The use of rituximab to prevent severe delayed haemolytic transfusion reaction in immunized patients with sickle cell disease.

BACKGROUND: Delayed haemolytic transfusion reaction (DHTR) is mainly caused by an immune response to transfused red blood cells (RBCs). Immunized patients have a high risk of producing antibodies in response to further transfusion. Controlling the immune response to RBCs is therefore a major goal in sickle cell disease (SCD). STUDY DESIGN: We report an observational study of eight alloimmunized SCD patients with history of severe DHTR who were treated with rituximab before a new transfusion to prevent further immunization and DHTR. RESULTS: Five patients showed a good clinical outcome following transfusion preceded by preemptive treatment with rituximab. The remaining patients presented mild DHTR. In all patients, the results of post-transfusion screening tests were identical to those of pretransfusion tests; no newly formed antibodies were detected. CONCLUSION: These cases suggest that rituximab prevents at least occurrence of newly formed antibodies in high responders and minimizes the risk of severe DHTR. This study confirms that DHTR is complex in SCD and does not rely only on the classical antigens/antibodies conflict. Considering potentially serious adverse effect of rituximab, this treatment should be considered cautiously, and only when transfusion is absolutely necessary in patients with history of severe DHTR linked to immunization.

Anti-T haemolysins: the effects of sialic acid removal and 2-aminoethylisothiouronium bromide treatment of erythrocytes on immune lysis.

BACKGROUND AND OBJECTIVES: Intravascular haemolytic reactions are reported in red-cell T-activated patients after blood transfusion. The relationship between T antigen antibodies present in normal plasma and these reactions remains unclear. In this study, we assessed the haemolytic activity of T antibodies in vitro in comparison with anti-A/B antibodies. MATERIALS AND METHODS: We established a haemolysis assay based on treating target red-blood-cells (RBCs) with 2-aminoethylisothiouronium bromide (AET). Two hundred and seven blood donor sera were analysed for anti-T, anti-A/B haemolysins and anti-T agglutinins. RESULTS: Anti-T haemolysins were found in 4 (1·9%) blood donor sera using a standard haemolysis method and in 174 (84%) samples using AET-treated RBCs. Haemolysis correlated with agglutination titres (P<10(-7) ). With both methods, anti-T haemolysins were much weaker than anti-A and anti-B haemolysins. Gradual desialylation of RBCs showed a correlation between sialic acid level as indicated by agglutination with Sambucus nigra lectin and anti-T mediated haemolysis that was significantly increased (fold 2·4) independently of T antigen expression. CONCLUSION: These data indicate that, in vitro, anti-T-mediated haemolysis depends primarily on the degree of desialylation of target RBCs. They suggest that the haemolytic activity of T antibodies-containing human sera is usually weak and may only become significant in the very rare setting of a profound desialylation of RBCs.

[Study of 206 transfused sickle cell disease patients: immunization, transfusion safety and red blood cell supply]. / Etude d'une cohorte de 206 patients drépanocytaires adultes transfusés: immunisation, risque transfusionnel et ressources en concentrés globulaires.

BACKGROUND: Prevention of hemolytic transfusion reactions depends upon our capacity to prevent allo-immunization and conflicts between antigens of transfused red blood cells and antibodies produced by the recipient. In this study, we show that to secure transfusion of sickle cell disease patients, it is necessary to take into account their immunohematologic characteristics in the organization of transfusion. METHODS AND RESULTS: Immunohematological data of 206 chronically transfused patients have been collected as well as phenotypes of transfused units. In order to prevent allo-immunization against C and E antigens for patients typed D+C-E-c+e+ (56%), 26% of the transfused units were D-C-E-c+e+. We found that 47% of the patients had a history of allo-immunization, whereas only 15% produced an antibody the day of inclusion in the study. The non-detectable antibodies were frequently known as dangerous for transfusion. Finally, this study shows the frequency of anti-D in D+ patients and anti-C in C+ patients, pointing out the question of partial antigens. CONCLUSION: To insure optimal transfusion safety for sickle cell disease patients, three points have to be improved: blood donation within the Afro-Caribbean community living in France, access to history of immuno-hematological data, detection of variant antigens, especially within the RH blood system.

Rituximab for prevention of delayed hemolytic transfusion reaction in sickle cell disease.

Delayed hemolytic transfusion reaction (DHTR), a life-threatening transfusion complication in sickle cell disease (SCD), is characterized by a marked hemoglobin drop with destruction of both transfused and autologous red blood cells (RBCs) and exacerbation of SCD symptoms. One mechanism of RBCs destruction is auto-antibody production secondary to transfusion. As rituximab specifically targets circulating B cells, we thought that it could be beneficial in preventing this immune-mediated transfusion complication. We report the case of a SCD patient who previously experienced DHTR with auto-antibodies and who needed a new transfusion. DHTR recurrence was successfully prevented by rituximab administration prior transfusion, supporting the safe use of rituximab to prevent DHTR in SCD patients as a second line approach when other measures failed.

Involvement of p21 in mitotic exit after paclitaxel treatment in MCF-7 breast adenocarcinoma cell line.

It has been shown recently that expression of p21 is enhanced by paclitaxel. This cytotoxic compound induces mitotic spindle damage resulting in blockade of the mitotic cell cycle associated or not with apoptotic cell death. In the present study, we showed that, in MCF-7 cells, paclitaxel induced accumulation of p21 in cells with a G2/M DNA content, corresponding to cells either in abnormal mitosis or in an interphase-like state (decondensed chromatin) with multiple nuclei. In MCF-7 cells, the increase in p21 was subsequent to the mitotic arrest and was associated with the exit from abnormal mitosis leading to formation of cells with micronuclei. In this cell line, we noted a relationship between the elevation of p21 expression and the inhibition of p34cdc2 activity. High levels of p21 protein were also found to be associated with inactive p34cdc2/cyclin B protein complex after treatment with paclitaxel. Treatment with p21 antisense oligonucleotide partially blocked induction of p21 expression by paclitaxel and significantly reduced survival of MCF-7 cells exposed to this agent. In NIH-OVCAR-3 cells, which are deficient in basal and paclitaxel-induced p21 expression, paclitaxel led to a prolonged activation of p34cdc2 and a delayed mitotic exit associated with apoptotic cell death. These observations suggest that p21 is not required for the mitotic arrest in response to paclitaxel, but argue in favor of a role for this inhibitor in facilitating the exit from abnormal mitosis. This effectively enhances cell survival after paclitaxel-induced spindle damage.

Involvement of p21 in the PKC-induced regulation of the G2/M cell cycle transition.

Activation of protein kinase C (PKC) inhibits cell cycle progression at the G1/S and G2/M transitions. We found that phorbol 12-myristate 13-acetate (PMA) induced upregulation of p21, not only in MCF-7 cells arrested in the G1 phase as previously shown, but also in cells delayed in the G2 phase. This increase in p21 in cells accumulated in the G1 and G2/M phases of the cell cycle after PMA treatment was inhibited by the PKC inhibitor GF109203X. This indicates that PKC activity is required for PMA-induced p21 upregulation and cell cycle arrest in the G1 and G2/M phases of the cell cycle. To further assess the role of p21 in the PKC-induced G2/M cell cycle arrest independently of its G1 arrest, we used aphidicolin-synchronised MCF-7 cells. Our results show that, in parallel with the inhibition of cdc2 activity, PMA addition enhanced the associations between p21 and either cyclin B or cdc2. Furthermore, we found that after PMA treatment p21 was able to associate with the active Tyr-15 dephosphorylated form of cdc2, but this complex was devoid of kinase activity indicating that p21 may play a role in inhibition of cdc2 induced by PMA. Taken together, these observations provide evidence that p21 is involved in integrating the PKC signaling pathway to the cell cycle machinery at the G2/M cell cycle checkpoint.

Up-regulation of cdc2 protein during paclitaxel-induced apoptosis.

Microtubule damages induced by paclitaxel inhibit proteasome-dependent degradation of cyclin B, resulting in a sustained activation of cyclin B/cdc2 kinase and a cell cycle arrest in mitosis. It has been previously shown that this kinase activity is also required for paclitaxel-induced apoptosis. We found here that paclitaxel increased cdc2 mRNA and protein levels and led to an accumulation of cdc2 in the active dephosphorylated form in NIH-OVCAR-3 cells. The addition of cycloheximide inhibited the paclitaxel-induced increase in cdc2 protein level, further indicating that paclitaxel stimulates cdc2 synthesis. This increase in cdc2 synthesis is a consequence of paclitaxel-induced arrest in mitosis. Indeed, dual analysis of DNA and cdc2 protein contents indicated that cdc2 up-regulation occurred in cells arrested with a G2/M DNA content. Furthermore, no up-regulation of cdc2 protein was observed when paclitaxel-treated cells were prevented from entering mitosis by treatment with purvalanol A, a cyclin-dependent kinase (CDK) inhibitor, or stimulated to exit mitosis with 2-AP, a non-specific kinase inhibitor. In addition, when paclitaxel-induced apoptosis was inhibited by Bcl-2 over-expression, cdc2 up-regulation did not occur, leading to a lower level of activation of the cyclin B/cdc2 complex. Taken together, these results indicated that paclitaxel-induced cdc2 protein synthesis participates in a positive feedback loop designed to increase the activity of cyclin B/cdc2 kinase and thus may play a role in paclitaxel-induced apoptosis.

Use of human reconstructed epidermis to analyze the regulation of beta-defensin hBD-1, hBD-2, and hBD-3 expression in response to LPS.

Defensins have been identified as key elements of innate immunity against microbial infections. In the present study, human beta-defensin-2 (hBD-2) mRNA and peptide expression were evaluated by RT-PCR and Western blotting in normal human keratinocytes, in function of their stage of differentiation. In proliferating, non-differentiating keratinocytes generated in serum-free, low-calcium medium, a very low hBD-2 mRNA expression was found. A significantly higher expression was detected in high-calcium cultivated keratinocytes grown either as monolayers or as multilayers under submerged conditions. In an air-liquid interface culture of keratinocytes, allowing epidermis to be reconstructed, hBD-2 mRNA expression level was significantly higher than in the other conditions and displayed inter-individual variability as observed in native epidermis. The peptide was detected only in reconstructed epidermis. These results indicate that hBD-2 gene expression in normal human keratinocytes is dependent upon their stage of differentiation. The level of expression of hBD-1 mRNA was lower and that of hBD-3 was higher than that of hBD-2 in reconstructed epidermis. Exposure of reconstructed epidermis to bacterial lipopolysaccharide (LPS) resulted in an average 4-fold increase in hBD-2 mRNA 18 h after challenge, but not of hBD-1 and hBD-3 gene expression. These results show the selective regulation of hBD-2-encoding gene in an organotypic epidermal model, in response to LPS. They also provide evidence that in vitro reconstructed epidermis represents a useful model for studying regulation of expression of beta-defensins after skin challenge with pathogenic microorganisms in conditions as close as possible to the in vivo situation.

Phosphorylation and proteasome-dependent degradation of Bcl-2 in mitotic-arrested cells after microtubule damage.

Treatment of NIH-OVCAR-3 cells with paclitaxel, a microtubule-stabilizing agent, induces mitotic arrest and apoptosis, but also Bcl-2 phosphorylation. We report here that Bcl-2 phosphorylation precedes Bcl-2 down-regulation and that both events are closely associated with mitotic arrest, but are not sufficient for paclitaxel to trigger apoptosis. Indeed, when paclitaxel-treated cells were induced to exit mitosis in the presence of 2-aminopurine, Bcl-2 phosphorylation and Bcl-2 down-regulation were both inhibited. In contrast, when apoptosis was inhibited by a caspase inhibitor or Bcl-2 over-expression, Bcl-2 phosphorylation and down-regulation still occurred. Furthermore, we show that Bcl-2 is degraded in mitosis by the proteasome-dependent pathway since Bcl-2 down-regulation is inhibited by proteasome inhibitors such as MG132, Lactacystin and LLnL. Taken together these results indicate that mitotic spindle damage results in post-translational modifications of Bcl-2 by phosphorylation and degradation.