Antioxidant/oxidant status and cardiac function in bradykinin B(1)- and B(2)-receptor null mice.

Kinin-vasoactive peptides activate two G-protein-coupled receptors (R), B(1)R (inducible) and B(2)R (constitutive). Their complex role in cardiovascular diseases could be related to differential actions on oxidative stress. This study investigated impacts of B(1)R or B(2)R gene deletion in mice on the cardiac function and plasma antioxidant and oxidant status. Echocardiography-Doppler was performed in B(1)R (B(1)R(-/-)) and B(2)R (B(2)R(-/-)) deficient and wild type (WT) adult male mice. No functional alteration was observed in B(2)R(-/-) hearts. B(1)R(-/-) mice had significantly lowered fractional shortening and increased isovolumetric contraction time. The diastolic E and A waves velocity ratio was similar in all mice groups. Thus B(1)R(-/-) mice provide a model of moderate systolic dysfunction, whereas B(2)R(-/-) mice displayed a normal cardiac phenotype. Plasma antioxidant capacity (ORAC) was significantly decreased in both B(1)R(-/-) and B(2)R(-/-) mice whereas the vitamin C levels were decreased in B(2)R(-/-) mice only. Plasma ascorbyl free radical was significantly higher in B(1)R(-/-) compared to WT and B(2)R(-/-) mice. Therefore, the oxidative stress index, ascorbyl free radical to vitamin C ratio, was increased in both B(1)R(-/-) and B(2)R(-/-) mice. Hence, B(1)R and B(2)R deficiency are associated with increased oxidative stress, but there is a differential imbalance between free radical production and antioxidant defense. The interrelationship between the differential B(1)R and B(2)R roles in oxidative stress and cardiovascular diseases remain to be investigated.

Effect of tresperimus on ex vivo expansion of CD34+CD38(-)-enriched cord blood cells.

Tresperimus, an analogue of 15-deoxyspergualine (15-DSG), has been found, in rodents, to induce a potent state of tolerance after organ and bone marrow allografts. In a previous study, we have reported that tresperimus at the optimal concentration of 0.5 microgram/ml supports the clonogenic potential of human cord blood CD34+ cells. Dose dependent inhibition of clonogenesis was also observed with complete reversibility following drug withdrawal. In this study, we tested the effect of 0.5 microgram tresperimus/ml on ex vivo expansion of primitive human cord blood CD34+CD38- cells. Our findings revealed that the total number of expanded cells was decreased in the presence of tresperimus. However, the multipotential and erythroid colonies were significantly increased in the presence of tresperimus compared with control cultures done without the test drug. Progenitor cell morphology was comparable in both test and control cultures. The telomerase activity was consistently lower in tresperimus-treated hematopoietic progenitors than in control cultures. These results suggest that tresperimus preserves primitive CD34+CD38- cells in a state of high potentiality while limiting the total number of their differentiated progeny. Bearing in mind that the test drug supports the clonogenic potential of CD34+ cells, the overall findings emphasize the importance of assessing the effect of tresperimus on in vivo long-term hematopoiesis which could predict the potential clinical use of tresperimus in the prevention of graft-versus-host disease in recipients of allogeneic bone marrow.

Effect of tresperimus on in vitro human cord blood CD34+ cell differentiation.

Tresperimus, an analogue of 15-deoxyspergualin (15-DSG), is immunosuppressive and prevents lethal graft-versus-host disease following allogeneic bone marrow transplantation in mice. Here, we present an in vitro dose response study examining the ability of tresperimus to support clonogenesis in cultured CD34+ cord blood stem cells. Our findings revealed that only the lowest dose examined, 0.5 microg tresperimus/ml, supports normal myelopoiesis, erythropoiesis and megakaryopoiesis. Greater concentrations of the drug induced dose-dependent inhibition of clonogenesis. This latter effect was not due to apoptosis and was reversible by drug withdrawal. We conclude that tresperimus at 0.5 microg/ml supports the clonogenic potential of cord blood CD34+ cells. Dose-dependent inhibition of clonogenesis was completely reversible following drug withdrawal. These results may be of clinical interest as tresperimus is currently used in phase I-III studies for the prevention of graft versus host disease in recipients of allogeneic bone marrow.

LF15-0195 prevents the induction and inhibits the progression of rat anti-GBM disease.

BACKGROUND: LF15-0195 is a novel immunosuppressant that is currently in phase II clinical trials for the treatment of vasculitis. This study examined whether LF15-0195 could suppress the induction and progression of rat anti-glomerular basement membrane (anti-GBM) glomerulonephritis. METHODS: Rapidly progressive glomerulonephritis was induced in primed rats by the administration of anti-GBM serum. In the first experiment, LF15-0195 was given daily by subcutaneous injection (days 0 to 14) to treat the induction of anti-GBM disease analyzed at day 14. In a second experiment, rats received LF15-0195 as an intervention treatment from days 7 to 28 (continuous therapy) or days 7 to 12 (pulse therapy) to treat the progression of disease assessed at day 28. RESULTS: Continuous LF15-0195 treatment during the induction of anti-GBM disease (experiment 1) prevented proteinuria and loss of renal function, and markedly reduced histological kidney lesions and renal fibrosis. LF15-0195 also reduced kidney leukocyte infiltrate, urine excretion of interleukin-1beta (IL-1beta) and transforming growth factor-beta (TGF-beta), and the serum antibody response, but not kidney deposition of Ig and C3. When LF15-0195 treatment was initiated at day 7, both continuous and pulse therapy partially inhibited disease progression by suppressing the loss of renal function, interstitial macrophage and T-cell accumulation, tubular cell proliferation, and renal fibrosis. CONCLUSION: LF15-0195 prevents the induction and suppresses the progression of rat anti-GBM disease through multiple mechanisms of action, suggesting that this drug may have significant therapeutic potential in human glomerulonephritis. The similar efficacy of continuous and pulse intervention treatment in this model indicates that short-term LF15-0195 treatment may achieve optimal benefit without prolonged bone marrow suppression.

A soluble transforming growth factor-beta (TGF-beta ) type I receptor mimics TGF-beta responses.

Transforming growth factor-beta (TGF-beta) signaling requires a ligand-dependent interaction of TGF-beta receptors Tau beta R-I and Tau beta R-II. It has been previously demonstrated that a soluble TGF-beta type II receptor could be used as a TGF-beta antagonist. Here we have generated and investigated the biochemical and signaling properties of a soluble TGF-beta type I receptor (Tau beta RIs-Fc). As reported for the wild-type receptor, the soluble Tau beta R-I does not bind TGF-beta 1 on its own. Surprisingly, in the absence of TGF-beta1, the Tau beta RIs-Fc mimicked TGF-beta 1-induced transcriptional and growth responses in mink lung epithelial cells (Mv1Lu). Signaling induced by the soluble TGF-beta type I receptor is mediated via the obligatory presence of both TGF-beta type I and type II receptors at the cell surface since no signal was observed in Mv1Lu-derivated mutants for TGF-beta receptors R-1B and DR-26. The comparison between the structures of TGF-betas and a three-dimensional model of the extracellular domain of Tau beta RI has shown that five residues of the supposed binding site of TGF-beta 1 (Lys(31), His(34), Glu(5), Tyr(91), and Lys(94)) were found with equivalent biochemical properties and similar spatial positions.

[Multimeric receptors, new targets for the pharmaceutical industry]. / Les récepteurs multimériques, de nouvelles cibles pour l'industrie pharmaceutique.

Cytokines and growth factors are reported to play a pivotal role in many pathologic conditions. Some recombinant proteins have demonstrated powerful therapeutic activities for example in arthritis for anti-TNF (antibody and soluble receptor) or in cancer for interleukin2 and are now available for clinical use. However limitation in production, treatment condition for use and final high costs have encouraged pharmaceutical industry to develop research of small synthetic compounds which can replace or inhibit natural ligands. During the last 5 years a series of publication have demonstrated that small peptides (up to 20 amino-acids) and non-peptide synthetic compounds may replace in vitro as well as in vivo some cytokine and cell growth factors. Selection of these compounds have used new technique of screening including phage display and gene reporter assays. Currently selected compounds mainly act as agonist and stimulate transduction signals in the target cell as do natural ligands. These results have demonstrated that at least for some cytokine and cell growth factor protein biological action may be mimicked by small molecular weight synthetic compound. Next steps will deal with selection of drug candidates able to be studied at the clinical level.

Structure-immunosuppressive activity relationships of new analogues of 15-deoxyspergualin. 2. Structural modifications of the spermidine moiety.

A series of new analogues of 15-deoxyspergualin (DSG), an immunosuppressive agent commercialized in Japan, was synthesized and tested in a graft-versus-host disease (GVHD) model in mice. Various substitutions of the spermidine "D" region were made in order to determine its optimum structure in terms of in vivo immunosuppressive activity. Various positions of methylation were first investigated leading to the discovery of the monomethylated malonic derivative 56h in which the pro-R hydrogen of the methylene alpha to the primary amine of the spermidine moiety has been replaced by a methyl group. Synthesis of the similarly methylated analogue of the previously reported glycolic derivative LF 08-0299 afforded 60e which demonstrated a powerful activity at a dose as low as 0.3 mg/kg in the GVHD model and was much more potent than DSG in the demanding heart allotransplantation model in rats. The improvement of in vivo activity was supposed to be related to an increase of the metabolic stability of the methylated analogues compared to the parent molecules. Due to its very low active dose, compatible with a subcutaneous administration in humans, and its favorable pharmacological and toxicological profile, 60e was selected as a candidate for clinical evaluation.

Analysis of in vivo immunosuppressive and in vitro interaction with constitutive heat shock protein 70 activity of LF08-0299 (Tresperimus) and analogues.

LF08-0299 (Tresperimus) is a new immunosuppressive analogue of Gusperimus (15-deoxyspergualin or DSG). Despite the fact that its mechanism of action remains unknown, DSG has previously been demonstrated to bind specifically to Hsc70 protein, a constitutive or cognate member of heat shock protein 70 family. Herein we further explore whether immobilised LF08-0299 will selectively retain the heat shock protein Hsc70. We analysed the correlation between biological activity in vivo in the prevention of murine graft-vs-host disease (GVHD) and the ability in vitro in dissociating Hsc70 from LF08-0299 resin for LF08-0299 and structural analogues. The Hsc70 protein bound to the LF08-0299 immobilised specifically via the spermidine primary amino group and could be successfully eluted from the column by various analogues of LF08-0299. All immunosuppressants tested were able to competitively bind Hsc70 although some biological inactive compounds could as well. Our data suggests that LF08-0299 and its active analogue effects were not mediated directly through the interaction of molecules with Hsc70. The mechanism of action probably occurred by more than one step, the first being the binding of Hsc70.

Structure-immunosuppressive activity relationships of new analogues of 15-deoxyspergualin. 1. Structural modifications of the hydroxyglycine moiety.

A series of new analogues of 15-deoxyspergualin (DSG), an immunosuppressive agent currently commercialized in Japan, was synthesized and tested in a graft-versus-host disease (GVHD) model in mice. Using the general concept of bioisosteric replacement, variations of the hydroxyglycine central "C" region were made in order to determine its optimum structure in terms of in vivo immunosuppressive activity. By this way, the malonic derivative 13a was discovered as the first example of a new series of potent immunosuppressive agents encompassing a retro-amide bond linked to the hexyl-guanidino moiety. Structure-activity relationships of this series were studied by synthesizing compounds 13g-i and 13k-s. Variation of the "right-amide" of 13a led to the urea 19a and the carbamates 23 and 27a which proved to be equally active as DSG in our GVHD model. Finally 27a was found to be the most potent derivative, being slightly more active than DSG in a heart allotransplantation model in rats. Due to the absence of chiral center in its structure and to its improved chemical stability compared to DSG, 27a was selected as a candidate for clinical evaluation.

Chimeric extracellular domain type II transforming growth factor (TGF)-beta receptor fused to the Fc region of human immunoglobulin as a TGF-beta antagonist.

Transforming growth factor-beta (TGF-beta) type-I and type-II receptors form a ligand-dependent heteromeric signalling complexes, in which transforming growth factor-beta receptor type II (TbetaRII) trends to act as the primary receptor. In the present study, we used a chimeric soluble type-II receptor fused with the Fc regions of human immunoglobulin (TbetaRIIs-Fc) in order to obtain a putative TGF-beta antagonist. Biochemical studies revealed that TbetaRIIs-Fc shared the same properties as the wild-type receptor. The TbetaRIIs-Fc receptor displayed an affinity of 1370 +/- 363 pM which was similar to those of the wild-type TbetaRII when expressed alone in Cos-1 cells (1122 +/- 413 pM). Furthermore, the chimeric receptor showed the same selectivity for TGF-beta isoforms as the native receptor. Although both TGF-beta1 and TGF-beta3 were able to bind TbetaBRIIs-Fc, TGF-beta2 could not compete with the binding of TGF-beta1 to TbetaRIIs-Fc. It was noted that this type of fused Fc receptor could be used in FlashPlate screening for potent agonism and antagonism of TGFbeta. Moreover, biological activities of the chimeric receptor showed it to be a potent TGF-beta1-antiproliferative and TGF-beta1-extracellular matrix transcriptional inhibitor on responses in Mv1Lu cells. To conclude, our results clearly show that the TbetaRIIs-Fc chimeric receptor could be used as a potent TGF-beta antagonist. These data raised the possibility that this TbetaRIIs-Fc construct might act successfully as an antagonist of both TGF-beta1 and TGF-beta3 in vivo.

T cell repertoire expression in murine recipients of bone marrow transplant after LF 08-0299 (Tresperimus) administration.

LF 08-0299 (Tresperimus), a novel immunosuppressive compound, has been previously shown to prevent graft-versus-host disease in murine models. In this study, we investigated the influence of LF 08-0299 on the TCR Vbeta repertoire of irradiated F1 recipient mice reconstituted with either syngeneic or parental bone marrow cells. We showed that a partial blockade of thymic differentiation occurred in normal mice under treatment at the transition CD4-/CD8- to CD4+/CD8+, and that this blockade was fully reversible. Despite the effect on the thymus, normal T cell repertoire negative selection was preserved following syngeneic bone marrow transplantation. We further assessed whether LF 08-0299 administration could modify Vbeta T cell expression in irradiated recipients reconstituted with parental bone marrow cells. In our murine parental to F1 transplant model, abnormal TCR Vbeta3, Vbeta5, Vbeta6 and Vbeta11 expression was demonstrated in peripheral lymph nodes of irradiated recipients. Moreover, Vbeta6 and Vbeta3 T cell populations were overexpressed. Administration of LF 08-0299 modified the pattern of Vbeta T cell expression. The expansion of Vbeta6 T cells was selectively inhibited under LF 08-0299 therapy and, in contrast, Vbeta5 T cells were overexpressed. Lymph node histological analysis showed that LF 08-0299 administration fully prevented the graft-versus-host reaction occurring in untreated recipient mice.

Tolerance in a rat cardiac allograft model after short-term treatment with LF 08-0299. Absence of clonal deletion and evidence of CD4+ suppressor cells.

LF 08-0299 is a new immunosuppressive compound. In a fully mismatched rat cardiac allograft model (Dark Agouti [DA]-->Lewis [LEW]), long-term unresponsiveness was observed after LF 08-0299 short-term treatment (20 days). Survival of additional cardiac and skin DA allografts, and rejection of third-party (Brown Norway [BN]) skin allografts demonstrated induction of a donor-specific tolerance state. The aim of this study was to investigate mechanisms of cardiac acceptance in this model. LEW rats with long-term surviving heart grafts (LTS LEW) were examined for their immune proliferative and cytotoxic responses toward donors (DA) and third-party (BN) antigens. Normal proliferative responses were observed and limiting dilution analysis did not reveal a reduction of T cytotoxic cell precursors. In our model, tolerance exists despite the presence of cells reactive with donor alloantigens. In vivo adoptive transfer of serum from LTS LEW failed to transfer unresponsiveness, indicating that serum factors do not seem to be involved in tolerance maintenance. Transfer of spleen cells, obtained from LTS LEW, showed specific prolongation of DA cardiac allografts in syngeneic hosts. Moreover, these cells were able to induce the rejection of third-party BN grafts. These results suggest that although LTS LEW possessed suppressor cells, they remained immunocompetent in recognizing and responding to third-party alloantigens. Purified CD4+ cells transferred unresponsiveness to secondary hosts, but CD8+ cells did not. Taken together, these results suggest that tolerance to donor alloantigens after treatment with LF 08-0299 in the rat cardiac allograft model is most likely due to induction of specific CD4+ suppressor cell activity, rather than induction of suppressive serum factor and selective elimination of antidonor helper or cytotoxic cell precursors (clonal deletion).

Prevention of lethal graft-versus-host disease following allogeneic bone marrow transplantation in mice by short course administration of LF 08-0299.

We investigated the ability of LF 08--0299, a new immunosuppressive compound, to prevent murine graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). A short term LF 08--0299 treatment at optimal dosage protected more than 75% of recipient mice from lethal GVHD induced either across minor antigens alone or the full H2 barrier. Furthermore, LF 08--0299 still prevented lethal GVHD when treatment was delayed to 10 days post-BMT. Long-term LF 08--0299-treated survivors were free of clinical signs of GVHD, and histopathologic examination of liver, skin, and intestines was normal, demonstrating that recipient mice did not develop chronic GVHD. We assessed the immunocompetence of long-term surviving recipient mice. Results from MLR and CTL assays were weak whereas responses against unrelated H2 antigens were reduced but still preserved. Moreover, in vivo transfer experiments demonstrated that spleen cells from long-term survivors were unable to induce lethal GVHD in irradiated recipients of host origin, while spleen cells injected in irradiated recipients of a host-unrelated H2 were fully competent to induce a lethal GVHD. Together these results indicate that stable chimeric recipient mice were specifically tolerant to host antigens. We further showed that while LF 08--0299 can protect recipient mice from lethal GVHD, it also preserved a graft-versus-leukemia effect when mice were inoculated with P815 tumor cells. These data suggest that LF 08--0299 may be a novel pharmaceutical agent that would prevent GVHD in human unrelated bone marrow transplantation.

Use of [35S]methionine-labelled rat lymphoblasts in micro-cytotoxic and limiting dilution assays.

When only limited numbers of effector cells are available for in vitro T cytotoxic determinations, standard assays cannot be performed. 51Cr is still the most commonly used marker of target cells in cytotoxicity assays but since the incorporation of this marker is low, especially in non-tumor cells such as lymphoblasts, larger numbers of both target and effector cells are required. Here we report the use of [35S]methionine-labelled rat ConA blasts in cytotoxic, micro-cytotoxic and limiting dilution assays. Regardless of whether [35S]methionine or 51Cr targets were employed, cytotoxic activities were identical when large numbers of target cells (10(4)) were used. The high uptake of [35S]methionine by ConA blasts (9 cpm/cell) permitted the use of a small number of target cells without any loss of sensitivity. Therefore, the number of effectors and targets required was dramatically reduced, especially with high E : T ratios such as 100 : 1. The use of low number of [35S]methionine-labelled rat ConA blasts as targets was also suitable for the measurement of alloreactive T cell precursor frequencies. This technique illustrates the possibility of studying T cytotoxicity in animal species lacking tumor target cell lines under experimental conditions where the availability of effector cells is limited and the optimal use of such cells becomes critical.