Transferrin-mediated uptake of aluminium by human parathyroid cells results in reduced parathyroid hormone secretion.

BACKGROUND: The present study investigates whether aluminium-transferrin (Al-Tf) uptake by Tf receptor-mediated endocytosis induces hypoparathyroidism and thus might contribute to the increasing prevalence of adynamic bone disease (ABD) in the current dialysis population. METHODS AND RESULTS: Human parathyroid glands as well as in vitro cultured human parathyroid cells were shown to express Tf receptors. Five-day-old cultures of parathyroid cells were incubated for 48 h in serum-free DMEM/F12 supplemented with 12 microM apo-Tf: 12 microM Tf to which 150 microg/l Al or 150 microg/l Al-citrate (Al-ci) was bound. The amount of Al taken up by the parathyroid cells either as Al-Tf or Al-ci did not differ. However, incubation of cell cultures with Al-Tf showed a significant proportional decrease (mean+/-SEM, -23.1+/-4.5%) in iPTH secretion as compared to the reference apo-Tf cultures. Al-ci did not suppress PTH secretion (+3.4+/-6.5%). The Al uptake after incubation with Al-Tf was found to be dose-dependent. With regard to iPTH secretion, a tendency toward a dose response relationship was observed. Northern blot analysis of parathyroid cells incubated in 12 microM apo-Tf or 12 microM Al-Tf demonstrated that the PTH mRNA synthesis was unaffected by the Tf-mediated uptake of Al. These observations suggest an effect of Al on PTH release rather than on PTH synthesis. Since the cytoskeleton can play an important role in the release of secretory vesicles, the influence of Al on the structure of actin, beta-tubulin and vimentin was investigated by confocal microscopy. Comparison of cultures incubated with apo-Tf and Al-Tf revealed no difference in the organization of these cytoskeletal proteins in relation to the inhibitory effect of Al-Tf on PTH secretion. CONCLUSION: In summary, data in the present paper demonstrate that the (i) human parathyroid gland/parathyroid cells exhibit Tf receptors; (ii) Al-Tf complex is taken up by the parathyroid gland in a dose-dependent manner; and (iii) uptake of Al by Tf receptor-mediated endocytosis reduces the secretion of PTH but not its synthesis. These in vitro findings allow us to suggest that Tf receptor-mediated uptake of Al might, besides other factors such as vitamin D, high calcium dialysate or CaCO(3) intake, play a role in the development of hypoparathyroidism associated with ABD. The exact mechanism by which Al-Tf suppresses iPTH secretion remains to be elucidated.

Bispecific antibody-mediated lysis of primary cultures of ovarian carcinoma cells using multiple target antigens.

We have shown previously that a bispecific antibody (BsAb) directed against both germ-cell alkaline phosphatase (GCAP) and the CD3 complex on mouse T cells could effectively eliminate GCAP-positive tumor cells in vivo using an immunocompetent mouse model. However, some GCAP-negative tumor cells were still able to grow, suggesting that BsAb therapy, when used in a clinical setting, could benefit from targeting several tumor markers to prevent outgrowth of tumor cells lacking a targeted marker. To test this hypothesis, we developed an in vitro model based on primary human ovarian carcinoma (OC) cultures and BsAbs directed against human T cells and several tumor markers [placental alkaline phosphatase (PLAP), GCAP, folate-binding protein (FBP) and CA19.9]. OC cells, isolated from primary tumors, were co-cultured with human peripheral blood mononuclear cells in the presence or absence of various concentrations of BsAbs against PLAP/GCAP, FBP and CA19.9 administered separately or in combination. Results derived from 18 primary OC samples showed that the combination treatment was better than or equally effective as the best single BsAB treatment in 60% of cases. Sometimes targeting FBP, PLAP/GCAP or CA19.9 alone was superior to targeting all simultaneously. Combining each BsAb with a low dose of IL-2 was always beneficial. These results indicate that before using a specific BsAb in the clinic, it is important to determine the optimal BsAb for each patient using this in vitro assay on cells from the removed tumor mass.

Bispecific antibody-mediated lysis of placental and germ cell alkaline phosphatase targeted solid tumors in immunocompetent mice.

Recently, an immunocompetent in vivo mouse model was developed based on germ cell alkaline phosphatase (GCAP) transgenic (FVB/N x C3H) mice in which both placental alkaline phosphatase (PLAP)+ and GCAP+ solid MO4 tumors develop. A bispecific anti-PLAP/GCAP anti-mouse CD3 antibody (Ab) 7E8 x 7D6, previously shown to induce efficient dose-dependent T-cell proliferation and PLAP+ tumor cell lysis in the presence of recombinant IL-2 and the anti-mouse CD3 Ab 7D6, was used in this report in in vivo lysis experiments targeting GCAP+ tumors grown in GCAP+ transgenic mice. Mice received injections i.v. twice a week with PBS (group 1) or with 10 micrograms of the bispecific Ab 7E8 x 7D6, either alone (group 2) or combined with 1 microgram of the anti-CD3 Ab 7D6 (group 3), starting 7 days after the tumor inoculation. A fourth group received a local treatment with mouse splenocytes precoated with 10 micrograms 7E8 x 7D6 and 1 microgram 7D6. In between Ab injections, groups 2, 3, and 4 received 10(4) units recombinant IL-2 (i.v.) every day. Two weeks of treatment with the bispecific Ab either alone or combined with 7D6 resulted in a significant decrease of GCAP+ tumor cells in groups 2 and 3 (4 +/- 3% and 10 +/- 11% GCAP+ cells/tumor) as compared to the nontreated tumors (95 +/- 5% GCAP+ cells), although tumor volumes were not significantly different (12 +/- 15 cm3 and 14 +/- 11 cm3 versus 16 +/- 7 cm3). Apparently, the elimination of GCAP+ cells from the tumor seemed to favor conditions enabling the outgrowth of the few GCAP- cells originally present in the tumor inoculate. In contrast, tumor volumes in group 4 (local treatment) were significantly smaller (P < 0.03; 5 +/- 10 cm3, 8 +/- 11% GCAP+ cells) as compared to the nontreated group, probably due to the presence of higher amounts of Ab and infiltrated activated T cells (567 +/- 322 CD5+ cells/mm2) capable of secreting cytostatic cytokines like tumor necrosis factor alpha and IFN-gamma as compared to groups 2 and 3 (266 +/- 135 and 198 +/- 86 CD5+ cells/mm2, respectively). In summary, this study clearly demonstrated that bispecific antibodies specifically concentrate cytotoxic T cells into a solid tumor in vivo, with subsequent elimination of the targeted tumor cell.

Requirement of monocytes and T-helper cells during development of tumor cell cytotoxicity in targeted T cells.

In cocultures of human placental alkaline phosphatase(PLAP)-positive MO4 tumor cells and human peripheral blood mononuclear cells (PBMC), also containing a heteroconjugate (7E8-OKT3) synthesized between the anti-PLAP monoclonal antibody 7E8 and the anti-CD3 antibody OKT3, and supplemented with low levels of recombinant interleukin-2 (rIL-2), T cells are progressively activated, resulting in tumor cell lysis. To unravel the contribution of PBMC subsets to the generation of this targetable cytotoxicity, PBMC subsets were studied after their isolation by cell sorting, either from fresh PBMC or from PBMC pre-activated with OKT3 and rIL-2. Whereas no targetable cytotoxicity was found in Fc-receptor-bearing CD3- cells, tumor cells were lysed by CD3+ T cells (mostly CD8+) isolated from pre-activated PBMC. When isolated from fresh PBMC, neither the CD8+ T cell subset, nor the total CD3+ T cell population developed significant targetable cytotoxicity, even in the presence of rIL-2. Thus, additional cell types are essential for the CD8+ T cell activation. Indeed, CD4+ T cells isolated from pre-activated but not from fresh PBMC were capable of eliciting cytotoxicity in fresh CD8+ T cells. The non-targeted monocytes were found to be the activators of the CD4+ T cells. In summary, targeting T cells to the surface of a tumor cell is not sufficient per se to achieve activation and lysis. The progressive tumor cell lysis by targeted T cells seems to be initiated by non-targeted monocytes activating CD4+ T cells, these cells in turn promoting CD8+ T cell activation, necessary for the development of cytotoxicity.

Transgenic mice expressing the tumor marker germ cell alkaline phosphatase: an in vivo tumor model for human cancer antigens.

We have generated a series of transgenic mouse lines harboring the entire human germ cell alkaline phosphatase (GCAP) gene linked to progressively longer sequences of flanking DNA. A 450-bp promoter sequence directs the expression of GCAP to the intestine and endothelial cells, while a 5' sequence of 1.7 kb directs GCAP expression to the spermatogenic lineage and to the eight-cell through the blastocyst stage of preimplantation development. The expression of GCAP in these FVB/N transgenic mice induces a cellular immune tolerance to GCAP. When mouse fibrosarcoma MO4 cells (C3H derived), stably transfected with the cloned GCAP gene, were injected s.c. in nontransgenic control (C3H x FVB/N) hybrid mice, GCAP-positive tumor cells were rejected. However, when GCAP-expressing transgenic (C3H x FVB/N) hybrid mice were challenged with these cells, GCAP-positive tumors developed. Tumors also developed in the transgenic hybrid mice upon injection of MO4 cells transfected with the highly homologous placental alkaline phosphatase (PLAP) cDNA in spite of the presence in PLAP of 10 amino acids that are different from the corresponding residues in GCAP. These GCAP transgenic mice will allow the study of the immune response associated with the repeated administration of conjugated or derivatized anti-GCAP and anti-PLAP monoclonal antibodies. They will also enable evaluation of the therapeutic potential of bifunctional antibodies for T-cell recruitment and destruction of GCAP/PLAP-producing tumor cells.

Tumor-cell lysis by in-situ-activated human peripheral-blood mononuclear cells.

A heteroconjugate (HC) was synthesized between OKT3 and monoclonal antibody (MAb) 7E8, which specifically reacts with the tumor marker placental alkaline phosphatase (PLAP). Similarly to OKT3, in vitro, the HC induced a dose-dependent proliferation response of human peripheral-blood mononuclear cells (PBMCs) and, in concert with rIL-2, it progressively activated T cells over a 4-day period. In co-cultures of continuously activated PBMCs and MO4 tumor cells (non-MHC-restricted mouse fibroblasts transfected with the cDNA for PLAP), the HC (25 ng/ml), again acting in concert with rIL-2, induced specific lysis of the MO4 cells. This process occurred progressively over 2 to 3 days and was monitored from the release in the supernatant fluid of cellular 3H-L-leucine, but also from analyses involving the remaining non-lysed cancer cells, i.e., by estimates of their protein content, by measurements of their viability, and most accurately by determinations of their PLAP content. Antibody 7E8 by itself induced a weak tumor-cell lysis (ADCC), potentiated by the addition of rIL-2. However, after 7 days of PBMC-preactivation with the HC and rIL-2, antibody 7E8 no longer mediated any ADCC, whereas the HC-dependent lysis was further potentiated. The observed proliferation of T cells and development of cytotoxicity at low concentrations of HC and rIL-2 support the idea that a moderate but continuous T-cell activation combined with T-cell targeting is sufficient for the induction of progressive and efficient tumor-cell lysis.