Induction of a non-apoptotic mode of cell death associated with autophagic characteristics with steroidal maleic anhydrides and 7ß-hydroxycholesterol on glioma cells.

Steroidal maleic anhydrides were prepared in one step: lithocholic, chenodeoxicholic, deoxicholic, ursocholic, and hyodeoxicholic acid derivatives. Their capability to induce cell death was studied on C6 rat glioma cells, and 7ß-hydroxycholesterol was used as positive cytotoxic control. The highest cytotoxicity was observed with lithocholic and chenodeoxicholic acid derivatives (23-(4-methylfuran-2,5-dione)-3α-hydroxy-24-nor-5ß-cholane (compound 1a), and 23-(4-methylfuran-2,5-dione)-3α,7α-dihydroxy-24-nor-5ß-cholane (compound 1b), respectively), which induce a non-apoptotic mode of cell death associated with mitochondrial membrane potential loss and reactive oxygen species overproduction. No cells with condensed and/or fragmented nuclei, no PARP degradation and no cleaved-caspase-3, which are apoptotic criteria, were observed. Similar effects were found with 7ß-hydroxycholesterol. The cell clonogenic survival assay showed that compound 1b was more cytotoxic than compound 1a and 7ß-hydroxycholesterol. Compound 1b and 7ß-hydroxycholesterol also induce cell cycle modifications. In addition, compounds 1a and 1b, and 7ß-hydroxycholesterol favour the formation of large acidic vacuoles revealed by staining with acridine orange and monodansylcadaverine evocating autophagic vacuoles; they also induce an increased ratio of [LC3-II / LC3-I], and modify the expression of mTOR, Beclin-1, Atg12, and Atg5-Atg12 which is are autophagic criteria. The ratio [LC3-II / LC3-I] is also strongly modified by bafilomycin acting on the autophagic flux. Rapamycin, an autophagic inducer, and 3-methyladenine, an autophagic inhibitor, reduce and increase 7ß-hydroxycholesterol-induced cell death, respectively, supporting that 7ß-hydroxycholesterol induces survival autophagy. Alpha-tocopherol also strongly attenuates 7ß-hydroxycholesterol-induced cell death. However, rapamycin, 3-methyladenine, and α-tocopherol have no effect on compounds 1a and 1b-induced cell death. It is concluded that these compounds trigger a non apoptotic mode of cell death, involving the mitochondria and associated with several characteristics of autophagy.

Human monoclonal IgMs with anti-Mycoplasma pneumoniae antibody activity.

We have previously reported that IgM antibodies to Pep13 P1, the major immunogenic peptide of Mycoplasma pneumoniae (MP) P1 cytoadhesin involved in microorganism cytoadherence, is a part of the natural antibody repertoire expressed early in life. Hence, Pep13P1 belongs to the panel of self and non-self antigens recognized by the primitive B cell repertoire. Considering that antibody activity of human monoclonal IgM associated with lymphoproliferative diseases is representative of the immune repertoire, we analyze, in this study, the antibody reactivity to P1 of twenty human monoclonal IgMs. Interestingly, we show that 25% of them are of anti-Pep13P1 specificity: one is a MIgM with reactivity against intermediate filaments, two are MIgMs with anti-MAG specificity and two IgMs with previously unknown antibody activity. Our results indicate that anti-P1 IgM antibodies are parts of the autoreactive than the heteroreactive B cell repertoire and Pep13P1 may have structural similarities with an unknown self antigen as the corresponding physiologic ligand.

[Immunochemical analysis of human monoclonal IGM with antibody cold agglutinin activity during Mycoplasma pneumoniae infection]. / Analyse immunochimique d'une IgM monoclonale humaine à activité anticorps agglutinine froide au cours d'une infection à Mycoplasma pneumoniae.

Mycoplasma pneumoniae (MP) infection is associated with the emergence of anti-I cold agglutinins of IgM isotype, rarely complicated by hemolytic anemia. The mechanism by which the autoimmune disease occurs is still considerably controversed. We describe here a case of MP infection complicated by severe autoimmune hemolytic anemia with monoclonal anti-I IgM. IgM anti-I antibodies were purified by adsorption-elution on OI+ red cells. The red blood cell antibodies were found to be tightly associated with IgG and anti-MP specificity probably as immune complexes. The significance of these results is discussed.

Immunoregulatory role for a public IgM idiotype in the induction of autoimmune diseases in Mycoplasma pneumoniae infection.

Mycoplasma pneumoniae (MP) infection is associated with the emergence of various autoimmune disorders and autoantibody production. The most common autoantibodies induced are of anti-I specificity and express cold agglutinin (CA) activity. However, the mechanisms by which the microbial infection triggers the appearance of these autoantibodies are still unknown. To investigate these mechanisms, we used BALB/c mice as experimental models. In this paper, we show that BALB/c mice polyclonal antisera to MP react with human CA IgMs, and reciprocally, that BALB/c mice polyclonal antisera to human IgM CA react with MP. However, antibodies directed against MP and against CA IgM triggered by both immunizations represent two separate sets of antibodies. This was also confirmed using monoclonal antibodies derived from the immunized mice. Among these MAb we selected a monoclonal antibody MAb1D3 which reveals a cross-reactive idiotope (CRI) shared by human CA and other MIgMs with various autoantibody activities (anti-MAG and anti-IF). The CRI defined by MAb1D3 is a recurrent interspecies idiotope that is expressed by post infectious IgM antibodies to MP. Hence, we present in this study new data showing that the concomitant appearance of CAs and anti-MP IgM antibodies during acute MP infection is the consequence of a common idiotypic regulation of antibodies to infectious and to self antigens.

A human monoclonal IgG reactive with a private idiotope of a monoclonal IgM with autoantibody activity against myelin-associated glycoprotein.

One hundred and thirteen sera from patients with monoclonal IgG were tested for reactivity against a panel of 13 human monoclonal IgM having various autoantibody activities: 6 to myelin-associated glycoprotein (MAG), 2 to vimentin intermediate filament protein and 5 to red blood cell antigens [cold agglutinins with specificity directed to I antigen (3 cases), i antigen (1 case) or Pr antigen (1 case)]. One IgG was found to react with a monoclonal IgM with anti-MAG activity. This reactivity was characterized as idiotypic and directed against a private idiotope of the monoclonal IgM. This work provides further evidence for the existence of anti-idiotypic antibody activity of monoclonal Ig occurring in human B cell neoplasias.