covering the necrotic zone and ectodermal cells overlying such mesenchymal cells. Both ectodermal and mesenchymal expression in limb bud were rapidly suppressed by local treatment of retinoic acid which can generate mirror-image duplication of digits. This indicates that retinoic acid alters the marginal presumptive non-cartilage forming mesenchyme cell lineage through suppression of Msx-1 expression.

Retinoic acid induces polarizing activity but is unlikely to be a morphogen in the chick limb bud.

Retinoic acid is a putative morphogen in limb formation in the chick and other vertebrates. In chick limb formation, it is thought that retinoic acid is released from the zone of polarizing activity (ZPA) and the concentration gradient of retinoic acid formed from the posterior to the anterior provides positional cues for digit formation. Implantation of a bead containing retinoic acid at the anterior margin of the limb bud induces a mirror-image symmetrical duplication of the digit pattern similar to that observed when the ZPA is grafted into the anterior margin of the host limb bud. Also, the level of endogenous retinoic acid (25 nM on average) is higher in the posterior one third of the limb bud. We found that when the bead containing either retinoic acid or an analogue but not the ZPA, was implanted in the anterior margin of the chick limb bud, expression of the retinoic acid receptor type-beta gene was induced around the bead within 4 h. These results indicate that exogenous retinoic acid is not identical with the ZPA morphogen. As the anterior tissue exposed to retinoic acid has polarizing activity, we conclude that the primary function of exogenous retinoic acid is to induce polarizing activity in the limb bud.

Synthetic retinoids, retinobenzoic acids, Am80, Am580 and Ch55 regulate morphogenesis in chick limb bud.

The retinobenzoic acids Am80, Am580 and Ch55 are synthetic stable analogs of retinoic acid (RA), and show very strong differentiation-inducing activity in human myelogeneous leukemia cell line HL-60. To examine the effects of these synthetic retinoids on limb pattern formation, AG1-X2 beads containing these retinoids were applied to the anterior margin of stage 19-20 chick wing buds. By implanting the beads with 1 microgram/ml retinoids, normal wings were formed and extra digits 2 or 32 were rarely formed. As the retinoid concentrations increased from 10 micrograms/ml to 100 micrograms/ml duplicated limbs 3234, 43234, 432234, 4334 were progressively produced. At higher concentrations, 1 mg/ml, the wings often truncated, although duplication occurred in some embryos. These synthetic analogs seem to have the same degree of morphogenetic potential as RA, since the activity index of these retinoids was similar to that of RA. Since these synthetic retinoids hardly bind to CRABP (cellular retinoic acid-binding protein), it may be possible that the retinoids and RA may affect limb-pattern formation without the interaction with CRABP. It is known that limb buds cannot develop distal structures when the posterior region including all ZPA (zone of polarizing activity) is removed. When beads containing the above mentioned retinoids were implanted to the anterior margin of wing buds from which the posterior one third region including all ZPA had been removed, distal growth of the wing buds and the formation of digit elements were observed. Some of the wing buds produced a completely reverse digit pattern 432. From these results, we discussed the roles of RA in limb development and pattern formation.

Distribution of retinoids in the chick limb bud: analysis with monoclonal antibody.

Retinoic acid induces anteroposterior duplicate formation in developing chick limb bud, and it may be a natural morphogen involved in limb pattern formation. Retinoic acid is produced from retinol locally in the limb bud via retinal, and thus, to elucidate the distribution of these retinoids in the limb bud seems to be important for the understanding of the morphogen formation. We produced a monoclonal antibody against the retinoids with BSA-RA (bovine serum albumin-retinoic acid) conjugate for antigen, and investigated the distribution of retinoids in the chick limb bud. The antibody predominantly bound to retinoic acid, but weakly to retinol and retinal. Retinoids appeared in the limb bud at stage 18 and were distributed through stages 20-24, when the pattern formation in distal mesoderm was in progress. Initially they were found evenly in the whole mesoderm, but disappeared gradually from core mesoderm and remained only in the region of peripheral mesoderm at stage 24. At stage 26, retinoids were detected only in ectoderm. These results support the idea that the retinoids actually play roles in limb pattern formation and suggest that the retinoids in the peripheral mesoderm are important for pattern formation. Further, the role of retinoids in epidermis development at later limb bud stages is also suggested.

Magnesium concentration in brains from multiple sclerosis patients.

Magnesium (Mg) concentrations were studied in the brains of 4 patients with definite multiple sclerosis (MS) and 5 controls. The magnesium contents were determined by inductively coupled plasma emission spectrometry in autopsy samples taken from 26 sites of central nervous system tissues, and visceral organs such as liver, spleen, kidney, heart and lung. The average Mg content in the CNS tissues, as well as visceral organs except for spleen, of MS patients showed a significantly lower value than that seen in control cases. The most marked reduction of Mg content was observed in CNS white matter including demyelinated plaques of MS samples. Whether or not these significantly lower Mg contents found in CNS and visceral organs of MS patients may play an essential role in the demyelinating process remain unclear, requiring further studies on MS pathogenesis from the point of metal metabolism.