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Biomedical subjects

Andrew M Knight

Publications and source records attributed to Andrew M Knight.

6 recordsLinked to original sources
PubMed2005

Lack of induced co-stimulation as a result of complement receptor 2 (CR2) ligation on mouse splenic B cells.

B cells act as efficient antigen-presenting cells if they acquire antigen via membrane-bound Ig [termed the B cell receptor (BCR)]. Ligation of the BCR leads to antigen internalization, processing and presentation to CD4+ T cells in association with MHC class II molecules. Ligation of the BCR also leads to the generation of activation signals. One short-term consequence of this is the up-regulation of co-stimulatory molecule expression by the B cell, allowing full T cell activation. Other antigen receptors expressed by B cells can also mediate efficient antigen presentation to CD4+ T cells. Ligating one such receptor, complement receptor 2 (CR2), has also been described to induce co-stimulatory molecule expression. If correct, this may have serious consequences for ensuring the specificity of the resultant B cell response. We have therefore investigated the effects of ligating both the BCR and CR2 independently of each other, as well as with reagents to cross-link the two receptors, in order to clarify these findings. In contrast to the effects seen upon BCR ligation, we find no evidence for co-stimulatory molecule up-regulation following CR2 ligation. As antigen presentation in the absence of co-stimulation may lead to the induction of tolerogenic or regulatory signals being delivered to T cell populations, these findings imply that the role of CR2 in B cell-mediated antigen presentation is different from that of the BCR.

Animals↗
PubMed2005

Multiple-channel scaffolds to promote spinal cord axon regeneration.

As molecular, cellular, and tissue-level treatments for spinal cord injury are discovered, it is likely that combinations of such treatments will be necessary to elicit functional recovery in animal models or patients. We describe multiple-channel, biodegradable scaffolds that serve as the basis for a model to investigate simultaneously the effects on axon regeneration of scaffold architecture, transplanted cells, and locally delivered molecular agents. Poly(lactic-co-glycolic acid) (PLGA) with copolymer ratio 85:15 was used for these initial experiments. Injection molding with rapid solvent evaporation resulted in scaffolds with a plurality of distinct channels running parallel along the length of the scaffolds. The feasibility of creating scaffolds with various channel sizes and geometries was demonstrated. Walls separating open channels were found to possess void fractions as high as 89%, with accessible void fractions as high as 90% through connections 220 microm or larger. Scaffolds degraded in vitro over a period of 30 weeks, over which time-sustained delivery of a surrogate drug was observed for 12 weeks. Primary neonatal Schwann cells were distributed in the channels of the scaffold and remained viable in tissue culture for at least 48 h. Schwann-cell containing scaffolds implanted into transected adult rat spinal cords contained regenerating axons at one month post-operation. Axon regeneration was demonstrated by three-dimensional reconstruction of serial histological sections.

Absorbable Implants↗
PubMed2005

Efficient production of complement (C3d)3 fusion proteins using the baculovirus expression vector system.

Proteins fused to activated complement (C) fragments elicit enhanced immunogenicity. This "natural adjuvant" effect may have important implications when considering novel vaccination approaches. Here we describe both the construction of a novel fusion protein, consisting of a well characterized test antigen fused to multiple copies of the activated complement component (C3d)3, as well as an efficient method for its expression and production in insect cells. Using the inherent biological advantages of the baculovirus expression system, as well as applying specific infection and harvesting modifications, we have optimized the efficiency of protein production. Our modifications allow purification of fusion proteins directly from cell supernatant in a single anion exchange chromatographic step. This alleviates the requirement for the inclusion of protein affinity tags. The integrity of the purified recombinant protein was evaluated by SDS PAGE analysis, reactivity with antibodies, as well as in vivo by administration as an immunogen.

Adjuvants, Immunologic↗
PubMed2004

Distinct sequences in the cytoplasmic domain of complement receptor 2 are involved in antigen internalization and presentation.

B cells express randomly rearranged surface Ig that forms part of a multiprotein complex known as the B cell receptor (BCR). Recognition of Ag via this receptor results in its capture, internalization, proteolysis and presentation to CD4+ T cells. The recognition of Ag by CD4+ T cells is critical for the selection of individual B cells, leading to the eventual secretion of a high affinity version of the BCR as an effective circulating Ab. B cells also express other receptors that recognize Ags associated with components of innate immunity. One of these receptors, CR2, binds Ags coated with activated complement components. Studies have shown that cross-linking CR2 and the BCR with complement-tagged Ags leads to enhanced Ag presentation by B cells. In addition, Ags targeted to B cell CR2 in the absence of BCR coligation are also efficiently presented to T cells. In this report, we identify several distinct sequences within the cytoplasmic domain of mouse CR2 (mCR2) that are essential for mCR2-mediated Ag presentation in both the presence and the absence of BCR cross-linking. The finding that distinct sequences in the cytoplasmic domain of mCR2 are essential for BCR-independent Ag presentation leads us to propose a novel role for CR2.

Amino Acid Sequence↗
PubMed2004

Lowering the affinity between antigen and the B cell receptor can enhance antigen presentation.

The B cell receptor (BCR) enables antigen-specific B cells to bind, internalize and target antigens for processing into small peptide fragments. These epitopes are then expressed on the plasma membrane in association with MHC class II molecules for recognition by CD4+ T cells. The affinity of the interaction between the BCR and antigen plays an important part in determining T cell epitope generation. In this report we provide evidence that the efficiency of antigen presentation by specific B cells does not need to be directly proportional to antigen/BCR affinity. We show that increased presentation can result from lowering the affinity of the antigen/BCR interaction. This finding suggests a novel mechanism by which B cells can recruit T cell help and obtain survival signals. Activation of these cells may have consequences for the generation of the B cell repertoire.

Animals↗