Epidermolysis bullosa simplex is a group of blistering skin disorders caused by defects in one of the keratin genes, KRT5 and KRT14. Previously reported KRT5 and KRT14 mutations are clustered in several hotspots, namely the rod ends of the 1A and 2B domains and in the non-helical linker region L12. Therefore, genomic KRT5 and KRT14 mutation analysis was initially limited to these hotspots. In this study we describe the screening of nine EBS patients for mutations in the hotspots. In two patients, with the Koebner and the Weber-Cockayne subtypes of epidermolysis bullosa simplex respectively, we could, however, not identify any mutation in one of the hotspot domains of KRT5 and KRT14. Therefore, it appeared to be necessary to screen the entire genes for mutations. For KRT5, a complete genomic mutation detection system was previously described. We now developed a complete genomic mutation detection system for KRT14. For the amplification of the KRT14 genes, we make use of restriction sites to exempt the keratin 14 pseudogene sequence from polymerase chain reaction amplification. Using the complete genomic mutation detection system for both KRT5 and KRT14, we identified four novel KRT5 mutations (IVS1-1G>C, K404E, A438D, E475K), two of which are outside the KRT5 hotspot domains, and three novel KRT14 mutations (IVS4+1G>A, L408M, L130P).
A patient with recessive epidermolysis bullosa simplex due to a previously described homozygous KRT14 1842-2A-->C splice-site mutation was re-examined, because we unexpectedly found signs of revertant mosaicism. The germline mutation resulted in different aberrant transcripts containing premature termination codons, all leading to truncated keratin 14 proteins. Basal keratinocytes in skin and in culture completely lacked keratin 14 and intermediate filaments. From this keratin 14-/- patient we started cultures from a new skin biopsy and here, we serendipitously found keratinocytes that spontaneously expressed keratin 14. This biopsy had been taken from an area of skin that was clinically affected, because blisters could simply be evoked by gentle rubbing. Immunofluorescence and electron microscopy of additional biopsies from this skin area revealed a mosaic expression of keratin 14 and reappearance of intermediate filaments in basal keratinocytes. Immunoblotting showed a revertant keratin 14 polypeptide with seemingly normal molecular weight. DNA analysis of exon 2 and its flanking intron borders showed no additional mutations in the genomic KRT14 sequence. Analysis of mRNA isolated from mosaic skin keratinocytes revealed an additional in-frame transcript (1844T-->G, 1845Delta6) that codes for an abnormal keratin 14 polypeptide with a two residue deletion and one amino acid change. The re-expression of a revertant, albeit abnormal, keratin 14 polypeptide, so-called partial revertant mosaicism, accounts for the antibody staining pattern and for the reappearance of intermediate filaments, which however, are semifunctional and not able to revert the clinical phenotype. The combination of a keratin 14-positive and a keratin 14-negative cell population in epidermis as well as in cultured keratinocytes suggests that the cellular reversion might be caused by an endogenous factor. We hypothesize that a second somatic modulating factor in the genome that affects the processing of the mutant KRT14 pre-mRNA may underlie this phenomenon.
The effectiveness of cellular immunotherapy of solid tumors is often hampered by the lack of specific infiltration of immune effector cells into the tumor mass. Therefore, we studied the potential of tumor antigen-specific antibodies to elicit tumor-specific myeloid cell activation, to induce or enhance tumor infiltration by immune cells. To this end, we developed an in vitro model system using the human myeloid cell line MonoMac-6. Incubation of IFN-␥-primed MonoMac-6 cells with serum-opsonized zymosan or EGP-2-directed, mouse IgG2a-opsonized, EGP-2-positive tumor cells resulted in the production of ROS and TNF-␣ and induced E-selectin and ICAM-1 expression on HUVECs. FcR-mediated MonoMac-6 cell activation was strictly dependent on the activation of MonoMac-6 cells with IFN-␥. In addition, no myeloid cell activation was observed in the presence of human serum or using tumor antigen-specific mouse antibody subclasses other than IgG2a, suggesting the crucial involvement of CD64 (Fc␥R1) in the effects observed. However, serum-inhibited myeloid cell activation was completely restored employing a 2-step targeting approach in which tumor cell opsonization with mouse anti-EGP-2 antibodies was followed by incubation with human antimouse Ig antibodies. Moreover, using this 2-step approach, not only anti-EGP-2-directed mouse IgG2a but also mouse IgG1 antibodies effectively induced tumor-specific myeloid cell activation. In conclusion, we describe a method to induce efficient and tumorspecific activation of myeloid cells based on the sequential use of mouse tumor antigen-specific and human antimouse Ig antibodies. Targeted myeloid cell activation may provide a means to aid in the induction of a tumor-directed immune response and as such, the method described here could be of clinical significance.
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