Paediatric age-dependent serum transferrin isoform distribution studied by HPLC.

Background: Human serum transferrin shows different transferrin isoforms with e.g. a varying amount of glycosylation, resulting in asialo-, mono-, di-, up to octasialotransferrin.

Defining the directionality and quality of influenza virus-specific CD8+ T cell cross-reactivity in individuals infected with hepatitis C virus.

Cross-reactivity of murine and recently human CD8(+) T cells between different viral peptides, i.e., heterologous immunity, has been well characterized.

T cell receptor engagement of peptide-major histocompatibility complex class I does not modify CD8 binding.

Activation of cytotoxic T cells is initiated by engagement of the T-cell receptor (TCR) with peptide-major histocompatibility class I complexes (pMHCI). The CD8 co-receptor also binds to pMHCI, but at a distinct site, and allows the potential for tripartite TCR/pMHCI/CD8 interactions, which can increase T cell antigen sensitivity. There has been a substantial interest in the effect of the pMHCI/CD8 interaction upon TCR/pMHCI engagement, and several conflicting studies have examined this event, using the soluble extracellular domains of CD8 and the TCR, by surface plasmon resonance.

Insights into post-translational processing of beta-galactosidase in an animal model resembling late infantile human G-gangliosidosis.

G(M1)-gangliosidosis is a lysosomal storage disorder caused by a deficiency of ss-galactosidase activity. Human GM1-gangliosidosis has been classified into three forms according to the age of clinical onset and specific biochemical parameters. In the present study, a canine model for type II late infantile human GM1-gangliosidosis was investigated 'in vitro' in detail.

Systematic identification of C. elegans miRISC proteins, miRNAs, and mRNA targets by their interactions with GW182 proteins AIN-1 and AIN-2.

MicroRNAs (miRNAs) regulate gene expression for diverse functions, but only a limited number of mRNA targets have been experimentally identified. We show that GW182 family proteins AIN-1 and AIN-2 act redundantly to regulate the expression of miRNA targets, but not miRNA biogenesis. Immunoprecipitation (IP) and mass spectrometry indicate that AIN-1 and AIN-2 interact only with miRNA-specific Argonaute proteins ALG-1 and ALG-2 and with components of the core translational initiation complex.

Use of peptide-major histocompatibility complex tetramer technology to study interactions between Staphylococcus aureus proteins and human cells.

In this study, we report the use of peptide-major histocompatibility complex tetramer technology to study the interactions that occur between Staphylococcus aureus proteins and human leukocytes. We demonstrated that this technology can be used to study the activity of superantigens such as toxic shock syndrome toxin 1 and also found that despite similarities to known proteins (i.e.

Coreceptor CD8-driven modulation of T cell antigen receptor specificity.

The CD8 coreceptor modulates the interaction between the T cell antigen receptor (TCR) and peptide-major histocompatibility class I (pMHCI). We present evidence that CD8 not only modifies the affinity of cognate TCR/pMHCI binding by altering both the association rate and the dissociation rate of the TCR/pMHCI interaction, but modulates the sensitivity (triggering threshold) of the TCR as well, by recruiting TCR/pMHCI complexes to membrane microdomains at a rate which depends on the affinity of MHCI/CD8 binding. Mathematical analysis of these modulatory effects indicates that a T cell can alter its functional avidity for its agonists by regulating CD8 expression, and can rearrange the relative potencies of each of its potential agonists.

Crystallization and preliminary X-ray structural studies of a Melan-A pMHC-TCR complex.

Melanocytes are specialized pigmented cells that are found in all healthy skin tissue. In certain individuals, diseased melanocytes can form malignant tumours, melanomas, which cause the majority of skin-cancer-related deaths. The melanoma-associated antigenic peptides are presented on cell surfaces via the class I major histocompatibility complex (MHC).

Ethylmalonic encephalopathy: clinical and biochemical observations.

Ethylmalonic encephalopathy (EE) is a rare, recently defined inborn error of metabolism which affects the brain, gastrointestinal system and peripheral blood vessels and is characterized by a unique constellation of clinical and biochemical features. A 7-month-old male, who presented with psychomotor retardation, chronic diarrhea and relapsing petechiae is described with the objective of highlighting the biochemical and neuroradiological features of this disorder as well as the effect of high-dose riboflavin therapy. Urinary organic acid analysis revealed markedly increased excretion of ethylmalonic acid, isobutyrylglycine, 2-methylbutyrylglycine and isovalerylglycine.

The HLA A*0201-restricted hTERT(540-548) peptide is not detected on tumor cells by a CTL clone or a high-affinity T-cell receptor.

Tumor-associated human telomerase reverse transcriptase (hTERT) is expressed in >85% of human tumors but not in most normal cells. As a result, this antigen has received considerable attention from those interested in cancer immunotherapy. Specifically, there has been strong interest in MHC class I-associated peptides derived from hTERT because these are expressed on the cell surface and thus may enable the targeting of tumor cells.

Availability of a diversely avid CD8+ T cell repertoire specific for the subdominant HLA-A2-restricted HIV-1 Gag p2419-27 epitope.

HLA-A2-restricted CTL responses to immunodominant HIV-1 epitopes do not appear to be very effective in the control of viral replication in vivo. In this study, we studied human CD8+ T cell responses to the subdominant HLA-A2-restricted epitope TV9 (Gag p24(19-27), TLNAWVKVV) to explore the possibility of increasing its immune recognition. We confirmed in a cohort of 313 patients, infected by clade B or clade C viruses, that TV9 is rarely recognized.

Different T cell receptor affinity thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte activation and tetramer binding properties.

T cells have evolved a unique system of ligand recognition involving an antigen T cell receptor (TCR) and a coreceptor that integrate stimuli provided by the engagement of peptide-major histocompatibility complex (pMHC) antigens. Here, we use altered pMHC class I (pMHCI) molecules with impaired CD8 binding (CD8-null) to quantify the contribution of coreceptor extracellular binding to (i) the engagement of soluble tetrameric pMHCI molecules, (ii) the kinetics of TCR/pMHCI interactions on live cytotoxic T lymphocytes (CTLs), and (iii) the activation of CTLs by cell-surface antigenic determinants. Our data indicate that the CD8 coreceptor substantially enhances binding efficiency at suboptimal TCR/pMHCI affinities through effects on both association and dissociation rates.

Human TCR-binding affinity is governed by MHC class restriction.

T cell recognition is initiated by the binding of TCRs to peptide-MHCs (pMHCs), the interaction being characterized by weak affinity and fast kinetics. Previously, only 16 natural TCR/pMHC interactions have been measured by surface plasmon resonance (SPR). Of these, 5 are murine class I, 5 are murine class II, and 6 are human class I-restricted responses.

Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class I invariant region.

CD8(+) cytotoxic T lymphocytes (CTL) are key determinants of immunity to intracellular pathogens and neoplastic cells. Recognition of specific antigens in the form of peptide-MHC class I complexes (pMHCI) presented on the target cell surface is mediated by T cell receptor (TCR) engagement. The CD8 coreceptor binds to invariant domains of pMHCI and facilitates antigen recognition.

CD8 exerts differential effects on the deployment of cytotoxic T lymphocyte effector functions.

Cytotoxic T lymphocytes (CTL) are equipped with a range of effector functions that contribute both to the control of intracellular pathogens and dysregulated cellular proliferation and to the development of certain immunopathologies such as autoimmune disease. Qualitative analyses of various CTL responses have revealed substantial heterogeneity in the diversity of functions that are mobilized in response to antigen. Here, we studied the influence of the CD8 co-receptor, which is known to enhance antigen recognition by CTL, on the secretion of eight different cytokines and chemokines by human CTL clones using flow cytometric bead array.

Potent T cell agonism mediated by a very rapid TCR/pMHC interaction.

The interaction between T cell receptors (TCR) and peptide-major histocompatibility complex (pMHC) antigens can lead to varying degrees of agonism (T cell activation), or antagonism. The P14 TCR recognises the lymphocytic choriomeningitis virus (LCMV)-derived peptide, gp33 residues 33-41 (KAVYNFATC), presented in the context of H-2D(b). The cellular responses to various related H-2D(b) peptide ligands are very well characterised, and P14 TCR-transgenic mice have been used extensively in models of virus infection, autoimmunity and tumour rejection.

Functional and biophysical characterization of an HLA-A*6801-restricted HIV-specific T cell receptor.

HLA-A*6801 exhibits several unusual features. First, it is known to bind weakly to CD8 due to the presence of an A245V substitution in the alpha3 domain. Second, it is able to accommodate unusually long peptides as a result of peptide 'kinking' in the binding groove.

Inherited metabolic disease in companion animals: searching for nature's mistakes.

Inborn errors of metabolism are caused by genetic defects in intermediary metabolic pathways. Although long considered to be the domain of human paediatric medicine, they are also recognised with increasing frequency in companion animals. The diagnosis of diseased animals can be achieved by searching for abnormal metabolites in body fluids, although such screening programmes have, until now, not been widely available to the small animal clinician.

Agr interference between clinical Staphylococcus aureus strains in an insect model of virulence.

Repression of virulence by Staphylococcus aureus strains from different Agr groups has been demonstrated in vitro and is proposed as a means of competitive interference. Here, using the insect Manduca sexta, we show for the first time that this interference also occurs in vivo within a mixed population.

The cytotoxic T cell response to peptide analogs of the HLA-A*0201-restricted MUC1 signal sequence epitope, M1.2.

The mucin MUC1 molecule is overexpressed on a variety of adenocarcinomas and is thus, a potential target for immunotherapy. Of the MUC1 peptides that bind to HLA-A*0201(A2), M1.2 (LLLLTVLTV) from the signal sequence appears to be the most immunogenic in humans.

Argininosuccinate lyase deficiency (ASL) and carbohydrate-deficient transferrin (CDT): experience with four independent CDT analysis methods--misleading results given by the %CDT TIA assay.

Background: Chronic liver disease can cause false-positive carbohydrate-deficient transferrin (CDT) results mimicking chronic alcohol abuse. We tested whether argininosuccinate lyase deficiency (ASL), a genetic disorder of the urea cycle with hepatomegaly and biochemical hepatitis, causes increased CDT results and whether this depends on the analytical method.

Methods: Seven serum samples from four ASL patients without alcohol abuse were analyzed by capillary electrophoresis, HPLC, particle-enhanced immunonephelometry with monoclonal CDT antibodies, and microcolumn CDT and non-CDT fractionation followed by a turbidimetric immunoassay with transferrin antibodies (%CDT TIA).

Anti-coreceptor antibodies profoundly affect staining with peptide-MHC class I and class II tetramers.

The T cell coreceptors CD8 and CD4 bind to invariable regions of peptide-MHC class I (pMHCI) and class II (pMHCII) molecules, respectively, and facilitate antigen recognition by a number of mechanisms. It is established that some antibodies (Ab) specific for the CD8 molecule, which stabilizes TCR/pMHCI interactions, can alter the binding of pMHCI tetramers to cell surface TCR. In contrast, the extremely weak pMHCII/CD4 interaction does not stabilize TCR/pMHCII interactions or contribute to cognate tetramer binding; consequently, it is assumed that anti-CD4 Ab do not affect pMHCII binding.

T-cell responses directed against multiple HLA-A*0201-restricted epitopes derived from Wilms' tumor 1 protein in patients with leukemia and healthy donors: identification, quantification, and characterization.

Purpose: Antigens derived from the Wilms' tumor (WT1) protein, which is overexpressed in leukemias, are attractive targets for immunotherapy. Four HLA-A*0201-restricted WT1-derived epitopes have been identified: WT37, WT126, WT187, and WT235. We determined the natural immunogenecity of these antigens in patients with hematologic malignancies and healthy donor.