Neelam Tejpal

Synergistic interactions of cyclosporine and rapamycin to inhibit immune performances of normal human peripheral blood lymphocytes in vitro

Rapamycin, an actinomycete macrolide lactone that inhibits cytokine-induced immunoactivation, and cyclosporine, an endecapeptide that prevents transcription of lymphokine messenger RNA, display mutually synergistic interactions in vitro and in vivo. Using the rigorous median-effect analysis to dissect the nature of immunosuppressive drug interactions, rapamycin significantly augmented the inhibitory effects of cyclosporine and/or dexamethasone upon human peripheral blood lymphocyte activation by phytohemagglutinin, anti-CD3 monoclonal antibody, and mixed lymphocyte reaction. Furthermore, the addition of rapamycin potentiated the activity of cyclosporine to reduce cytotoxic cell generation and precursor frequency during in vitro alloactivation, using cell-mediated lympholysis and limiting dilution analyses, respectively. Similarly, cyclosporine potentiated the inhibitory effects of rapamycin upon proliferation of IL-2 (CTLL-2) and IL-6 (MH60.BSF-2) lymphokine-dependent cell lines. Lineweaver-Burk plots of the Michaelis-Menton equation suggested rapamycin inhibits IL-2 signal transduction in competitive, and IL-6 signal transduction in noncompetitive fashion, suggesting distinctive components of the various cytokine-receptor mechanisms. In vivo the cyclosporine/rapamycin combination exerted synergistic immunosuppression of rejection reactions in rats toward heterotopic cardiac allografts, using concentrations at which drugs were individually ineffective. These observations suggest that cyclosporine and rapamycin may be combined at significantly reduced doses to achieve unprecedented levels of immunosuppressive efficacy.

Immunosuppressive effects of FTY720 alone or in combination with cyclosporine and/or sirolimus.

BACKGROUND We examined the ability of FTY720, a novel immunosuppressant that prolongs the survival of allografts in experimental animal models, to potentiate the immunosuppressive effects of cyclosporine (CsA) and/or sirolimus (SRL) in vitro and in vivo. METHODS FTY720 alone (10-5000 ng/ml) or in combination with other drugs was added to human peripheral blood lymphocytes (PBLs) undergoing stimulation in vitro with phytohemagglutinin (PHA) or OKT3 monoclonal antibody. The combination index (CI) values were calculated to evaluate the nature of the interactions between FTY720 and CsA and/or SRL: CI values <1 reflect synergistic, CI=1, additive, and CI>1, antagonistic interactions. In addition, Wistar Furth (RT1u) rat recipients of Buffalo (RT1b) heart allografts were treated with FTY720 alone or in combination with other agents. FTY720 alone was also tested to block small bowel or liver allograft rejection in rats. RESULTS FTY720 alone produced only modest inhibition of the proliferation of human PBL stimulated with PHA or OKT3 monoclonal antibody. In combination with CsA or SRL, however, FTY720 produced synergistic effects, namely, CI values of 0.58 and 0.36, respectively. A 14-day course of FTY720 (0.05-8.0 mg/kg/day) by oral gavage prolonged heart allograft survival in dose-dependent fashion. Although a 14-day oral course of CsA (1.0 mg/kg/day) alone was ineffective (mean survival time=7.0+/-0.7 vs. 6.4+/-0.6 days in treated vs. untreated hosts), treatment with a combination of 1.0 mg/kg/day CsA and 0.1 mg/kg/day FTY720 extended allograft survival to 62.4+/-15.6 days (P<0.001; CI=0.15). Similarly, a 14-day oral course of 0.08 mg(kg/day SRL alone was ineffective (6.8+/-0.6 days; NS), but the combination of SRL with 0.5 mg/kg/day FTY720 extended the mean survival time to 34.4+/-8.8 days (CI=0.28). The CsA/SRL (0.5/0.08 mg/kg/day) combination acted synergistically with FTY720 (0.1 mg/kg/day) to prolong heart survivals to >60 days (CI=0.18). CONCLUSIONS FTY720 potentiates the immunosuppressive effects of CsA and/or SRL both in vitro (by inhibiting of T-cell proliferative response) and in vivo (by inhibiting allograft rejection).

The synergistic interactions in vitro and in vivo of brequinar sodium with cyclosporine or rapamycin alone and in triple combination

The rigorous median-effect analysis was used to assess the interactions between cyclosporine and drugs that inhibit nucleotide synthesis pathways. Using in vitro proliferation assays wherein human lymphocytes were triggered by phytohemagglutin, anti-CD3 monoclonal antibody, or mixed lymphocyte reactions, CsA was shown to display additive interactions with 6-mercaptopurine (6-MP), mizorbine (MZB), and mycophenolic acid (MPA), and a synergistic interaction with brequinar (BQR). In the in vitro assays, BQR contributed a further synergistic effect to the double-drug combination CsA/rapamycin (RAPA). Of the four inhibitors of nucleotide synthesis pathways, only BQR noncompetitively inhibited IL-2-stimulated proliferation of the CTLL-2 cell line. Using the in vivo assay of heterotopic Buffalo (BUF, RT-1b) cardiac allografts in Wistar-Furth (WFu, RT-1u) hosts, oral administration of BQR displayed about 100% bioavailability--which, like the bolus intravenous (i.v.) mode, was eight-fold more effective than continuous i.v. infusions. Furthermore median-effect analysis of serial amounts of orally administered BQR demonstrated that it contributes synergistically to the immunosuppressive effects of intravenously delivered CsA/RAPA (0.5/0.01 mg/kg/day). The degree of synergism was proportionate to the extent of the immunosuppression. These findings document the potency of the CsA/RAPA/BQR triple-drug combination and suggest that the synergistic effects may permit dose reductions of each component, thereby mitigating toxicities resulting from the large amounts of individual agents necessary to achieve allo-unresponsiveness.

The Mannich Base NC1153 Promotes Long-Term Allograft Survival and Spares the Recipient from Multiple Toxicities

JAK3 is a cytoplasmic tyrosine kinase with limited tissue expression but is readily found in activated T cells. Patients lacking JAK3 are immune compromised, suggesting that JAK3 represents a therapeutic target for immunosuppression. Herein, we show that a Mannich base, NC1153, blocked IL-2-induced activation of JAK3 and its downstream substrates STAT5a/b more effectively than activation of the closely related prolactin-induced JAK2 or TNF-α-driven NF-κB. In addition, NC1153 failed to inhibit several other enzymes, including growth factor receptor tyrosine kinases, Src family members, and serine/threonine protein kinases. Although NC1153 inhibited proliferation of normal human T cells challenged with IL-2, IL-4, or IL-7, it did not block T cells void of JAK3. In vivo, a 14-day oral therapy with NC1153 significantly extended survival of MHC/non-MHC mismatched rat kidney allografts, whereas a 90-day therapy induced transplantation tolerance (>200 days). Although NC1153 acted synergistically with cyclosporin A (CsA) to prolong allograft survival, it was not nephrotoxic, myelotoxic, or lipotoxic and did not increase CsA-induced nephrotoxicity. In contrast to CsA, NC1153 was not metabolized by cytochrome P450 3A4. Thus, NC1153 prolongs allograft survival without several toxic effects associated with current immunosuppressive drugs.

15 AU81, a prostacyclin analog, potentiates immunosuppression and mitigates renal injury due to cyclosporine

15 AU81, a synthetic prostacyclin analog, inhibits human mixed lymphocyte culture responses in a dose-dependent manner at concentrations within a range [0.1 to 10.0 micrograms/ml] similar to that of cyclosporine, methylprednisolone, antilymphocyte serum, and prostaglandin E1 and E2 analogs. Using the median-effect analysis to dissect immunosuppressive interactions, 15 AU81 and cyclosporine were shown to produce synergistic effects on human peripheral blood lymphocyte activation upon phytohemagglutinin or anti-CD3 monoclonal antibody stimulation. In vivo, 15 AU81 potentiated the effect of cyclosporine to prolong the survival of rabbit renal allografts using concentrations of each agent that were individually ineffective. Furthermore, addition of 15 AU81 mitigated functional damage of rabbit kidneys due to high-dose cyclosporine therapy. These observations suggest that the addition of 15 AU81 may broaden the therapeutic window of cyclosporine by potentiating immunosuppression and mitigating nephrotoxic effects.

Abrogation of chronic rejection in rat model system involves modulation of the mTORC1 and mTORC2 pathways

Background Current immunosuppressive regimens fail to avert chronic rejection (CR) of transplanted organs; however, selective targeting of actin-cytoskeletal regulators decreases T-cell motility and abrogates CR in rat model system. Administration of mutated class I major histocompatibility complex molecules or selective targeting of the RhoA pathway, which controls T-cell cytoskeletal activity, using Y27632 (a selective Rock1 inhibitor) resulted in reduced T-cell infiltration and abrogation of CR as judged from the neointimal index (13.9±19.7 vs. 45±37.5; P<0.001) and the number of affected vessels (30% vs. 60%; P<0.01). Here, we examined the role of mammalian target of rapamycin (mTOR) pathway in inhibition of CR. Methods A mutated class I major histocompatibility complex molecule that eliminates CR was delivered into ACI recipients of Wistar-Furth hearts at the time of transplantation with subtherapeutic cyclosporine (10 mg/kg on days 0–2). Controls included untreated and cyclosporine A–treated (10 mg/kg on days 0–2) heart allograft recipients. Results Western blotting and immunostaining showed that rat heart allografts with abolished CR exhibited down-regulation of the RAPA-sensitive mTORC1 components such as mTOR and Raptor and down-regulation of the RAPA-insensitive mTORC2 elements Rictor and Sin1. The mTOR regulator Deptor and its downstream target Rac1 were also inhibited. Conclusions Abrogation of CR in rat model system involves modulation of two mTOR pathways: a RAPA-sensitive mTORC1 pathway regulating cellular proliferation and a RAPA-insensitive mTORC2 pathway regulating T-cell motility. Selective targeting of T-cell actin cytoskeletal pathways shows potential for pathway-targeted immunosuppression therapies.

Rapid estimation of whole blood everolimus concentrations using architect sirolimus immunoassay and mathematical equations: Comparison with everolimus values determined by liquid chromatography/mass spectrometry

United States Food and Drug Administration (FDA) in 2010 approved the use of immunosuppressant drug everolimus, which requires therapeutic drug monitoring in whole blood. Taking advantage of structural similarity between sirolimus and everolimus we attempted to rapidly estimate everolimus concentration from apparent sirolimus concentration obtained by using Architect sirolimus immunoassay and mathematical equations (both polynomial and linear). Mathematical equations were derived by curve‐fitting methods based on observed apparent sirolimus concentration and true everolimus concentration determined by a liquid chromatography combined with mass spectrometry (LC/MS) method using eight everolimus standards (concentration range 1–30 ng/mL) prepared in whole blood. In order to determine the validity of our approach, we analyzed 12 specimens from patients receiving everolimus using both Architect sirolimus assay and LC/MS method. We observed good correlation between calculated everolimus values and true everolimus values as determined by LC/MS. However, if a patient is switched from sirolimus to everolimus, then sirolimus immunoassay can roughly estimate everolimus concentration plus any residual sirolimus present in whole blood and it is not possible to calculate everolimus concentration. J. Clin. Lab. Anal. 25:207–211, 2011.

Allograft Rejection Requires STAT5a/b-Regulated Antiapoptotic Activity in T Cells but Not B Cells

STATs play key roles in immune function. We examined the role of STAT5a/b in allograft rejection. STAT5a/b-deficient mice showed a 4-fold increased survival time of heart allografts (p < 0.01). Unlike wild type, purified STAT5a/b−/− T cells transferred to Rag1−/− recipients failed to mediate heart allograft rejection until supplemented with STAT5a/b−/− B cells. In vitro, STAT5a/b−/− T cells did not proliferate in response to Con A or alloantigens but entered apoptosis within 48 h (95%). Activated STAT5a/b−/− T cells showed increased expression of proapoptotic (caspases, DNA repair genes, TNF/TNFR-associated factor family genes) and decreased antiapoptotic mRNAs in microarrays, while Western blots confirmed reduced antiapoptotic Bcl-2 and elevated proapoptotic Bax protein expression. Interestingly, at 24 h postactivation, STAT5a/b+/+ and STAT5a/b−/− T cells produced similar levels of IL-2, IL-4, IL-10, and IFN-γ mRNA; ELISPOT assay showed an equivalent number of IL-4- and IFN-γ-producing T cells in both STAT5a/b+/+ and STAT5a/b−/− splenic populations. Sera from STAT5a/b+/+ and STAT5a/b−/− rejectors had donor-specific IgM, IgG1, IgG2a, and IgG2b Ab, while STAT5a/b deficiency had no impact on B cell survival or proliferation in response to LPS. Compared with allografts from STAT5a/b+/+ recipients, heart allografts from STAT5a/b−/− recipients had markedly reduced infiltration by CD4 and CD8 T cells but increased infiltration by B cells and dense endothelial deposition of C4d, a marker of humoral rejection. Thus, activated STAT5a/b−/− T cells produce cytokines prior to entering apoptosis, thereby promoting differentiation of B cells yielding donor-specific IgM and IgG Ab that mediate allograft rejection.

Allochimeric class I MHC protein-induced tolerance by partial TCR engagement requires activation of both CTL4- and common γ-chain-dependent cytokine signals

Background. The various toxicities associated with the general immune suppression resulting from current clinical immunosuppressive therapies continue to plague transplant recipients as well as jeopardize allograft survival. Methods. The present study utilized allochimeric class I MHC antigens (&agr;1hu70-77-RT1.Aa) bearing only four donor RT1.Au polymorphic amino acids (a.a.; His70, Val73, Asn74, and Asn77) superimposed on the recipient RT1.Aa background to induce transplantation tolerance in the rat cardiac transplant model. Results. Oral delivery of &agr;1hu70-77-RT1.Aa protein alone (days 0–6) induced tolerance, as evidenced by inhibition of both acute and chronic rejection processes. Delivery of &agr;1hu70-77-RT1.Aa with therapeutic doses of cyclosporine (CsA) also prevented chronic rejection, otherwise readily developed after treatment with CsA alone. A polymerase chain reaction (PCR)-based analysis showed that tolerant recipients had reduced numbers of interleukin (IL)-2/interferon (IFN)-&ggr;-producing T helper (Th)1 cells and elevated numbers of IL-4/IL-10-producing Th2 cells. Adoptive transfer experiments revealed that potent regulatory T cells mediated tolerance. The same T cells displayed diminished T cell receptor (TCR)-driven signaling via extracellular regulated kinase, AP-1, and NF-&kgr;B, as well as the common &ggr;-chain (&ggr;c) cytokine-receptor-induced signaling by Janus kinase 3 (Jak3)/stimulators and activators of transcription Stat/5 pathways. Tolerance induction was prevented in vivo by inhibition of signal 2 by CTL4Ig or of signal 3 by either rapamycin, which disrupts the mammalian target of rapamycin, or AG490, which inhibits Jak3. Finally, partial or complete tyrosine phosphorylation of Zap70 was observed in alloantigen-specific T cell clones in response to tolerogenic versus immunogenic peptides, respectively. Conclusions. Tolerance induction by allochimeric proteins is achieved by partial TCR activation in the presence of signals 2 and 3, resulting in a skewed Th2 phenotype.