Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20-30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150-300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides

Evidence that rapamycin rescue therapy delays rejection of major (MHC) plus minor (non-MHC) histoincompatible heart allografts in rats

The capacity of delayed onset of rapamycin (RAPA) therapy to block process of destruction was examined in rats undergoing heart allograft rejection. Untreated Wistar Furth (WFu; RT-1u) recipients reject Buffalo (BUF; RT-1b) heart allograft with a mean survival time (MST) of 6.5 +/- 0.5 days. A 14-day i.v.infusion of 0.8 mg/kg RAPA begun on the day of transplantation prolonged the survival to 74.1 +/- 20.2 days (P < 0.001), 0.2 mg/kg to 32.2 +/- 10.0 days (P < 0.001), and 0.08 mg/kg to 36.4 +/- 11.8 days (P < 0.001). When RAPA therapy (0.8 mg/kg) was begun 3 or 4 days after transplantation, the grafts survived 85.2 +/- 31.1 (P < 0.001), and 70.2 +/- 43.3 (P < 0.005) days, respectively. Therapy initiated on day 5 was much less effective; most transplants were rejected within 10 days; one graft survived 32 and two grafts 60 days (MST = 17.6 +/- 20.0, NS). A 0.2 mg/kg RAPA dose prolonged graft survival with initial use on days 3 (31.6 +/- 12.2 days; P < 0.001) or 4 (31.4 +/- 8.1 days; P < 0.001) but not on day 5. The 0.08 mg/kg RAPA prolonged hearts only when started on day 3 (47.2 +/- 2.7 days; P < 0.001) but not on days 4 or 5. WFu recipients treated with a subtherapeutic dose of cyclosporine (1 mg/kg; 9.1 +/- 1.5 days) displayed prolonged heart allograft function when treated subsequently with RAPA (0.8 or 0.08) beginning from days 4, 5, or 6 postgrafting. These in vivo results are supported by in vitro experiments. The frequency of BUF alloreactive elements among normal WFu LN cells (fTc) was 337 +/- 139/10(6) T cells in limiting dilution assay. Addition of RAPA (1 muMol) at the beginning of culture significantly reduced (P < 0.025) the fTc to 17 +/- 6.6/10(6), or alternatively on days 4 or 6 to 37.3 +/- 20.0/10(6) and 58.6 +/- 21.8/10(6), respectively. Thus, both in vivo and in vitro data demonstrate that delayed RAPA therapy may interrupt alloimmune reaction

The mechanism of unresponsiveness to allografts induced by rapamycin and rapamycin/cyclosporine treatment in rats

The mechanisms by which rapamycin (RAPA) and/or cyclosporine induce unresponsiveness to allografts were investigated in a rat model. Buffalo (BUF, RT-1b) heart allografts were rejected by Wistar-Furth (WFu, RT-1u) recipients at a mean survival time (MST) of 6.5 +/- 0.5 days. A 14-day course of RAPA (0.8 mg/kg) delivered intravenously by an osmotic pump prolonged BUF allograft survival to 76.1 +/- 23.4 days (P < 0.001). Adoptive transfer of 30-50 x 10(6) spleen and lymph node T cells that had been isolated on day 40 postgrafting from CsA- or RAPA/CsA-treated hosts into lightly irradiated (6 Gray) secondary WFu recipients prolonged BUF graft survival from 9.8 +/- 1.2 to 29.2 +/- 11.0 (P < 0.01) and 58.2 +/- 38.9 days (P < 0.004), respectively. T cells transferred from animals treated with RAPA alone failed to prolong graft survival. In contrast, sera isolated on day 40 postgrafting from WFu primary hosts treated with RAPA alone or with the RAPA/CsA combination, but not with CsA alone, extended the survival of BUF hearts: 3 ml serum from RAPA-treated hosts prolonged BUF heart survival to 76.6 +/- 31.3 days (P < 0.002) and from RAPA/CsA-treated hosts to 47.1 +/- 12.8 days (P < 0.001). The effect of serum was immunologically specific: it did not prolong the survival of third-party outbred Sprague Dawley heart allografts. Although the IgM fraction (0.2 mg) purified from the serum of RAPA-treated recipients was ineffective (10.6 +/- 0.8 days; NS), an equal amount of the IgG fraction significantly (P < 0.002) prolonged BUF heart allograft survival to 26 days (n = 4). Thus, hosts treated with RAPA or a RAPA/CsA combination develop IgG antibodies that mediate the unresponsive state toward allogeneic heart allograft