Real-time measurement of antigenic peptide binding to empty and preloaded single-chain MHC class I molecules

Cytotoxic T lymphocytes (CTL) recognize peptides in association with major histocompatibility complex (MHC) class I proteins, but how peptides bind to class I is not well understood. We used a fluorescence technique to measure antigenic peptide binding to a soluble, single-chain Kd (SC-Kd) molecule in which the Kd heavy chain was connected by a 15-residue link to β2-microglobulin. Peptides were covalently labeled at their N terminus with dansyl, and binding of dansylated Kd-restricted peptides to SC-Kd resulted in significant fluorescence enhancement, which could be inhibited by unmodified Kd-restricted peptides. Real-time binding of a dansylated peptide could be followed by monitoring the fluorescence at 530 nm. The dansylated Plasmodium berghei circumsporozoite (PbCS) 263–260 peptide bound to “empty” SC-Kd with an association rate constant of 1140 M−1s−1, and the subsequent spontaneous dissociation of the SC-Kd-peptide complex was slow. The dissociation increased dramatically after addition of excess unlabeled PbCS 253–260 peptide, but with a slower association constant for unlabeled peptide, 77 Msup\u3e−1s−1. Thus, the Kd-peptide complex on the surface of antigen-presenting cells should be stable, but high concentrations of peptides in the endoplasmic reticulum (ER) lumen would allow for peptide exchange on Kd before export to the surface. The apparent activation energy for PbCS 253–260 peptide binding to SC-Kd was 6.78 ± 0.64 kcal/mole, similar to values previously reported for antigen-antibody interactions

Purification and ligand binding of a soluble class I MHC molecule consisting of the first three domains of H-2Kd fused to b2-microglobulin expressed in the baculovirus/insect cell system

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule

Interactions between ants and aphidophagous and coccidophagous ladybirds

Aphidophagous and coccidophagous coccinellids come into conflict with homopteran-tending ants for access to food. Antagonistic interactions between coccinellids and ants may be competitive or non-competitive. Competitive interactions occur when coccinellids attack aphids or coccids that are being tended by ants for honeydew. Non-competitive interactions include all interactions away from ant-tended homopteran colonies. We here review observations and studies of such interactions. We note that most competitive interactions occur at times when untended aphids/coccids are scarce. We describe the chemical and physical defences that coccinellids use against ant aggression and consider whether these have evolved as general anti-predator deterrents or specifically in response to ants. Myrmecophilous coccinellids are then considered, with particular focus on the two most studied species, Coccinella magnifica and Platynaspis luteorubra. We note that the myrmecophily of the two species has the same adaptive rationale-to enable the ladybirds to prey on ant-tended aphids at times of aphid scarcity-but that it is based on different traits to facilitate life with ants. Finally, we consider the role of ants in the evolution of habitat specialisation in some coccinellids

Efficacy and safety of two rituximab biosimilars for treating immune thrombocytopenia: a reference-product matched study

The emergence of rituximab biosimilars offers the prospect of significant savings to the healthcare system. However, these drugs have never been evaluated for treating immune thrombocytopenia (ITP). This was an observational, matched study. We included adults who received a rituximab biosimilar for ITP. Each rituximab-naïve biosimilar patient was matched with two controls from the historic ITP-ritux registry. For non-naïve patients, we compared the response to the biosimilar with that observed with the reference product. Response status was defined according to international criteria. We included 107 patients; 55 receiving Rixathon™ and 52 Truxima™. Three months after the first infusion of rituximab biosimilars, the overall response rate was 47/74 (63.5%) versus 76/142 (53.5%) for the matched controls receiving the reference product (p = .13). The 3-month overall response rate was 76.5% for Rixathon™ versus 51.5% for the matched control group (p = .01) and 21/40 (52.5%) for Truxima™ versus 41/74 (55.4%) for the matched controls (p = .81). For non-naïve patients, the response pattern was similar to that observed previously with the reference product. Safety was analogous to that observed with the reference product. Rituximab biosimilars seemed safe and effective for ITP treatment