A comprehensive analysis of peptides presented by HLA-A1.

Human leukocyte antigen (HLA)-A1 is one of the most common Caucasian HLA-A alleles. Here, we describe the comprehensive analysis of the HLA-A*01:01 ligand repertoire with the identification of 4735 naturally processed and presented peptides derived from 2477 source proteins. We found HLA-A*01:01 bound an equivalent number of ligands of 9 or 10 amino acids in length as well as being remarkably tolerant of even longer peptides. Indeed close to half of the HLA-A1 bound peptides identified ranged between 11 and 13 amino acids in length. These longer peptides contained the strong canonical motif of and acidic E/D residue at position 3 (P3) and Y at the C-terminus (CΩ), a motif that was still apparent in peptides of up to 18 amino acids in length. The identification of this large database of natural ligands will facilitate the refinement of predictive algorithms particularly with respect to longer peptide ligands.

A comprehensive analysis of constitutive naturally processed and presented HLA-C*04:01 (Cw4)-specific peptides.

The human B lymphoblastoid cell line C1R is widely regarded as human leukocyte antigen-A (HLA-A)/HLA-B negative and is therefore frequently exploited as a recipient cell line to study HLA class I functions. However, the normal levels of HLA-C*04:01 often hamper the investigation of introduced HLA class I allomorphs, which is particularly evident in sensitive applications such as mass spectrometry. Here we describe the comprehensive analysis of endogenous HLA-C*04:01 ligands expressed on the surface of C1R cells to (i) define a large sequence dataset of HLA-C*04:01 ligands, to (ii) refine the HLA-C*04:01 peptide-binding motif and (iii) to provide a resource that allows discrimination between peptides bound to introduced HLA class I subtypes and to the endogenous HLA-C*04:01 molecules.

Indicators of dynamic stability in transtibial prosthesis users.

An improved understanding of factors related to dynamic stability in lower-limb prosthesis users is important, given the high occurrence of falls in this population. Current methods of assessing stability are unable to adequately characterize dynamic stability over a variety of walking conditions. F-Scan Mobile has been used to collect plantar pressure data and six extracted parameters were useful measures of dynamic stability. The aim of this study was to investigate dynamic stability in individuals with unilateral transtibial amputation based on these six parameters. Twenty community ambulators with a unilateral transtibial amputation walked over level ground, uneven ground, stairs, and a ramp while plantar pressure data were collected. For each limb (intact and prosthetic) and condition, six stability parameters related to plantar center-of-pressure perturbations and gait temporal parameters, were computed from the plantar pressure data. Parameter values were compared between limbs, walking condition, and groups (unilateral transtibial prosthesis users and able-bodied subjects). Differences in parameters were found between limbs and conditions, and between prosthesis users and able-bodied individuals. Further research could investigate optimizing parameter calculations for unilateral transtibial prosthesis users and define relationships between potential for falls and the dynamic stability measures.

Transglutaminase-catalyzed transamidation: a novel mechanism for Rac1 activation by 5-hydroxytryptamine2A receptor stimulation.

Transglutaminase (TGase)-induced activation of small G proteins via 5-hydroxytryptamine (HT)(2A) receptor signaling leads to platelet aggregation (Cell 115:851-862, 2003). We hypothesize that stimulation of 5-HT(2A) receptors in neurons activates TGase, resulting in transamidation of serotonin to a small G protein, Rac1, thereby constitutively activating Rac1. Using immunoprecipitation and immunoblotting, we show that, in rat cortical cell line A1A1v, serotonin increases TGase-catalyzed transamidation of Rac1. This transamidation occurs in both undifferentiated and differentiated cells. Treatment with a 5-HT(2A/2C) receptor agonist 2,5-dimethoxy-4-iodoamphetamine, but not the 5-HT(1A) receptor agonist 5-hydroxy-2-dipropylamino tetralin, increases transamidation of Rac1 by TGase. In A1A1v cells, 5-HT(2A) receptors mediate the transamidation reaction because expression of 5-HT(2C) receptors was not detectable and the selective 5-HT(2A) receptor antagonist blocked transamidation. Time course studies demonstrate that transamidation of Rac1 is significantly elevated after 5 and 15 min of serotonin treatment, but returns it to control levels after 30 min. The activity of Rac1 is also transiently increased following serotonin stimulation. Inhibition of TGase by cystamine or small interfering RNA reduces TGase modification of Rac1, and cystamine also prevents Rac1 activation. Serotonin itself is bound to Rac1 by TGase following 5-HT(2A) receptor stimulation as demonstrated by coimmunoprecipitation experiments and a dose-dependent decrease of serotonin-associated Rac1 by cystamine. These data support the hypothesis that Rac1 activity is transiently increased due to TGase-catalyzed transamidation of serotonin to Rac1 via stimulation of 5-HT(2A) receptors. Activation of Rac1 via TGase is a novel effector and second messenger of the 5-HT(2A) receptor-signaling cascade in neurons.

Endogenous and exogenous factors contributing to the surface expression of HLA B27 on mutant APC.

We have examined the expression of HLA B*2705 in the mutant cell line 721.220, which lacks endogenous HLA A and B alleles and expresses a defective tapasin molecule. Several peptide sensitive mAbs distinguish between HLA B*2705 expressed on the surface of 721.220 cells (B27.220) and 721.220 cells co-transfected with human tapasin (B27.220.hTsn). This differential staining defines subtle differences in the conformation of HLA B27, which most likely reflect changes in the repertoire of antigenic peptides bound to B27 in the presence and absence of wild type tapasin. HLA B27 molecules expressed on the surface of 721.220 display increased levels of "free" B27 heavy chain (HC-10 staining), an epitope that is dependent on TAP-translocated peptides. The conformation and stability of B27 molecules was examined by investigating the integrity of mAb epitopes and the half-lives of these complexes on cells cultured with and without serum. The decay of surface B27 epitopes occurred more rapidly in B27.220 and this effect was exaggerated in serum free media. Importantly, the decay of surface B27 molecules in B27.220.hTsn cells was characterized by an early increase in HC-10 staining when the cells were grown in serum free media. This decay of B27 molecules via HC-10 reactive intermediates was not observed in B27.220 cells, implying molecules on these cells may already have passed through this stage prior to surface expression. Taken together these observations indicate that tapasin has a significant contribution to the composition and stability of the B27-bound peptide repertoire.

The role of radial elastic properties in the development of aortic dissections.

PURPOSE: The response of the upper and lower thoracic aorta to radial tensile stresses was compared with the response to circumferential and longitudinal stresses to understand the role of tensile stress in the tearing phase of an aortic dissection. METHODS: Square tissue samples (1.6 by 1.6 cm) were cut from the upper and lower segments of six porcine thoracic aortas and were elongated in the radial direction with a tensile testing machine. The radial extensibility of the thoracic aorta was compared with adjacent tissue samples that were tested in tension in the circumferential and longitudinal directions based on Young's modulus (ie, the ratio of tensile stress to strain). RESULTS: The elastic properties of the thoracic aorta in the radial direction were markedly different from both the circumferential and longitudinal properties. The average Young's modulus (calculated immediately before failing) was significantly lower in the radial direction for both the upper and lower thoracic segments (61.4 +/- 4.3 kPa, SEM) than the Young's modulus of corresponding segments in the circumferential and longitudinal directions that were not tested to failure (151.1 +/- 8.6 kPa and 112.7 +/- 9.2 kPa, respectively; P <. 05). Sections 7 micrometer thick were collected from four samples obtained from one upper thoracic aorta that were strained at 0, 1.0, 2.5, and 4.0 and then stained either with Movat's pentachrome or with hematoxylin and eosin. Histological analysis of the samples stressed in the radial direction revealed that smooth muscle cells were torn loose from their attachments to each other and to adjacent elastin. CONCLUSION: Although the aorta normally functions under radial compressive stresses associated with lumen blood pressure, these results show that the aorta tears radially at a much lower value of stress than would have been predicted from previous studies that have reported longitudinal and circumferential Young's modulus. This could explain why dissections propagate readily once the initial tear occurs.

An evaluation of the effectiveness of a videotape programme on interobserver reliability in outcome assessment for fibromyalgia.

AIMS: A study was designed to assess the effects of a standardized instructional videotape on training senior medical students to acceptable levels of reliability in performing several commonly used observer dependent outcome measures in patients with fibromyalgia (FM). METHODS: During a single day, six third-year medical students independently examined six patients with FM in predetermined order using a Latin Square design, before and after viewing a standardized videotape which demonstrated methods for performing doiorimelry and for delecting skinfold tenderness and reactive hyperaemia. Reliability coefficients were calculated based on the variance components of the Analysis of Variance (ANOVA) table. RESULTS: Prestandardization reliability coefficients were <0.80 for 8 measures. Following standardization all reliability coefficients, but one, approximated or exceeded 0.80. CONCLUSIONS: An important and beneficial effect of the standardization procedure was noted for several outcome variables. Such improvements in observer agreement have important implications for training senior medical students to perform quantitative measurement in the longitudinal management of FM patients in clinical practice. The use of a videotape to achieve this goal has obvious cost and convenience advantages compared with personal onc-on-one or small group training procedures.