Transplantation of fetal liver cells corrects accumulation of lipids in tissues and prevents fatal neuropathy in cholesterol-storage disease BALB/c mice.

The cholesterol storage disease (CSD) BALB/c mouse represents a model of the Niemann-Pick type C (NPC) disease. It is characterized by the accumulation of unesterified cholesterol within various tissues, resulting in fatal neurological lesions. Transplantation of 6x10(6) fetal liver cells from normal allogeneic CBA mice into lethally irradiated CSD mice led to reconstitution of the recipient mice with donor cells. As a result of this stable chimerism, deposition of lipids in tissues was decreased, neuropathy was prevented, and survival was significantly prolonged (over 190 days on average in transplanted mice versus 70 days in untreated mice). Foamy cells containing unesterified cholesterol, observed by filipin staining, were numerous in most tissues from untreated CSD mice; they were significantly fewer in CSD mice treated with fetal liver transplantation at the age of 36-45 days.

Prolongation of skin allograft survival in mice following administration of ALLOTRAP.

Recently, Clayberger et al. demonstrated that ALLOTRAP, small synthetic peptides derived from a conserved region of the alpha 1 helix of certain HLA class I molecules, inhibited human CTL responses in vitro. In rats, ALLOTRAP 07 therapy combined with a subtherapeutic dose of cyclosporine led to the permanent acceptance of heart allografts. In the present study, the effect of ALLOTRAP on the survival of skin allografts in mice was studied. The tail skin of male C57B1/6 (H-2b) mice was grafted on the back of male CBA (H-2k) recipients. In untreated animals, the skin graft was rejected after 11.6 +/- 1.13 days (MST +/- SD). Cyclosporine administered orally for 5 days after transplantation prolonged graft survival to 13.1 +/- 2.13 days. ALLOTRAP 2702 prolonged graft survival to 16.57 +/- 2.15 days when administered orally for five days posttransplantation and to 18.86 +/- 0.38 when administered intraperitoneally until rejection. Thus, ALLOTRAP peptides derived from human MHC class I sequences, in addition to inhibiting human T cell responses in vitro, also prolong allograft survival in rats and mice.

Induction of hyperacute rejection of skin allografts by CD8+ lymphocytes.

BACKGROUND: Second-set rejection is generally regarded as a phenomenon mainly mediated by humoral cytotoxic antibodies, although a few discordant data have been presented. In the reported experiments, we have taken advantage of the absence of production of specific cytotoxic alloantibodies contrasting with the normal development of transplantation cellular immunity, in two murine models: chimeric mice and RAG mice. METHODS: Chimeras (BALB/c-->CBA) were obtained by transplantation of 2x10(7) fetal liver cells from BALB/c (H-2d) mice to lethally irradiated CBA (H-2k) mice. After hyperimmunization with third-party C57/ BL6 (B6) (H-2b) skin transplants and with injections of 2x10(7) B6 spleen cells, antibody production, and skin graft survival were analyzed. To identify further the factors or cells responsible for accelerated rejection of B6 skin transplants in hyperimmunized chimeras, transfer experiments were carried out involving the injection of serum, whole spleen cells, spleen T cells, spleen CD8+ T cells or spleen CD4+ T cells from chimeras into BALB/c mice that had received 6 Gy irradiation. The recipient mice were then grafted with B6 skin. Similarly, the immunodeficient RAG mice were used to construct a model of recipient animals with anti-H-2d hyperimmunized B6 T cells in the total absence of antibody. RESULTS: In chimeras, anti-B6 cytotoxic antibodies were not detectable in any of hyperimmunized chimeric mice, yet accelerated rejection of B6 skin transplant occurred: a graft survival of 8.6+/-0.5 days (d), comparable to 8.9+/-0.8 d survival in CBA control mice subjected to the same hyperimmunization procedure, and significantly shorter than that in nonhyperimmunized (BALB/c-->CBA) chimeras (11.6+/-0.5 d) or in non-hyperimmunized CBA control mice (12.1+/-0.6 d). High titers of anti-B6 cytotoxic antibodies were present in the serum of hyperimmunized CBA control mice. In transfer experiments, the graft survival was over 14 d in mice treated with irradiation alone, with irradiation + serum or with irradiation + CD4+ T cells. It was significantly shorter in mice treated with irradiation + whole spleen cells, with irradiation + T cells or with irradiation + CD8+ T cells (8.9+/-0.8 d). Similarly, in immunodeficient RAG mice, reconstitution of the T cell compartment with T cells from hyperimmunized B6 mice led to accelerated rejection of BALB/c skin allografts (11.4+/-1.1 d vs. 18.8+/-0.8 d when T cells were provided by nonimmunized mice). In a second transfer of cells from these reconstituted RAG mice into naive RAG mice, CD8+ T cells were shown to induce accelerated rejection of skin allografts (12.0+/-0.6 d) whereas CD4+ T cells were much less efficient (16.5+/-0.1 d). CONCLUSION: These data indicate that T cells, and especially the CD8+ subset, can be responsible for second-set rejection in the absence of anti-donor antibodies in chimeric and RAG mouse models. These sensitized CD8+ T cells are also likely to play an important role in normal mice, in addition to that of cytotoxic antibodies.

Two novel vitamin D analogues, KH 1060 and CB 966, prolong skin allograft survival in mice.

KH 1060 and CB 966, two novel analogues of 1,25-dihydroxyvitamin D3(1,25(OH)2D3), were found to significantly delay the rejection of allogeneic skin grafts in CBA (H-2k) recipient mice, transplanted with skin from C57Bl/6 (H-2b) donor mice. Graft survival was assessed in mice treated with KH 1060 or CB 966 until the day of rejection, in comparison to mice treated with vehicle, 1,25(OH)2D3 or cyclosporin A (CsA). The mean graft survival time in days was found to be: 16.6 +/- 0.5 (CsA, 20 mg/kg/day orally (p.o.); 13.9 +/- 0.6 and 15.5 +/- 0.6 (1,25(OH)2D3, 0.2 and 0.4 microgram/kg/day intraperitoneally (i.p.)); 14.3 +/- 0.2 and 18.7 +/- 0.5 (CB 966, 0.2 and 0.4 microgram/kg/day i.p.); 13.7 +/- 0.8, 15.7 +/- 1.7, 18.2 +/- 2.7 and 24.5 +/- 0.5 (KH 1060, 0.02, 0.1, 0.2 and 0.4 microgram/kg/day i.p.). Mean graft survival in days for control mice was: 10.3 +/- 0.2 (vehicle for injection, i.p.) and 11.5 +/- 0.2 (olive oil, p.o.). Serum calcium levels, measured on the day of rejection, rose moderately after treatment with either KH 1060 or CB 966. Combination of CsA (20 mg/kg/day p.o.) with KH 1060 (0.1 microgram/kg/day i.p.) resulted in an additive or synergistic effect: 23.4 +/- 1.1 days of skin allograft survival, compared to 15.7 +/- 1.7 days with KH 1060 alone, and 15.1 +/- 1.9 days with CsA alone. KH 1060 was the most active of the tested compounds and can therefore be regarded as a potent immunosuppressor in transplantation; it can be used in combination with CsA and is effective at doses which only marginally affect serum calcium levels.

Survey of experimental data on fetal liver transplantation.

Fetal liver transplantation has been shown to induce hematological and immunological reconstitution in irradiated rodents, dogs, horses, and sheep. Engraftment and reconstitution without GvHD has been readily obtained using histocompatible donors. When mismatched fetal donors were used, a comparatively larger number of donor cells was required, in addition to pre-treatment of host with higher doses of irradiation or irradiation plus chemotherapy. Stem cell suspensions devoid of any T lymphocyte can be transplanted across major histocompatibility barrier without inducing overt GvHD. The transplanted animals become tolerant to both donor and host grafts.

Identification of denatured enzyme proteins in sodium dodecyl sulfate polyacrylamide gels.

A simple modification of the immunological sandwich method of Muilerman et al. for the identification of denatured enzyme proteins in sodium dodecyl sulfate-polyacrylamide gels is described, enabling the method to be used in principle for any enzyme whose activity is not inhibited by binding to antibodies. An immunological sandwich consisting of denatured enzyme, antibodies, and native enzyme is formed on a nitrocellulose filter blot of the gel, the filter is divided into strips, and each strip is tested for enzyme activity. The presence of enzyme activity serves to identify the region in the gel containing denatured enzyme protein. Experiments with human lysosomal alpha-glucosidase as a model system are described. The method was applied to identify a protein of Mr 125,000 as the main component with UDPgalactose pyrophosphatase activity in a partially purified preparation of the enzyme from rat liver.

[Lysosomal enzyme deficiency: in vitro correlation of deficient cells using a conditioned medium or by co-culturing with non-deficient cells]. / Déficience en enzyme lysosomiale: correction in vitro de cellules déficitaires par du milieu conditionné ou par des co-cultures avec des cellules non déficitaires.

We demonstrate that lysosomal enzyme (alpha-L-fucosidase) can enrich deficient fibroblasts, with purified enzyme brought by the medium, or with an enzyme supply by various cell sources. The co-culture systems lead to a deficient cell correction, whatever donor cells are lymphocytes or lymphoblastoid cells. This correction arise only with alive cells, and is strongly inhibited by mannose-6-phosphate. Our results do not support the hypothesis that cell to cell contact independently of mannose-6-phosphate binding site is necessary for transfer of lysosomal enzyme from lymphocytes to fibroblasts. We suggest that the neighbourhood of cells leads to a phosphorylated precursor increase in the pericellular area, which creates an enzyme stabilizing effect favourable at its incorporation.

The place of fetal liver transplantation in the treatment of inborn errors of metabolism.

Over the last 16 years, 202 fetal tissue transplants have been performed in our department to treat 29 patients with severe inborn errors of metabolism without immunodeficiency, 26 patients with congenital and severe immunodeficiency diseases, and 2 patients with severe aplastic anaemia. The actuarial survival curve of patients with inborn errors of metabolism treated with fetal liver transplantation shows a 12-year survival of 77%. The condition of many of these patients has been improved by the treatment, but transplantation has had to be repeated in order to maintain clinical amelioration. Enzyme levels were not significantly and durably increased in peripheral blood but the quantities of substrates detected in sera and urines were significantly reduced and tissue deposits were stabilized.