Automated prediction of the clinical impact of structural copy number variations.

Copy number variants (CNVs) play an important role in many biological processes, including the development of genetic diseases, making them attractive targets for genetic analyses. The interpretation of the effect of these structural variants is a challenging problem due to highly variable numbers of gene, regulatory, or other genomic elements affected by the CNV. This led to the demand for the interpretation tools that would relieve researchers, laboratory diagnosticians, genetic counselors, and clinical geneticists from the laborious process of annotation and classification of CNVs. We designed and validated a prediction method (ISV; Interpretation of Structural Variants) that is based on boosted trees which takes into account annotations of CNVs from several publicly available databases. The presented approach achieved more than 98% prediction accuracy on both copy number loss and copy number gain variants while also allowing CNVs being assigned "uncertain" significance in predictions. We believe that ISV's prediction capability and explainability have a great potential to guide users to more precise interpretations and classifications of CNVs.

Verbal and Cross-Modal Ratings of Music: Validation and Application of an Icon-Based Rating Scale.

Can music be rated consistently using nonverbal descriptors such as colours and temperatures? 144 participants rated 6 experimenter-selected and 2 self-selected pieces of music along 15 bipolar icon (graphic) scales intended to portray emotions, and sensory experiences consisting of colour, temperature, shape, speed, texture, and weight. Participants also rated the same pieces using bipolar verbal scales which aimed to encompass the concepts represented by the icons (e.g., the word "red" for the colour red). Furthermore, the icons themselves were subjected to open-ended verbal labelling to validate the icon scale. Colour icons spontaneously evoked a cross-modal association on 67% of occasions: blue being cool, and red/orange being warm or hot, and the icon scale had overall good face validity. Music regularly and consistently evoked multisensory associations (using the icon scale) including shapes, colours, weight, and temperatures, in addition to emotions. Cross-modal perception is indicative of music's character rather than the enjoyment of the music. The icon scale provides new insights into music perception and for applications where language skill may limit participant expression.

Interleukin-8 enhances nonoxidative intracellular killing of Mycobacterium fortuitum by human granulocytes.

The results of this study show that recombinant interleukin-8 (IL-8) enhances the intracellular killing of Mycobacterium fortuitum by human granulocytes. This chemokine did not stimulate the phagocytosis of M. fortuitum by granulocytes at various bacterium-to-cell ratios. The killing process was not affected by the NADPH oxidase inhibitor diphenyleneiodonium bisulfate, which indicates that recombinant IL-8 stimulates oxygen-independent mycobactericidal mechanisms of granulocytes. IL-8 did not stimulate H2O2 production in granulocytes but primed the cells for enhanced H2O2 production upon stimulation with preopsonized M. fortuitum. In sum, the chemokine IL-8 not only is involved in the recruitment of granulocytes to the site of infection but also facilitates the elimination of microorganisms by increasing the efficiency of the bactericidal activity of granulocytes.

Impaired phagocytosis of Staphylococcus aureus by granulocytes and monocytes of AIDS patients.

In the present study the microbicidal activities of granulocytes and monocytes from AIDS patients (CDC group IV) were assessed and compared with those of healthy controls. The phagocytosis and intracellular killing of Staphylococcus aureus by patient and control cells were measured using a method in which the rate of intracellular killing can be assessed independently of the rate of phagocytosis. Both granulocytes and monocytes of AIDS patients showed a decreased phagocytosis of S. aureus in comparison to phagocytes of healthy individuals. The rates of intracellular killing of S. aureus by granulocytes and monocytes did not differ significantly between these patients with late-stage HIV infection and controls.

Inflammation-induced changes in expression and glycosylation of genetic variants of alpha 1-acid glycoprotein. Studies with human sera, primary cultures of human hepatocytes and transgenic mice.

The relative occurrence of genetic variants of human alpha 1-acid glycoprotein (AGP) in relation to changes in glycosylation was studied in sera of patients with burn injury, media of cytokine-treated primary cultures of human hepatocytes and Hep 3B cells, and sera of transgenic mice expressing the human AGP-A gene. It is concluded (i) that the glycosylation of AGP was not dependent on its genetic expression and (ii) that both the variants determined by the AGP-A gene as well as by the AGP-B/B' genes are increased after inflammation or treatment with interleukins 1 and 6.

Changes in the serum concentration and the glycosylation of human alpha 1-acid glycoprotein and alpha 1-protease inhibitor in severely burned persons: relation to interleukin-6 levels.

The relation between interleukin-6 (IL-6) levels and changes in serum concentrations and glycosylation (concanavalin A affinity) of two human acute-phase glycoproteins, alpha 1-acid glycoprotein (AGP) and alpha 1-protease inhibitor (PI), was studied in sequential serum samples of burn patients. The level of IL-6 was already increased at the first day following injury, and after a dip at day 2 or 3 rapidly reached a second maximal value at day 4 or 5. The serum concentrations of AGP and PI reached their maximal values after day 5 and remained at a high level throughout the total period studied (7 weeks). The concanavalin A reactivities of both acute-phase glycoproteins were found to be elevated only during the first 2-2.5 weeks. Maximal values were observed on day 2 and from day 7 to 16, following closely the rise and fall of the IL-6 serum level. After day 16, the concanavalin A affinity rapidly declined long before a decrease was observed in the serum concentrations of AGP and PI. Our previous in vitro studies have indicated an involvement of IL-6 in the induction of both secretion and increased concanavalin A affinity. This study indicates that IL-6 could play a causal role in the induction of both phenomena in vivo.

Con A-nonreactive human alpha 1-acid glycoprotein (AGP) is more effective in modulation of lymphocyte proliferation than Con A-reactive AGP serum variants.

Human alpha 1-acid glycoprotein (AGP) has been shown to modulate various cellular and humoral immune reactions in vitro. Using glycosidase-modified derivatives of AGP, the importance of its carbohydrate moiety with regard to these effects has been noted. In normal serum, three molecular AGP forms interacting differently with concanavalin A (Con A) are present. The ratio of these forms is often changed during various physiopathological conditions. In this study, we could show that differences exist between the three AGP forms with regard to their immunomodulatory effectiveness. At physiological concentrations, the Con A-nonreactive variant AGP-A induced a stronger inhibition of the anti-CD3 stimulated lymphocyte proliferation than the other forms. Interestingly, AGP-A was also found to be responsible for the stimulation of lymphocyte proliferation induced by low AGP concentrations in vitro. Both immunomodulatory effects of AGP were abrogated by desialylation of the glycoprotein. These results support an immunomodulatory role of AGP in conditions characterized by a changed ratio of the differently glycosylated AGP forms.

Interferon-gamma-activated human granulocytes kill ingested Mycobacterium fortuitum more efficiently than normal granulocytes.

Although shortly after the onset of a mycobacterial infection granulocytes are present at the site of inflammation, the role of granulocytes in the elimination of mycobacteria is not well understood. In vitro studies with, for example Mycobacterium tuberculosis or M. bovis, are hampered by the slow proliferation and clumping of the bacteria. To avoid these disadvantages, we developed a model using the atypical mycobacterium M. fortuitum. The present study concerned two questions: whether human granulocytes are able to phagocytose and intracellularly kill opsonized M. fortuitum and whether intracellular killing of these bacteria can be enhanced by treatment of the granulocytes with recombinant human interferon-gamma (rIFN-gamma). The results showed that normal granulocytes phagocytosed opsonized M. fortuitum rapidly, but did not kill these bacteria effectively. The intracellular killing of M. fortuitum was significantly enhanced by incubation of the granulocytes with rIFN-gamma for 18 h before the start of the killing assay. Since these rIFN-gamma-pretreated granulocytes did not release more O2- and H2O2 upon stimulation with phorbol 12-myristate 13-acetate or opsonized M. fortuitum than control granulocytes, non-oxidative killing mechanisms are probably involved in the enhanced killing of M. fortuitum.

Con A affinity of rat alpha 1-acid glycoprotein (rAGP): changes during inflammation, dexamethasone or phenobarbital treatment as detected by crossed affino immunoelectrophoresis (CAIE) are not only a reflection of biantennary glycan content.

Using crossed affino immunoelectrophoresis (CAIE), the secretion of the Con A most reactive form (CAIE-3) of rat alpha 1-acid glycoprotein (rAGP) has been shown to be increased in sera of Wistar and Sprague Dawley rats during inflammation and treatment with dexamethasone or phenobarbital. Primary hepatocyte cultures prepared from experimentally treated Wistar rats reflect these in vivo findings, since rAGP as present in corresponding secretion media shows similar changes in Con A reactivity. In this study, the relation of this increase towards the amount of biantennary glycans was investigated for both differently treated rat strains. For this purpose, metabolically labelled rAGP, secreted by isolated hepatocytes under the various conditions, was separated on Con A-Sepharose into four fractions. For each fraction of rAGP its behaviour in CAIE was established, revealing a positive correlation for Con A reactivity between the two methods. However, the enormous increase in Con A reactivity of rAGP in CAIE during inflammation and other conditions (increase in CAIE-3), could not be shown using Con A-Sepharose chromatography. Glycopeptides of each fraction were prepared and the amount of biantennary glycans was assessed. Contrary to expectations, an increase of the total amount of biantennary glycans of rAGP, secreted during conditions associated with an increase in CAIE-3 was not found. The independency of the results with regard to rat strain and procedures used underlined the generality of these findings. Consequently, not only the biantennary glycan content is responsible for the separation of rAGP in CAIE. The importance of other differences in glycosylation, e.g. sialylation, for the increase of rAGP CAIE-3 during various experimental conditions is discussed.

Effects of monocytic products, recombinant interleukin-1, and recombinant interleukin-6 on glycosylation of alpha 1-acid glycoprotein: studies with primary human hepatocyte cultures and rats.

Changes in the carbohydrate moieties of acute-phase glycoproteins (APGPs) often accompany the increase in their secretion by the liver during inflammation. In this study, we investigated whether factors known to regulate APGP gene expression are also involved in the altered glycosylation. For this purpose, the glycosylation pattern of alpha 1-acid glycoprotein (AGP) as secreted by human hepatocytes, cultured in the presence and absence of dexamethasone and monokines, was studied by crossed affino- (concanavalin A) immunoelectrophoresis (CAIE). The monokines rIL-1 and rIL-6, in the presence of dexamethasone, both stimulated AGP secretion and caused a change in glycosylation towards an increased Con A reactivity, including the appearance of two strongly reactive forms (D and E) normally not present. Dexamethasone alone did not influence either process. When tested in vivo in rats, rIL-6 also induced an increased presence of Con A-reactive forms of AGP in serum. In conclusion, the changes in secretion and glycosylation of AGP as seen during inflammation seem to be mediated by the same factor(s).

Glycosylation of three molecular forms of human alpha 1-acid glycoprotein having different interactions with concanavalin A. Variations in the occurrence of di-, tri-, and tetraantennary glycans and the degree of sialylation.

Human alpha 1-acid glycoprotein (AGP) was separated into a non-bound (AGP-A; 46%), a retarded (AGP-B; 39%) and a bound fraction (AGP-C; 15%) using concanavalin A (ConA)-Sepharose chromatography. The apparent molecular masses, as determined by SDS-PAGE, of the three fractions were 43.5, 42.3 and 41.2 kDa, respectively. The occurrence of N-linked di-, tri- and tetraantennary glycans on these three molecular forms (AGP-A, -B, and -C) was studied by sequential lectin-affinity chromatography of the 14C-labelled glycopeptides. These were obtained by extensive pronase treatment followed by N-[14C]acetylation of the peptide moieties. The glycopeptides of AGP-A did not bind to ConA-Sepharose whereas for AGP-B and AGP-C 18% and 44%, respectively, of the glycopeptides were bound as diantennary structures. Glycopeptide fractions of all three forms of AGP which were not bound to ConA-Sepharose were shown to contain equal amounts of both tri- and tetraantennary glycans by chromatography with Phaseolus vulgaris leukoagglutinating lectin (L-PHA). With the assumption that each molecule contains five glycosylation sites, it could be shown that AGP-A contains no diantennary structures whereas AGP-B and AGP-C contain one and two diantennary structures, respectively. In addition each of the molecular forms contains equal amounts of tri- and tetraantennary structures on the remaining glycosylation sites. The results of this study, therefore, exclude a uniformity of glycan chains in the three molecular forms of AGP. The degree of sialylation of each of the molecular forms was investigated by chromatography on L-PHA-agarose and Ricinus communis agglutinin-I--agarose both before and after desialylation of the glycopeptides. It was shown that about 90% of the biantennary glycans of both AGP-B and AGP-C were disialylated while the remainder were monosialylated. The degree of sialylation of the tri- and tetraantennary glycans was identical for the three molecular forms. In each case, one or more terminal galactose residues occurred on at least 20% of the tri- and 65% of the tetraantennary chains. It is suggested that the decrease in the exposure of galactose residues from AGP-A to AGP-C is related to the concomittant decrease in branching of the glycans of the three molecular forms. The relevance of these findings to studies on the function of AGP during inflammatory and liver diseases is discussed.

Glycosylation of four acute-phase glycoproteins secreted by rat liver cells in vivo and in vitro. Effects of inflammation and dexamethasone.

We have studied the role of the liver in the relative increase of Concanavalin A (Con A)-reactive molecular forms of various positive rat acute-phase glycoproteins (APGPs) occurring in serum during inflammation. Secretion media of hepatocytes isolated from inflamed rats showed a 2 to 5-fold increase of the total amounts of four APGPs studied in comparison to secretion media of control hepatocytes. These changes were in analogy with those observed for corresponding sera, except for alpha 1-antitrypsin. All the different Con A-reactive molecular forms were present in the media, with exception of the most reactive form of ceruloplasmin. In vitro and in vivo, dexamethasone augmented the secretion of three APGPs, and especially of the Con A-most reactive forms. The in vitro effect of dexamethasone--augmented secretion of Con A-reactive molecular forms of alpha 1-acid glycoprotein and haptoglobin--was comparable with the results obtained for hepatocytes isolated from inflamed rats. In vivo, dexamethasone treatment resulted in an even higher increase of the serum concentration of the Con A-most reactive forms of both APGPs than experimental inflammation did. Although an extrahepatic contribution cannot be excluded, these results suggest that alterations in the Con A reactivity of APGPs as observed during the acute-phase of inflammation have their origin in the liver. A change in the Con A reactivity of glycoprotein indicates a modulation of its glycosylation. Since dexamethasone can affect these changes in vivo and in vitro, glucocorticoids most probably are involved in the regulation of the glycosylation of the APGPs during biosynthesis in the liver.

Cross-reactivity among birch pollen, vegetables and fruits as detected by IgE antibodies is due to at least three distinct cross-reactive structures.

Sera of patients suffering from birch pollinosis were studied in the radio-allergo-sorbent test (RAST) for the presence of IgE antibodies to various allergens of vegetable origin. The sera selected were positive in the RAST for both birch pollen and fruits. IgE antibodies directed against at least three different cross-reacting determinants in birch pollen were detected. In addition to periodate-susceptible cross-reacting determinants, which are found on a number of glycoproteins, two non-related periodate-resistant determinants were found in birch pollen, with molecular weights of 20 and 18 kD, respectively. The 20-kD component appears to be responsible for the co-occurrence of the binding of IgE to allergens of fresh fruits, whereas the 18-kD component appears to cause the cross-reactivity among grass pollen, potato and fruits.

In vitro studies in nickel allergy: diagnostic value of a dual parameter analysis.

A comparison was made between the diagnostic value of assaying nickel-induced lymphocyte proliferation (lymphocyte transformation test, LTT) and migration inhibition factor (MIF) production in nickel contact sensitivity. Although lymphocyte proliferation was significantly increased in the group of patients with skin test reactivity to nickel, positive LTT were also frequently found in skin test-negative subjects: in 63% of subjects with and in 30% of subjects without a history of metal allergy. This would limit the value of the LTT as an in vitro correlate of skin test reactivity. However, in certain patients positive lymphocyte transformation may reveal nickel sensitization at a time of undetectable skin reactivity. Data obtained with the macrophage migration inhibition test (MMIT) showed a good correlation with nickel patch test reactions. Accurate determination of MIF became feasible by using cells from the human monocytoid cell line U937 as target cells in a microdroplet agarose assay. Using this MMIT, positive reactions occurred in 13% of the healthy controls and false-negative reactions were found in 26% of patients with positive skin test reactivity to nickel. As LTT and MMIT data appeared to be only weakly correlated in the individuals tested, a dual parameter analysis was performed. An excellent correlation [p = 1.8 (10(-8]] was found between skin test and in vitro reactivity for individuals with matching in vitro results (60% of all individuals tested). In those individuals with discordant in vitro data, skin testing will remain indispensable for diagnosing nickel allergy.