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

Rapamycin inhibits production of cytotoxic but not noncytotoxic antibodies and preferentially activates T helper 2 cells that mediate long-term survival of heart allografts in rats

Rapamycin (RAPA) induces unresponsiveness toward heart allografts by at least two mechanisms: selective production of noncytotoxic IgG2c-blocking Ab and preferential activation of Th2 cells. RAPA (0.8 mg/kg/day) delivered via a 14-day osmotic pump to Wistar Furth (WF; RT-1u) recipients prolongs Buffalo (BUF; RT-1b) heart allograft survival from a mean survival time (MST) of 6.5 +/- 0.5 days to 75.0 +/- 18.9 days (n = 18; p < 0.001), with 6 of 18 grafts beating for more than 100 days. Recipient sera or their IgG but not IgM fraction, obtained after postgrafting day 40, passively transfer the unresponsive state to sublethally irradiated secondary recipients in a dose-dependent and immunologically-specific fashion. Sera obtained after untreated WF hosts rejected BUF hearts contained IgG moieties of all subclasses that bound to class I MHC BUF epitopes. In contrast, the unresponsive sera contained predominantly non-C'-fixing IgG2c and only marginal amounts of activated (C') fixing IgG1, IgG2a, and IgG2b Ab. The transcription of IL-2, IL-4, and IL-10 mRNAs was assessed using a PCR method. There were similar increases in the levels of IL-2, IL-4, and IL-10 mRNA in heart allografts from both untreated and RAPA-treated recipients on day 5 postgrafting. In contrast, on days 60 and 300 postgrafting heart allografts from RAPA-treated unresponsive recipients showed increased levels of IL-10 and IL-4 but not of IL-2 mRNA, suggesting preferential activation of Th2 cells. Thus, RAPA treatment selectively inhibits the synthesis of C'-binding of IgG subclasses, spares the non C-binding blocking IgG2c Ab, and preferentially activates Th2 cells

Immunosuppressive effects of defibrotide

The effect of defibrotide (DF) alone or in combination with CsA was examined using in vitro proliferation assays with human PBLs and in vivo heterotopic heart allografts in rats. DF alone (12.5-50 mg/ml) inhibited in vitro PBL proliferation more effectively after PHA (50-56%) or OKT3 (50-95%), than after alloantigenic (25-30%), stimulation. Furthermore, the combination of DF (1-4 mg/ml) with CsA (10-40 ng/ml) caused an 85.2-86.8% reduction in proliferative responses after OKT3 stimulation. Median-effect analysis documented that the combination index for DF and CsA was consistently lower than 0.3 at various concentration ratios of the 2 agents. Combination index values below 1.0 reflect drug synergism; those equal to 1.0 show additive, and above 1.0, antagonistic, interactions. Daily intraperitoneal injections of DF (150 mg/kg) failed to prolong the survival of Buffalo (RT-1b) heart allografts in Wistar-Furth (RT-1u) recipients, namely mean survival times of 7.0 +/- 0.7 days with, vs. 6.5 +/- 0.5 days without, DF treatment. Similarly, intravenous or intra-arterial infusion of DF (280 mg/kg) delivered directly into the heart allograft by a 7-day osmotic pump was ineffective. However, a course of local, but not systemic, DF (280 mg/kg) combined with a 14-day i.v. administration of a subtherapeutic dose of CsA (1 mg/kg) significantly prolonged heart allograft survival to 22.8 +/- 5.0 days (P < 0.001). Thus, in vitro DF is immunosuppressive alone at high concentrations, and in combination with CsA at low concentrations. Continuous infusion of DF into the graft combined with systemic administration of CsA prolonged transplant survival in vivo. These findings suggest that high tissue levels of DF potentiate the immunosuppressive effects of Cs

Kinetics of in vitro immune responses of T and B cells during tolerance induction by sirolimus

The purpose of the study presented herein was to examine immune performances of rat heart allograft recipients immunosuppressed with sirolimus (SRL, rapamycin; Rapamune, Wyeth-Ayerst, Princeton, NJ)