We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.

Abstract-Direct binding of apolipoprotein (apo)B-containing lipoproteins to proteoglycans is the initiating event inatherosclerosis, but the processes involved at later stages of development are unclear. Here, we investigated the importance of the apoB-proteoglycan interaction in the development of atherosclerosis over time and investigated the role of lipoprotein lipase (LPL) to facilitate low-density lipoprotein (LDL) retention at later stages of development. Atherosclerosis was analyzed in apoB transgenic mice expressing LDL with normal (control LDL) or reduced proteoglycan-binding (RK3359-3369SA LDL) activity after an atherogenic diet for 0 to 40 weeks. The initiation of atherosclerosis was delayed in mice expressing RK3359-3369SA LDL, but they eventually developed the same level of atherosclerosis as mice expressing control LDL. Retention studies in vivo showed that although higher levels of 131 I-tyramine cellobiose-labeled control LDL ( 131 I-TC-LDL) were retained in nonatherosclerotic aortae compared with RK3359-3369SA 131 I-TC-LDL, the retention was significantly higher and there was no difference between the groups in atherosclerotic aortae. Lower levels of control 125 I-TC-LDL and RK3359-3369SA 125 I-TC-LDL were retained in atherosclerotic aortae from ldlr Ϫ/Ϫ mice transplanted with lpl Ϫ/Ϫ compared with lpl ϩ/ϩ bone marrow. Uptake of control LDL or RK3359-3369SA LDL into macrophages with specific expression of human catalytically active or inactive LPL was increased compared with control macrophages. 1 We tested the response-toretention hypothesis 2 in an earlier study using genetically modified mice that expressed human recombinant lowdensity lipoproteins (LDLs) with reduced proteoglycanbinding activity and provided direct evidence showing that subendothelial retention of apolipoprotein (apo)B100-containing lipoproteins is the initiating event in atherogenesis. 3 Furthermore, we demonstrated that the atherogenicity of LDL is linked to their proteoglycan-binding activity. 3 Lipoproteins associate with artery wall proteoglycans via both direct and indirect interactions. Direct binding between LDL and proteoglycans involves an ionic interaction between basic amino acids in apoB100 (Site B; residues 3359 to 3369) and negatively charged sulfate groups on the glycosaminoglycan (GAG) chains of proteoglycans. 4,5 Indirect binding between LDL and proteoglycans is facilitated by apoE, 3 which is found in human atherosclerotic plaques together with apoB. 6 ApoE binds vascular proteoglycans, and apoEenrichment of proteoglycan-binding-defective LDL (in which Site B has been inactivated by mutagenesis) partly restores proteoglycan binding. 3 Mouse LDL contains significant amounts of apoE, and consequently proteoglycan-bindingdefective LDL isolated from mouse plasma displays Ϸ30% of normal proteoglycan binding. 3 Subendothelial retention of LDL via indirect binding to GAGs can also be facilitated by lipoprotein lipase (LPL). 7 We have shown previously that the binding between LPL and LDL is mediated throug...

Patients with bladder pain syndrome/interstitial cystitis had increased mRNA levels of IL-17A, 10 and 6, and iNOS. IL-17A might be important in the inflammatory process. To our knowledge the increase in IL-17A is a novel finding that may have new treatment implications.

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