New phenotypic, functional and electrophysiological characteristics of KG-1 cells

Myeloid dendritic cells (DC) are representatives of a rare and phenotypically diverse population of professional antigen presenting cells possessing high functional heterogeneity and flexibility. Here we studied the phenotypic, functional and electrophysiological characteristics of KG-1 cells, an erythroleukemia model cell line, which shares morphological and physiological similarities with immature and mature myeloid DC. We compared the expression of internalizing receptors and other cell surface molecules, antigen uptake and migration of unstimulated and activated KG-1 cells with the characteristics of immature and mature DC. Unstimulated KG-1 cells were less potent in capturing extracellular materials than immature DC. In contrast to monocyte-derived DC KG-1 cells stimulated by PMA and ionomycin ceased to migrate along the MIP-3beta chemokine gradient despite their high expression of CCR7 chemokine receptor and MDR, a transporter implicated in DC migration. Moreover, we determined the ion channel repertoire of KG-1 cells before and after treatment with PMA and ionomycin by using the patch-clamp technique. We found that both unstimulated and activated KG-1 cells expressed time- and voltage-independent, ChTx sensitive intracellular Ca(2+)-gated potassium conductance suggesting the presence of K(Ca) channels in their membranes. Based on our results we propose that KG-1 cells resemble myeloid DC but also possess unique phenotypic, functional and electrophysiological characteristics.     

CAS (cellular apoptosis susceptibility) gene expression in ovarian carcinoma: Correlation with 20q13.2 copy number and cyclin D1, p53, and Rb protein expression

We immunohistochemically analyzed cellular apoptosis susceptibility (CAS) protein expression and compared it with 20q13.2 copy number and the expression of cell cycle-associated proteins retinoblastoma (Rb), cyclin D1, and p53 and prognosis on paraffin-embedded tissue from 69 ovarian carcinomas (OCs). CAS protein reactivity was present in 100%, Rb in 54%, cyclin D1 in 47%, and p53 in 49%. Significant reciprocal correlation was observed between high levels of CAS and histologic type, FIGO (International Federation of Obstetrics and Gynecology) stage III and grade 3, residual tumor (>2 cm), 20q13.2 (ZNF217 gene) amplification (>4 copies in >20% cells), and high expression of cyclin D1 (all P < .05). No association was found between cyclin D1, p53, or Rb levels with clinicopathologic factors. In univariate analysis, residual tumor, FIGO stage and grade, ZNF217 amplification, and CAS levels predicted outcome (all P < .05). In multivariate analysis, stage, grade, amount of residual tumor, and ZNF217 amplification showed independent prognostic value (all P < .05). In OC, alteration of CAS and ZNF217 genes, both located at 20q13, is frequent and relevant prognostically. Cyclin D1, Rb, and p53 seem to have a secondary role.     

Alpha-thalassemia/mental retardation syndrome in a 45,X male

An unbalanced Y;autosome translocation leading to a male with a 45,X karyotype is rare with about 30 published cases. A male with a 45,X karyotype as a result of a unique, submicroscopic, unbalanced Y;16 translocation is presented with alpha-thalassemia/mental retardation syndrome.     

GeneID in Drosophila

GeneID is a program to predict genes in anonymous genomic sequences designed with a hierarchical structure. In the first step, splice sites, and start and stop codons are predicted and scored along the sequence using position weight matrices (PWMs). In the second step, exons are built from the sites. Exons are scored as the sum of the scores of the defining sites, plus the log-likelihood ratio of a Markov model for coding DNA. In the last step, from the set of predicted exons, the gene structure is assembled, maximizing the sum of the scores of the assembled exons. In this paper we describe the obtention of PWMs for sites, and the Markov model of coding DNA in Drosophila melanogaster. We also compare other models of coding DNA with the Markov model. Finally, we present and discuss the results obtained when GeneID is used to predict genes in the Adh region. These results show that the accuracy of GeneID predictions compares currently with that of other existing tools but that GeneID is likely to be more efficient in terms of speed and memory usage.     

Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections

The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples.     

Sublingual immunotherapy for hazelnut food allergy: a randomized, double-blind, placebo-controlled study with a standardized hazelnut extract

BACKGROUND: Food allergy may be life-threatening, and patients affected need to receive accurate diagnoses and treatment. Hazelnut has often been implicated as responsible for allergic reactions, and trace quantities can induce systemic reactions. OBJECTIVE: The aim of this study was to evaluate the efficacy and tolerance of sublingual immunotherapy with a standardized hazelnut extract in patients allergic to hazelnut. METHODS: This was a randomized, double-blind, placebo-controlled study. Inclusion criteria were a history of hazelnut allergy and positive skin prick test and double-blind placebo-controlled food challenge results. Patients were then randomly assigned into 2 treatment groups (hazelnut immunotherapy or placebo). Efficacy was assessed by double-blind, placebo-controlled food challenge after 8 to 12 weeks of treatment. Blood samples were drawn for measurement of specific IgE, IgG(4), and serum cytokines before and after treatment. RESULTS: Twenty-three patients were enrolled and divided into 2 treatment groups. Twenty-two patients reached the planned maximum dose at 4 days. Systemic reactions were observed in only 0.2% of the total doses administered. Mean hazelnut quantity provoking objective symptoms increased from 2.29 g to 11.56 g (P = .02; active group) versus 3.49 g to 4.14 g (placebo; NS). Moreover, almost 50% of patients who underwent active treatment reached the highest dose (20 g), but only 9% in the placebo. Laboratory data showed an increase in IgG(4) and IL-10 levels after immunotherapy in only the active group. CONCLUSION: Our data confirm significant increases in tolerance to hazelnut after sublingual immunotherapy as assessed by double-blind, placebo-controlled food challenge, and good tolerance to this treatment.     

Retinoids induce Fas(CD95) ligand cell surface expression via RARgamma and nur77 in T cells

Cells from the CD4+ murine T hybridoma line IP-12-7 enter the apoptotic suicide program via the Fas ligand (FasL)/Fas-mediated pathway upon TCR stimulation. This stimulus regulates the sensitization of the Fas death pathway and the cell surface appearance of preformed FasL. The apoptosis is dependent on new mRNA and protein synthesis and involves up-regulation of nur77.Two groups of nuclear receptors for retinoic acids (RA) have been identified: retinoic acid receptors (RAR) and retinoid X receptors. IP-12-7 cells express RARalpha and RARgamma. Here we show that,in the IP-12-7 T cells, RA also induced the expression and DNA binding of nur77, and the cell surface appearance of FasL. The induction was mediated via RARgamma. Despite the induced expression of cell surface FasL, only two structurally related RARgamma-selective compounds, CD437 and CD2325, initiated apoptosis in these cells. The lack of apoptosis induction by natural RA was related to the inability of RARgamma to sensitize the Fas death-pathway. Cell surface FasL, however, was able to induce cell death in Fas-bearing target cells. Natural RA also induced the expression of FasL in phytohemagglutinin-activated peripheral murine T cells. It is proposed that therapeutically administered RA might induce apoptosis in Fas-sensitive cells via induction of FasL expression in activated Tcells.     

The prion protein in human neuromuscular diseases

The basis of human prion diseases affecting the nervous system is accumulation of a disease-associated conformer (PrPSc) of the normal cellular prion protein (PrPC). Earlier studies demonstrated increased expression of PrPC in inclusion body myositis (IBM), dermato-, and polymyositis, as well as neurogenic muscle atrophy. To define the spectrum and reliability of PrPC immunoreactivity, its expression was examined systematically in a series of pathologically characterized muscular disorders by means of immunohistochemistry, confocal laser microscopy, and immunogold electron microscopy. Anti-PrPC immunolabelling of rimmed vacuoles was observed in IBM, inclusions of myofibrillary myopathy, targets, regenerating, and atrophic fibres, mononuclear cells, in addition to ragged red fibres in mitochondrial myopathies, and focal sarcolemmal immunostaining in non-diseased controls. Quantitative analysis demonstrated that, in neurogenic muscle lesions, anti-PrPC staining detects a significantly broader spectrum of fibres than anti-vimentin or anti-NCAM. In dystrophic muscle, PrPC expression was mainly restricted to regenerating fibres. In IBM, PrPC expression was not confined to rimmed vacuoles or vacuolated fibres and only a small percentage (7.1%) of rimmed vacuoles were PrPC positive. Ultrastructurally, PrPC was observed in the cytoplasm of lymphocytes, in the myofibrillar network of targets, and in rimmed vacuoles. Knowledge of disease circumstances with altered expression of PrPC is important in the setting of a potentially increased chance for extraneural PrPC-PrPSc conversion. In addition, our observations suggest that PrPC may have a general stress-response effect in various neuromuscular disorders.