Clusterin facilitates apoptotic cell clearance and prevents apoptotic cell-induced autoimmune responses.

Clusterin (Clu), an extracellular chaperone, exhibits characteristics of soluble innate immunity receptors, as assessed by its ability to bind some bacteria strains. In this study, we report that Clu also binds specifically to late apoptotic cells but not to live, early apoptotic, or necrotic cells. Histones, which accumulate on blebs during the apoptotic process, represent privileged Clu-binding motifs at the surface of late apoptotic cells. As a consequence, Clu potentiates, both in vitro and in vivo, the phagocytosis of late apoptotic cells by macrophages. Moreover, the increased phagocytosis of late apoptotic cells induced by Clu favors the presentation and cross-presentation of apoptotic cell-associated antigens. Finally, we observed that, in a model of apoptotic cell-induced autoimmunity, and relative to control mice, Clu(-/-) mice develop symptoms of autoimmunity, including the generation of anti-dsDNA antibodies, deposition of immunoglobulins and complement components within kidneys, and splenomegaly. These results identify Clu as a new molecule partner involved in apoptotic cell efferocytosis and suggest a protective role for Clu in inflammation and autoimmune diseases.

Improved vitrification method allowing direct transfer of goat embryos.

The aim of this study was to design a vitrification method suited to field embryo transfer experiments in goat. In a first experiment, a standard vitrification protocol, previously designed for sheep embryos was compared to slow freezing of goat embryos. No significant difference was observed on kidding rate (48% versus 69%, respectively), nor on embryo survival rate (35% versus 45%). Second experiment: all embryos were vitrified. After warming, embryos were either transferred directly (direct transfer), or after in vitro dilution of the cryoprotectants (conventional transfer). The kidding rate was not affected by the transfer method (38% versus 23%, respectively). However, embryo survival rate tended to be higher after direct transfer (26% versus 14%). Third experiment: OPS vitrification was compared to standard vitrification. The kidding rate was not affected (22% versus 39%, respectively), but the embryo survival rate was lower after OPS (14% versus 28%). Fourth experiment: 0.4M sucrose was added with cryoprotectants in vitrification. The kidding rate after direct transfer was significantly enhanced after addition of sucrose (56% versus 27%, respectively), whereas embryo survival rate was not significantly affected (32% versus 18%). Fifth experiment: vitrification with sucrose supplementation was compared to slow freezing. No significant difference was observed after direct transfer on kidding rate (52% versus 31%, respectively), but embryo survival rate tended to be higher after vitrification (34% versus 21%). In conclusion, our results indicate that addition of 0.4M sucrose in association with direct transfer improves significantly the viability of goat vitrified embryos.

Prolongation of heart xenograft survival in a hamster-to-rat model after therapy with a rationally designed immunosuppressive peptide.

BACKGROUND: Modification of the aminoacid sequence of peptides derived from the HLA class I heavy chain in combination with computer rational design resulted in the development of a peptide, RDP1258, with enhanced immunosuppressive activity. METHODS: We evaluated the activity of this peptide, analyzing infiltrate by immunohistology and cytokine transcripts by reverse transcriptase-polymerase chain reaction method, in a hamster-to-rat xenograft model where recipients were treated with cobra venom factor (CVF) and peptide. RESULTS: Although CVF or peptide alone had no effect, a combination of CVF/peptide RDP1258 resulted in a significant prolongation of graft survival (7.9+/-1 vs. 4.5+/-0 and 3.5+/-0 days, P<0.001). This effect was associated with an increased expression of heme oxygenase 1 (HO-1) in spleen, a significant reduced graft infiltrate, and a decrease of tumor necrosis factor-alpha mRNA transcripts (P<0.05) compared with CVF-treated recipients (1.6+/-0.07 vs. 3.3+/-0.3%, P=0.001) on day 3 after transplantation. CONCLUSION: These observations are consistent with the observation that up-regulation of HO-1 results in inhibition of immune effector functions and suggest that the peptide acts, at least partially, through HO-1 regulation.

RDP1258, a new rationally designed immunosuppressive peptide, prolongs allograft survival in rats: analysis of its mechanism of action.

Peptides derived from the HLA class I heavy chain (a.a. 75-84) have been shown to modulate immune responses in vitro and in vivo in a non-allele-restricted fashion. In vivo studies in rodents have demonstrated prolonged allograft survival following peptide therapy. The immunomodulatory effect of these peptides has been correlated with peptide-mediated modulation of heme oxygenase 1 activity (HO-1). Recently, we used a rational approach for designing novel peptides with enhanced immunosuppressant activity. These peptides were also more potent inhibitors of HO-1 activity in vitro. Here we evaluated one of these peptides, RDP1258, for its ability to prolong heterotopic heart graft survival in rats. The peptide mediated effect on HO-1 was analyzed in vitro and in vivo. Peptide RDP1258 was shown to inhibit rat HO-1 in vitro in a dose-dependent fashion. However, RDP1258, like other HO-inhibitors, when administered to rats, secondarily resulted in an up-regulation of splenic HO-1 activity. Up-regulation of HO-1 was associated with prolonged heart allograft survival (6.6 +/- 0.6 vs. 2/14 > 100 days and 12/14 16.2 +/- 1.7 days; p < 0.001). The analysis of graft infiltrating cells on day 5 after transplantation showed a significant decrease in the number of graft infiltrating cells in RDP1258-treated recipients compared to untreated ones (14.8 vs. 32.7%; p < 0.01). In addition, grafts from peptide-treated animals showed significantly decreased expression of TNF-alpha mRNA and increased levels of iNOS mRNA. Our results are consistent with the recent observation that up-regulation of HO-1 results in the inhibition of several immune effector functions. Modulation of HO-1 activity may enable the development of novel immunomodulatory strategies in humans.

Hourly distribution of time of parturition in the domestic goat.

Data on 2589 records collected during 12 years in one breed of dairy goats that were maintained in stables were analysed to examine the influence of various factors on the time of parturition. Parturitions were observed from mid-January to the end of April. Hourly frequencies of birth showed an unimodal distribution with maximum births at midday and minimum births around midnight with 90.6% of deliveries occurring between 0600 and 2000 h. The distribution showed a noticeable homogeneity when the following parameters were taken into account: litter-size, litters with live or stillborn kids, time of the year and length of gestation. In two other herds, 117 and 166 records obtained after 4 and 3 years of observation were studied. As in the first herd, births were more frequent between 0700 to 1900 h (day; 72.3 and 70.2 %, respectively) than between 1900 and 0700 h (night; P<0.001). These observations on the time of birth of the domestic goat suggest a regulation of the birth process by circadian events which themselves remain unknown.