Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides

We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.     

Childhood adversities as risk factors for alexithymia and other aspects of affect dysregulation in adulthood

BACKGROUND: Affect regulation is assumed to be a biologically based function that can become disrupted by inadequate parenting and by traumatic experiences. We studied the relation between the perceived parental parenting style, and sexual and physical abuse, with alexithymia, dissociation, anxiety and depression. METHODS: In a cross-sectional study psychiatric outpatients were administered a structured interview on childhood physical and sexual abuse and they completed a number of questionnaires about the parenting styles of their parents, and about alexithymia, dissociation and mood pathology. RESULTS: Maternal and paternal parenting styles were moderately correlated with alexithymia and depression. The paternal parenting style was also correlated with dissociation. Optimal parenting of one of the parents had a buffering effect on the degree of alexithymia, but not on the severity of other forms of affect dysregulation. The effect of sexual or physical abuse did not add to that of parental parenting style in terms of predicting affect dysregulation. However, a positively perceived maternal parenting style was found to have a buffering effect in terms of the degree of alexithymia, if sexual abuse had also taken place. CONCLUSIONS: Perceived parenting does appear to be of some significance in the development of alexithymia. Optimal parenting of one of the parents may protect against the development of alexithymia when the parenting of the other parent is perceived as non-optimal. However, it is likely that other factors besides parental care and sexual or physical abuse play an important role in the development of an adequate affect regulation.     

Neural networks involved in mathematical thinking: evidence from linear and non-linear analysis of electroencephalographic activity

Using linear and non-linear methods, electroencephalographic (EEG) signals were measured at various brain regions to provide information regarding patterns of local and coordinated activity during performance of three arithmetic tasks (number comparison, single-digit multiplication, and two-digit multiplication) and two control tasks that did not require arithmetic operations. It was hypothesized that these measures would reveal the engagement of local and increasingly complex cortical networks as a function of task specificity and complexity. Results indicated regionally increased neuronal signalling as a function of task complexity at frontal, temporal and parietal brain regions, although more robust task-related changes in EEG-indices of activation were derived over the left hemisphere. Both linear and non-linear indices of synchronization among EEG signals recorded from over different brain regions were consistent with the notion of more "local" processing for the number comparison task. Conversely, multiplication tasks were associated with a widespread pattern of distant signal synchronizations, which could potentially indicate increased demands for neural networks cooperation during performance of tasks that involve a greater number of cognitive operations.     

Age, gender and litter-related variation in T-lymphocyte cytokine production in young pigs

The capacity of farm animals to produce cytokines could be an important determinant of robustness and health. From research in rodents and humans it appears that the production and the balance of T helper 1 (Th1) and T helper 2 (Th2)-type cytokines influences susceptibility to autoimmune and infectious diseases. It is known that pigs show a large variation in many immune response parameters. So far the extent of individual variation in the production of Th1- and Th2-type cytokines in commercial outbred pigs has not been reported. In the current experiment we determined mRNA expression, as well as protein production of cytokines in 32 pigs from eight litters. From each litter two male and two female pigs were tested at 2, 5 and 8 weeks of age. Two Th1-type cytokines, interleukin (IL)-2 and interferon (IFN)-gamma, and two Th2-type cytokines, IL-4 and IL-10, were measured after phytohaemagglutinin (PHA)-stimulation of blood mononuclear cells. Cytokine production and the Th1/Th2-ratio were highly variable. The variation in cytokine protein production was moderately consistent across ages, i.e. pigs that produced high levels of cytokine at 2 weeks of age tended to do so as well at 5 and 8 weeks of age. Cytokine production tended to increase with age, and gilts and boars differed in their IL-2/IL-4 ratio. Unexpectedly, age, gender and litter effects often differed for mRNA and protein production data. We hypothesize that cytokine production is a consistent trait in pigs, especially at the protein production level. Future investigations in more animals and across a wider age range are necessary.     

Both Fc receptors and lymphocyte-function-associated antigen 1 on human Tγ lymphocytes are required for antibody-dependent cellular cytotoxicity (killer cell activity)

A monoclonal antibody, designated CLB-LFA-1/1, directed to the human lymphocyte-function-associated antigen 1 (LFA-1) was raised by immunization of mice with the peripheral blood lymphocytes of a Tγ lymphocytosis patient. The monoclonal antibody was selected by inhibition of the natural killer cell and the antibody-dependent killer cell activity of the patient's Tγ lymphocytes. In addition, the monoclonal antibody was shown to inhibit the cytotoxic activity of T cell clones specific for either class I or class II HLA molecules. The antigen recognized by CLB-LFA-1/1 consisted of three polypeptide chains with molecular weights of 180000 (α), 155000 and 94000 (β). The antibody reacted with T cells, B cells, monocytes and granulocytes, and stained normal Tγ cells and Tγ cells of patients with Tγ lymphocytosis two- to threefold stronger than normal T cells. It was shown that LFA-1 and the Fc receptor on Tγ cells did not comodulate and it is therefore concluded that Fc receptors and LFA-1 are independent membrane structures, both required for the killer cell activity of Tγ cells.     

Responses of calves to acute stress: individual consistency and relations between behavioral and physiological measures

The present study examined the consistency over time of individual differences in behavioral and physiological responsiveness of calves to intuitively alarming test situations as well as the relationships between behavioral and physiological measures. Twenty Holstein Friesian heifer calves were individually subjected to the same series of two behavioral and two hypothalamo-pituitary-adrenocortical (HPA) axis reactivity tests at 3, 13 and 26 weeks of age. Novel environment (open field, OF) and novel object (NO) tests involved measurement of behavioral, plasma cortisol and heart rate responses. Plasma ACTH and/or cortisol response profiles were determined after administration of exogenous CRH and ACTH, respectively, in the HPA axis reactivity tests. Principal component analysis (PCA) was used to condense correlated measures within ages into principal components reflecting independent dimensions underlying the calves' reactivity. Cortisol responses to the OF and NO tests were positively associated with the latency to contact and negatively related to the time spent in contact with the NO. Individual differences in scores of a principal component summarizing this pattern of inter-correlations, as well as differences in separate measures of adrenocortical and behavioral reactivity in the OF and NO tests proved highly consistent over time. The cardiac response to confinement in a start box prior to the OF test was positively associated with the cortisol responses to the OF and NO tests at 26 weeks of age. HPA axis reactivity to ACTH or CRH was unrelated to adrenocortical and behavioral responses to novelty. These findings strongly suggest that the responsiveness of calves was mediated by stable individual characteristics. Correlated adrenocortical and behavioral responses to novelty may reflect underlying fearfulness, defining the individual's susceptibility to the elicitation of fear. Other independent characteristics mediating reactivity may include activity or coping style (related to locomotion) and underlying sociality (associated with vocalization).     

Hyperpipecolic acidaemia: a diagnostic tool for peroxisomal disorders

Peroxisomal disorders include a complex spectrum of diseases, characterized by a high heterogeneity from both the clinical and the biochemical points of view. Specific assays are required for the study of peroxisome metabolism. Among these, pipecolic acid evaluation is considered as a supplementary test. We have established the diagnostic role of pipecolic acid in 30 patients affected by a peroxisomal defect (5 Zellweger syndromes, 10 Infantile Refsum diseases, 1 neonatal adrenoleukodystrophy, 6 patients affected by a peroxisomal biogenesis disorder with unclassified phenotype, 1 case of rhizomelic chondrodysplasia punctata (RCDP), 2 acyl-CoA oxidase deficiencies, 2 bifunctional enzyme deficiencies, 2 Refsum diseases, and 1 beta-oxidation deficiency). Pipecolic acid was increased in all generalized peroxisomal disorders, while normal pipecolic acid with abnormal very long chain fatty acid concentrations was strong evidence for a single peroxisomal enzyme deficiency. Unexpectedly, hyperpipecolic acidaemia was found also in a child affected by RCDP and in two patients with Refsum disease. In six patients the suggestion of a peroxisomal disorder was raised by the fortuitous finding of a pipecolic acid peak in amino acid chromatography, routinely performed as a general metabolic screening. For all patients, pipecolic acid proved to be a useful parameter in the biochemical classification of peroxisomal disorders.     

Determination of capillary leakage due to recombinant interleukin-2 by means of noninvasive conductivity measurements

Summary One of the most common side effects of treatment with recombinant interleukin-2 (IL-2) is capillary leakage. Its genesis is not completely understood. The aim of the study was to determine whether capillary leakage can be monitored by means of a non-invasive conductivity technique and to study its starting point. Eight patients with advanced renal cell cancer were studied in a medium care section of the Department of Medical Oncology, University Hospital over 4 days during treatment sessions of continuous, intravenously administered IL-2 (mean dose of 15.6 × 10⁶ IU · m^–2 · day ^–1). The fluid shift from the intravascular to the extra- and intracellular compartments was monitored by means of noninvasive conductivity measurements. Changes in blood volume were calculated from serial erythrocyte counts. The clinical parameters of capillary leakage (oliguria, positive fluid balance, and gain in mass) were recorded. The mean gain in mass was 9% after 4 days of IL-2 treatment. The extracellular fluid volume increased significantly [46 (SD 23.2)%; P < 0.01], whereas the intracellular fluid volume did not change. The increase in blood volume (BV) amounted to 7% (P < 0.05). The decline in albumin concentration was significantly more than the increase in BV [38 (SD 4.3) %; P < 0.01], indicating capillary albumin leakage. The main changes were observed after the 2nd day of treatment. From this study, it is suggested that conductivity measurements are a suitable method to monitor capillary leakage induced by IL-2, and could be used to detect the exact onset and severity of this leakage. The leakage started within the first 24 h of treatment and was detected as a fluid shift from the intravascular to the extracellular space, while the intracellular compartment remained stable. These measurements could be useful during intervention studies with the aim of preventing this adverse effect of IL-2.     

RSV-induced bronchiolitis but not upper respiratory tract infection is accompanied by an increased nasal IL-18 response

The aim of this study was to investigate potential differences in the local nasal immune response between bronchiolitis and upper respiratory tract infection induced by respiratory syncytial virus (RSV). Nasal brush samples were obtained from 14 infants with RSV bronchiolitis and from 8 infants with RSV upper respiratory tract infection. The samples were taken during infection (acute phase) and 2-4 weeks later (convalescent phase). Cytospin preparations were stained immunohistochemically for T cells, macrophages, and eosinophils. Staining also took place for intercellular adhesion molecule-1 (ICAM-1), T-helper 1 (Th1)-like (interleukin-12 [IL-12], interferon-gamma [IFN-gamma]), Th2-like (IL-4, IL-10), and proinflammatory cytokines (IL-6, IL-8, IL-18). During both RSV-induced bronchiolitis and upper respiratory tract infection, cellular inflammation was observed. This was characterised by an increase in the numbers of nasal macrophages, which tended to be higher in bronchiolitis than in upper respiratory tract infection. Numbers of T lymphocytes and ICAM-1 positive cells increased during both bronchiolitis and upper respiratory tract infection. There were no differences between numbers in the groups. Interestingly, a distinct nasal proinflammatory cytokine response was observed in RSV-induced bronchiolitis. This is characterised by an increase in the number of IL-18 positive cells. This increase is specific for bronchiolitis, as a similar increase could not be detected in RSV-induced upper respiratory tract infection. Numbers of IL-6 and IL-12 positive cells were higher in both bronchiolitis and upper respiratory tract infection, and there were no differences between the groups. By contrast, the number of IL-8, IFN-gamma, IL-4, and IL-10-positive cells remained constant. In conclusion, clear differences were found in nasal immune responses of children with RSV-induced upper respiratory tract infection or bronchiolitis. The induction of a strong IL-18 response was typical for bronchiolitis, as this could not be observed in RSV-induced upper respiratory tract infection, and could explain the eosinophilia that is observed frequently during bronchiolitis.