Negative and positive selection of antigen-specific cytotoxic T lymphocytes affected by the α3 domain of MHC I molecules

THE α1 and α2 domains of major histocompatibility complex (MHC) class I molecules function in the binding and presentation of foreign peptides to the T-cell antigen receptor and control both negative and positive selection of the T-cell repertoire. Although the α3 domain of class I is not involved in peptide binding, it does interact with the T-cell accessory molecule, CDS. CDS is important in the selection of T cells as anti-CDS antibody injected into perinatal mice interfers with this process. We previously used a hybrid class I molecule with the α1/α2 domains from L^d and the α3 domain from Q7^b and showed that this molecule binds an L^d-restricted peptide but does not interact with CD8-dependent cytotoxic T lymphocytes. Expression of this molecule in transgenic mice fails to negatively select a subpopulation of anti-L^d cytotoxic T lymphocytes. In addition, positive selection of virus-specific L^d-restricted cytotoxic T lymphocytes does not occur. We conclude that besides the α1/α2 domains of class I, the α3 domain plays an important part in both positive and negative selection of antigen-specific cells

In vivo measurement of bending stiffness in fracture healing

BACKGROUND: Measurement of the bending stiffness a healing fracture represents a valid variable in the assessment of fracture healing. However, currently available methods typically have high measurement errors, even for mild pin loosening. Furthermore, these methods cannot provide actual values of bending stiffness, which precludes comparisons among individual fractures. Thus, even today, little information is available with regards to the fracture healing pattern with respect to actual values of bending stiffness. Our goals were, therefore: to develop a measurement device that would allow accurate and sensitive measurement of bending stiffness, even in the presence of mild pin loosening; to describe the course of healing in individual fractures; and help to evaluate whether the individual pattern of bending stiffness can be predicted at an early stage of healing. METHODS: A new measurement device has been developed to precisely measure the bending stiffness of the healing fracture by simulating four-point-bending. The system was calibrated on aluminum models and intact tibiae. The influence of pin loosening on measurement error was evaluated. The system was tested at weekly intervals in an animal experiment to determine the actual bending stiffness of the fracture. Transverse fractures were created in the right tibia of twelve sheep, and then stabilized with an external fixator. At ten weeks, bending stiffness of the tibiae were determined in a four-point-bending test device to validate the in-vivo-measurement data. RESULTS: In-vivo bending stiffness can be measured accurately and sensitive, even in the early phase of callus healing. Up to a bending stiffness of 10 Nm/degree, measurement error was below 3.4% for one pin loose, and below 29.3% for four pins loose, respectively. Measurement of stiffness data over time revealed a significant logarithmic increase between the third and seventh weeks, whereby the logarithmic rate of change among sheep was similar, but started from different levels. Comparative measurements showed that early individual changes between the third and fourth weeks can be used as a predictor of bending stiffness at seven weeks (r = 0.928) and at ten weeks (r = 0.710). CONCLUSION: Bending stiffness can be measured precisely, with less error in the case of pin loosening. Prediction of the future healing course of the individual fracture can be assessed by changes from the third to the fourth week, with differences in stiffness levels. Therefore, the initial status of the fracture seems to have a high impact on the individual healing course

Further investigation of the role of HLA-DPB1 in adult Hodgkin's disease (HD) suggests an influence on susceptibility to different HD subtypes

It has been suggested in a number of studies that susceptibility to adult Hodgkin's disease (HD) is influenced by the HLA class II region, and specifically by alleles at the HLA-DPB1 locus. Since HD is diagnostically complex, it is not clear whether different HLA-DPB1 alleles confer susceptibility to different HD subtypes. To clarify this we have extended a previous study to type DPB1 alleles in 147 adult HD patients from a single centre. We have analysed patients with nodular sclerosing (NS), mixed cellularity (MC) or lymphocyte predominant (LP) HD, and gender in relation to HLA-DPB1 type, in comparison with 183 adult controls. The results confirmed previously reported associations of DPB1*0301 with HD susceptibility (relative risk (RR) = 1.42; 95% confidence interval (CI) 0.86-2.36) and DPB1*0201 with resistance to HD (RR = 0.49; CI 0.27-0.90). However, analysis by HD subtype and gender showed that *0301-associated susceptibility was confined to females with HD (RR = 2.46; CI 1.02-5.92), and *0201-associated resistance to females with NS-HD (RR = 0.28; CI 0.10-0.79). Susceptibility to NS-HD was also associated in females with *1001 (RR = 11.73; CI 1.32-104.36), and resistance with *1101 (RR = 0.08; CI 0.01-0.65). In contrast, susceptibility to LP-HD was associated in males with *2001 (RR = 32.14; CI 3.17-326.17), and to MC-HD with *3401 (RR = 16.78; CI 2.84-99.17). Comparison of DPB1-encoded polymorphic amino-acid frequencies in patients and controls showed that susceptibility to MC-HD was associated with Leucine at position 35 of DPB1 (RR = 8.85; CI 3.04-25.77), Alanine-55 (RR = 15.17; CI 2.00-115.20) and Valine-84 (RR = 15.94; CI 3.55-71.49). In contrast, Glutamic acid 69 was significantly associated with resistance to MC-HD (RR = 0.14; CI 0.03-0.60). Certain DPB1 alleles and individual DPbeta1 polymorphic amino acid residues may thus affect susceptibility and resistance to specific HD subtypes. This may be through their influence on the binding of peptides derived from an HD-associated infectious agent, and the consequent effect on immune responses to the agent

Pharmacokinetics and pharmacodynamics of fenoldopam mesylate for blood pressure control in pediatric patients

<p>Abstract</p> <p>Background</p> <p>Fenoldopam mesylate, a selective dopamine1-receptor agonist, is used by intravenous infusion to treat hypertension in adults. Fenoldopam is not approved by the FDA for use in children; reports describing its use in pediatrics are limited. In a multi-institutional, placebo controlled, double-blind, multi-dose trial we determined the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics and side-effect profile of fenoldopam in children.</p> <p>Methods</p> <p>Seventy seven (77) children from 3 weeks to 12 years of age scheduled for surgery in which deliberate hypotension would be induced were enrolled. Patients were randomly assigned to one of five, blinded treatment groups (placebo or fenoldopam 0.05, 0.2, 0.8, or 3.2 mcg/kg/min iv) for a 30-minute interval after stabilization of anesthesia and placement of vascular catheters. Following the 30-minute blinded interval, investigators adjusted the fenoldopam dose to achieve a target mean arterial pressure in the open-label period until deliberate hypotension was no longer indicated (e.g., muscle-layer closure). Mean arterial pressure and heart rate were continuously monitored and were the primary endpoints.</p> <p>Results</p> <p>Seventy-six children completed the trial. Fenoldopam at doses of 0.8 and 3.2 mcg/kg/min significantly reduced blood pressure (p < 0.05) during the blinded interval, and doses of 1.0–1.2 mcg/kg/min resulted in continued control of blood pressure during the open-label interval. Doses greater than 1.2 mcg/kg/min during the open-label period resulted in increasing heart rate without additional reduction in blood pressure. Fenoldopam was well-tolerated; side effects occurred in a minority of patients. The PK/PD relationship of fenoldopam in children was determined.</p> <p>Conclusion</p> <p>Fenoldopam is a rapid-acting, effective agent for intravenous control of blood pressure in children. The effective dose range is significantly higher in children undergoing anesthesia and surgery (0.8–1.2 mcg/kg/min) than as labeled for adults (0.05–0.3 mcg/kg/min). The PK and side-effect profiles for children and adults are similar.</p

Composite likelihood estimation of demographic parameters

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background: Most existing likelihood-based methods for fitting historical demographic models to DNA sequence polymorphism data to do not scale feasibly up to the level of whole-genome data sets. Computational economies can be achieved by incorporating two forms of pseudo-likelihood: composite and approximate likelihood methods. Composite likelihood enables scaling up to large data sets because it takes the product of marginal likelihoods as an estimator of the likelihood of the complete data set. This approach is especially useful when a large number of genomic regions constitutes the data set. Additionally, approximate likelihood methods can reduce the dimensionality of the data by summarizing the information in the original data by either a sufficient statistic, or a set of statistics. Both composite and approximate likelihood methods hold promise for analyzing large data sets or for use in situations where the underlying demographic model is complex and has many parameters. This paper considers a simple demographic model of allopatric divergence between two populations, in which one of the population is hypothesized to have experienced a founder event, or population bottleneck. A large resequencing data set from human populations is summarized by the joint frequency spectrum, which is a matrix of the genomic frequency spectrum of derived base frequencies in two populations. A Bayesia

Predicting Class II MHC-Peptide binding: a kernel based approach using similarity scores

BACKGROUND: Modelling the interaction between potentially antigenic peptides and Major Histocompatibility Complex (MHC) molecules is a key step in identifying potential T-cell epitopes. For Class II MHC alleles, the binding groove is open at both ends, causing ambiguity in the positional alignment between the groove and peptide, as well as creating uncertainty as to what parts of the peptide interact with the MHC. Moreover, the antigenic peptides have variable lengths, making naive modelling methods difficult to apply. This paper introduces a kernel method that can handle variable length peptides effectively by quantifying similarities between peptide sequences and integrating these into the kernel. RESULTS: The kernel approach presented here shows increased prediction accuracy with a significantly higher number of true positives and negatives on multiple MHC class II alleles, when testing data sets from MHCPEP [1], MCHBN [2], and MHCBench [3]. Evaluation by cross validation, when segregating binders and non-binders, produced an average of 0.824 A(ROC )for the MHCBench data sets (up from 0.756), and an average of 0.96 A(ROC )for multiple alleles of the MHCPEP database. CONCLUSION: The method improves performance over existing state-of-the-art methods of MHC class II peptide binding predictions by using a custom, knowledge-based representation of peptides. Similarity scores, in contrast to a fixed-length, pocket-specific representation of amino acids, provide a flexible and powerful way of modelling MHC binding, and can easily be applied to other dynamic sequence problems

The Role of relA and spoT in Yersinia pestis KIM5+ Pathogenicity

The ppGpp molecule is part of a highly conserved regulatory system for mediating the growth response to various environmental conditions. This mechanism may represent a common strategy whereby pathogens such as Yersinia pestis, the causative agent of plague, regulate the virulence gene programs required for invasion, survival and persistence within host cells to match the capacity for growth. The products of the relA and spoT genes carry out ppGpp synthesis. To investigate the role of ppGpp on growth, protein synthesis, gene expression and virulence, we constructed a ΔrelA ΔspoT Y. pestis mutant. The mutant was no longer able to synthesize ppGpp in response to amino acid or carbon starvation, as expected. We also found that it exhibited several novel phenotypes, including a reduced growth rate and autoaggregation at 26°C. In addition, there was a reduction in the level of secretion of key virulence proteins and the mutant was>1,000-fold less virulent than its wild-type parent strain. Mice vaccinated subcutaneously (s.c.) with 2.5×104 CFU of the ΔrelA ΔspoT mutant developed high anti-Y. pestis serum IgG titers, were completely protected against s.c. challenge with 1.5×105 CFU of virulent Y. pestis and partially protected (60% survival) against pulmonary challenge with 2.0×104 CFU of virulent Y. pestis. Our results indicate that ppGpp represents an important virulence determinant in Y. pestis and the ΔrelA ΔspoT mutant strain is a promising vaccine candidate to provide protection against plague

An Assay to Monitor HIV-1 Protease Activity for the Identification of Novel Inhibitors in T-Cells

The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR). The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP) gene only in the presence of an effective PR Inhibitor (PI). Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells

A Communal Bacterial Adhesin Anchors Biofilm and Bystander Cells to Surfaces

While the exopolysaccharide component of the biofilm matrix has been intensively studied, much less is known about matrix-associated proteins. To better understand the role of these proteins, we undertook a proteomic analysis of the V. cholerae biofilm matrix. Here we show that the two matrix-associated proteins, Bap1 and RbmA, perform distinct roles in the biofilm matrix. RbmA strengthens intercellular attachments. In contrast, Bap1 is concentrated on surfaces where it serves to anchor the biofilm and recruit cells not yet committed to the sessile lifestyle. This is the first example of a biofilm-derived, communally synthesized conditioning film that stabilizes the association of multilayer biofilms with a surface and facilitates recruitment of planktonic bystanders to the substratum. These studies define a novel paradigm for spatial and functional differentiation of proteins in the biofilm matrix and provide evidence for bacterial cooperation in maintenance and expansion of the multilayer biofilm