SUMMARYThe third complementarity-determining region (CDR3) of immunoglobulin variable genes for the heavy chain (V H ) has been shown to be shorter in length in hypermutated antibodies than in nonhypermutated antibodies. To determine which components of CDR3 contribute to the shorter length, and if there is an effect of age on the length, we analysed 235 cDNA clones from human peripheral blood of V H 6 genes rearranged to immunoglobulin M (IgM) constant genes. There was similar use of diversity (D) and joining (J H ) gene segments between clones from young and old donors, and there was similar use of D segments among the mutated and non-mutated heavy chains. However, in the mutated heavy chains, there was increased use of shorter J H 4 segments and decreased use of longer J H 6 segments compared to the non-mutated proteins. The overall length of CDR3 did not change with age within the mutated and non-mutated categories, but was signi®cantly shorter by three amino acids in the mutated clones compared to the non-mutated clones. Analyses of the individual components that comprise CDR3 indicated that they were all shorter in the mutated clones. Thus, there were more nucleotides deleted from the ends of V H , D, and J H gene segments, and fewer P and N nucleotides added. The results suggest that B cells bearing immunoglobulin receptors with shorter CDR3s have been selected for binding to antigen. A smaller CDR3 may allow room in the antibody binding pocket for antigen to interact with CDRs 1 and 2 as well, so that as the VDJ gene undergoes hypermutation, substitutions in all three CDRs can further contribute to the binding energy.

chemical characterization of the cutaneous cellular infiltrate in different areas of chronic leg ulcers.Current understanding of the immunological mechanisms involved in the pathogenesis of venous leg ulcers is insufficient. In this study the cellular composition of skin biopsies taken from the center, the edge, and 2 cm distant from the edge of venous leg ulcers was characterized quantitatively by immunohistochemical staining. In the epidermis the mean numbers of Langerhans cells (CD 1 a+) were four times lower at the edge of the ulcer compared to clinically intact epidermis 2 cm distant from the edge. In the dermis a statistically significant increase in the mean numbers of macrophages (CD68+) and neutrophils (NP57+) from the distant area towards the center of the ulcer was observed. No significant differences were observed in the distribution of T cells nor in the ratio of CD4+/CD8+ T-cell subsets between the different regions of the ulcer. About 30% of T lymphocytes were CD8+ in all microenvironments. The center and the edge of the ulcer were dominated by macrophages comprising 63% and 53% of the cells respectively, while T lymphocytes dominated the distant area. The area 2 cm distant from the edge was also heavily infiltrated by macrophages and neutrophils. B cells (CD22+) and NK cells (CD56+) were relatively rare in all areas, comprising less than 3% of the dermal infiltrate. In conclusion, local microenvironments each with a different cellular composition can be defined within venous leg ulcers.

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