[show abstract][hide abstract] ABSTRACT: The major limitation of conventional phage display is caused by its dependence on the Sec translocation pathway. All proteins displayed on filamentous phages must first be transported into the bacterial periplasm in an unfolded state via the Sec translocation machinery. Proteins that require a cytoplasmic environment and/or cytoplasmic components for folding, or that contain "stop transfer" signals, or reach their native state before they interact with the Sec proteins are not compatible with the Sec pathway. They can never be presented using conventional phage display. We have developed an alternative phage display system, termed the TPD system, which overcomes these limitations of conventional phage display by exploiting the properties of the twin-arginine translocation (Tat) pathway. The Tat pathway only exports folded proteins that have already attained their native conformation in the cytoplasm. We investigated the functional efficiency of the TPD system by displaying and panning for a mutant of the green fluorescent protein.
[show abstract][hide abstract] ABSTRACT: The etiology of Kawasaki disease (KD) is still unknown. Therefore, the diagnosis relies on clinical criteria only. Although a specific therapy for KD is not available, coronary complications can be significantly reduced with the help of intravenous immunoglobulin (IVIG) therapy. It is not clear how IVIG interacts with the immune system. Previously, we selected a large number of IgG and IgM Fab fragments specifically reacting with IVIG molecules by phage display and antiidiotypic panning from three patients with autoimmune thrombocytopenia, a patient with lupus, and a healthy individual. Sequencing revealed that the favored V(H) germline gene segments of these IVIG-bound Fabs were 3-23 or 3-30/3-30.5, the most frequently rearranged V(H) genes among human B cells. The binding pattern suggested a B cell superantigen-like, specific interaction of an IVIG subset with B cells that present B cell receptors derived from these two germline genes. The aim of the current study was to investigate whether treatment with IVIG influences this restricted interaction. Therefore we cloned and selected Fab fragments from a patient with KD before and after IVIG therapy. A favored selection of antibodies derived from both the 3-23 and the 3-30/3-30.5 germline gene segments as before was observed. Importantly, the reactivity with IVIG was significantly higher for clones from the library prepared after the IVIG treatment, providing the first in vivo functional evidence that a subset of IVIG may selectively activate B cells of this germline origin. This mechanism may add to the therapeutic effect of IVIG in the treatment of KD.
[show abstract][hide abstract] ABSTRACT: To perform a comparative analysis of 1) intravenous Ig (IVIG)-bound Fab fragments from a patient with autoimmune thrombocytopenia that had progressed to systemic lupus erythematosus (SLE) and 2) IVIG-selected Fabs from an SLE patient without thrombocytopenia.
IVIG preparations have been successfully used to treat certain cases of autoimmune thrombocytopenia and SLE. Specific interactions of IVIG with the components of the immune system are not well characterized. To investigate these, we had previously cloned a large number of phage-displayed IgG Fab fragments, derived from 3 patients with autoimmune thrombocytopenia, that were specifically bound by IVIG molecules during panning. Many of these Fabs reacted with platelets. Sequencing revealed that the most frequently used VH germline gene segments of all IVIG-bound Fabs were 3-23 and 3-30/3-30.5. One patient's autoimmune thrombocytopenia had progressed to SLE. Using the same cloning and panning procedures, we performed a comparative analysis of this patient's IVIG-bound Fab fragments and the IVIG-selected Fabs from an SLE patient without thrombocytopenia.
We observed an exclusive selection of antibodies derived from 3-23 and 3-30/3-30.5 germline segments. In contrast to the Fab fragments from the autoimmune thrombocytopenia patient who developed SLE, none of the IVIG-selected Fabs from the SLE patient without thrombocytopenia bound to thrombocytes.
Our results suggest a preferential interaction of a subfraction of IVIG-representative of normal Ig repertoires-with antibodies and B cell receptors derived from these 2 gene segments. Importantly, these are the most frequently rearranged VH germline genes among human B cells. This kind of interaction is characteristic of a B cell superantigen, since light chains, antigen specificity, and the high variation in the third complementarity-determining region 3 showed little influence on the selection of 3-23- or 3-30/3-30.5-derived Fabs by IVIG. However, at least some of the contact residues on Fabs for IVIG appear to be different from those for staphylococcal protein A and human immunodeficiency virus gp 120. The IVIG-selected Fabs may now be used to clone antibodies representative of this IVIG subfraction to study their possible regulatory influence on the B cell repertoire during normal development and disease.
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