Apoptosis is an important cellular mechanism for controlling cell viability and proliferation. With respect to eosinophils, cytokines prolong their survival, whereas corticosteroids reduce their survival in vitro. CD30, a member of the TNFR family, is expressed on the surface of many cell types, including Hodgkin’s lymphoma cells. CD30 is capable of inducing apoptosis after Ab treatment in some cell lines. To determine whether this surface structure is involved in apoptosis of human eosinophils, we examined its expression and the effect of anti-CD30 Ab treatment on the viability of eosinophils. Purified human eosinophils expressed low, but consistently detectable, levels of CD30. Immobilized, but not soluble, forms of anti-CD30 Abs (HRS-4 and Ber-H8) or recombinant mouse CD30 ligand exhibited an extremely rapid and intense survival-reducing effect on the eosinophils in the presence of exogenous IL-5; this effect was both concentration and time dependent. Furthermore, high concentrations of IL-5 could not reverse the reduced survival rates. After treatment with anti-CD30 Ab, gel electrophoresis of DNA extracted from the eosinophils demonstrated changes consistent with apoptosis. The immobilized F(ab′)2 of the anti-CD30 Ab failed to induce eosinophil apoptosis. The addition of anti-CD18 Ab also completely abrogated the induction of eosinophil apoptosis. Further examination using specific signal transduction inhibitors suggested the involvement of p38, mitogen-activated protein kinase kinase 1/2, and specific tyrosine kinase, but not NF-κB, in the induction of CD30-mediated eosinophil apoptosis. These data demonstrate that CD30 can modify eosinophil survival by causing an extremely rapid and intense induction of apoptosis through a tightly regulated intracellular signaling pathway.

Currently, there are no treatments for Alport syndrome, which is the second most commonly inherited kidney disease. Here we report the development of an exon-skipping therapy using an antisense-oligonucleotide (ASO) for severe male X-linked Alport syndrome (XLAS). We targeted truncating variants in exon 21 of the COL4A5 gene and conducted a type IV collagen α3/α4/α5 chain triple helix formation assay, and in vitro and in vivo treatment efficacy evaluation. We show that exon skipping enabled trimer formation, leading to remarkable clinical and pathological improvements including expression of the α5 chain on glomerular and the tubular basement membrane. In addition, the survival period was clearly prolonged in the ASO treated mice group. This data suggests that exon skipping may represent a promising therapeutic approach for treating severe male XLAS cases.

This article describes the design, synthesis, and biological evaluation of new indole-based cytosolic phospholipase A2α (cPLA2α, a group IVA phospholipase A2) inhibitors. A screening-hit compound from our library, (E)-3-{4-[(4-chlorophenyl)thio]-3-nitrophenyl}acrylic acid (5), was used to design a class of 3-(1-aryl-1H-indol-5-yl)propanoic acids as new small molecule inhibitors. The resultant structure-activity relationships studied using the isolated enzyme and by cell-based assays revealed that the 1-(p-O-substituted)phenyl, 3-phenylethyl, and 5-propanoic acid groups on the indole core are essential for good inhibitory activity against cPLA2α. Optimization of the p-substituents on the N1 phenyl group led to the discovery of 56n (ASB14780), which was shown to be a potent inhibitor of cPLA2α via enzyme assay, cell-based assay, and guinea pig and human whole-blood assays. It displayed oral efficacy toward mice tetradecanoyl phorbol acetate-induced ear edema and guinea pig ovalbumin-induced asthma models.

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The majority of patients with myasthenia gravis (MG), an organ-specific autoimmune disease, harbor autoantibodies that attack the nicotinic acetylcholine receptor (nAChR-Abs) at the neuromuscular junction of skeletal muscles, resulting in muscle weakness. Single cell manipulation technologies coupled with genetic engineering are very powerful tools to examine T cell and B cell repertoires and the dynamics of adaptive immunity. These tools have been utilized to develop mAbs in parallel with hybridomas, phage display technologies and B-cell immortalization. By applying a single cell technology and novel high-throughput cell-based binding assays, we identified peripheral B cells that produce pathogenic nAChRAbs in patients with MG. Although anti-nAChR antibodies produced by individual peripheral B cells generally exhibited low binding affinity for the α-subunit of the nAChR and great sequence diversity, a small fraction of these antibodies bound with high affinity to nativestructured nAChRs on cell surfaces. B12L, one such Ab isolated here, competed with a rat Ab (mAb35) for binding to the human nAChR and thus considered to recognize the main immunogenic region (MIR). By evaluating the Ab in in vitro cell-based assays and an in vivo rat passive transfer model, B12L was found to act as a pathogenic Ab in rodents and presumably in humans.These findings suggest that B cells in peripheral blood may impact MG pathogenicity. Our methodology can be applied not only to validate pathogenic Abs as molecular target of MG treatment, but also to discover and analyze Ab production systems in other human diseases.