Urinary leukotriene and Bcl I polymorphism of glucocorticoid receptor gene in preschoolers with recurrent wheezing and high risk of asthma

BACKGROUND: Urinary leukotriene (LTE4) is an important marker of airway inflammation presence. A relationship between single nucleotide polymorphism in the glucocorticoid receptor (GCR) gene promoter (Bcl I polymorphism), development of asthma and sensitivity to glucocorticoids has been hypothesised. OBJECTIVE: To explore the possible association between the Bcl I polymorphism and baseline levels of urinary LTE4 in preschoolers with recurrent wheezing episodes. We prospectively enrolled and classified 86 preschoolers based on the risk of developing asthma (by the Asthma Predictive Index [API]). METHODS: At admission standardised questionnaires for demographics and respiratory illness characteristics were completed. The Bcl I polymorphism of the GCR was determined by a PCR-RFLP assay from blood samples, and urinary leukotriene was assessed from urine samples by an enzyme immunoassay. RESULTS: We enrolled 86 preschoolers (46 with positive API and 40 with negative API). There were no statistical differences in demographic, respiratory illnesses and wheezing episodes characteristics between both groups. Also, the prevalence of Bcl I polymorphism was similar between positive vs. negative API groups (34.8% vs. 38.9% for homozygote GG, 56.5% vs. 52.8% for heterozygote GC, 8.7% vs. 8.3% for homozygote CC, respectively, p = 0.94). However, urinary LTE4 (median [IQR]) was higher in preschoolers with positive than negative API (7.18 [5.57-8.96 pg/ml] vs. 6.42 [3.96-8.07 pg/ml], p = 0.02, respectively). CONCLUSIONS: In our population, wheezing preschoolers with positive API exhibit higher levels of urinary LTE4 than those with negative API; but there were no differences in Bcl I polymorphism of the GCR

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Urinary leukotriene and Bcl I polymorphism of glucocorticoid receptor gene in preschoolers with recurrent wheezing and high risk of asthma.

BACKGROUND: Urinary leukotriene (LTE4) is an important marker of airway inflammation presence. A relationship between single nucleotide polymorphism in the glucocorticoid receptor (GCR) gene promoter (Bcl I polymorphism), development of asthma and sensitivity to glucocorticoids has been hypothesised. OBJECTIVE: To explore the possible association between the Bcl I polymorphism and baseline levels of urinary LTE4 in preschoolers with recurrent wheezing episodes. We prospectively enrolled and classified 86 preschoolers based on the risk of developing asthma (by the Asthma Predictive Index [API]). METHODS: At admission standardised questionnaires for demographics and respiratory illness characteristics were completed. The Bcl I polymorphism of the GCR was determined by a PCR-RFLP assay from blood samples, and urinary leukotriene was assessed from urine samples by an enzyme immunoassay. RESULTS: We enrolled 86 preschoolers (46 with positive API and 40 with negative API). There were no statistical differences in demographic, respiratory illnesses and wheezing episodes characteristics between both groups. Also, the prevalence of Bcl I polymorphism was similar between positive vs. negative API groups (34.8% vs. 38.9% for homozygote GG, 56.5% vs. 52.8% for heterozygote GC, 8.7% vs. 8.3% for homozygote CC, respectively, p=0.94). However, urinary LTE4 (median [IQR]) was higher in preschoolers with positive than negative API (7.18 [5.57-8.96pg/ml] vs. 6.42 [3.96-8.07pg/ml], p=0.02, respectively). CONCLUSIONS: In our population, wheezing preschoolers with positive API exhibit higher levels of urinary LTE4 than those with negative API; but there were no differences in Bcl I polymorphism of the GCR.

Genetic variations in host IL28B links to the detection of peripheral blood mononuclear cells-associated hepatitis C virus RNA in chronically infected patients.

Hepatitis C virus (HCV) is mainly hepatotropic; however, several reports document the presence of genomic viral RNA in extrahepatic sites including peripheral blood mononuclear cells (PBMCs). In this study, the presence of HCV RNA was initially evaluated in the plasma and peripheral blood mononuclear cells (PBMCs) of 53 HCV-infected patients who were treated per protocol. PBMC-associated HCV RNA was detectable in 79% of patients. Early virological response to combined pegylated interferon-α (PegIFN) and ribavirin (RBV) therapy in patients with undetectable levels of PBMCs-associated HCV RNA was 100%, while it was 60% (P = 0.003) in those who had detectable levels of PBMC-associated HCV RNA. A sustained virological response was observed in 35% of patients with detectable PBMC-associated HCV RNA, but was 70% in patients with undetectable levels of PBMC-associated HCV RNA (P = 0.07). In a multivariate analysis incorporating parameters such as HCV genotype, viral load, presence of cirrhosis and absence of PBMC-associated HCV RNA, a significant relationship was observed between the detection of PBMC-associated HCV RNA and the sustained virological response (OR 19.4, 95% CI: 2.1-486.2, P = 0.0061). The association between single nucleotide polymorphism (SNP) in IL28B, known predictor of antiviral therapy outcome, and the occurrence of HCV RNA in PBMC in 84 chronically infected patients was then evaluated. Results suggest that the presence of a G allele in rs8099917, known to associate to a poor response to PegIFN/RBV therapy, also predicts an increased association of HCV RNA with PBMC (OR: 3.564; 95% CI: 1.114-11.40, P = 0.0437).

Hepatitis C virus quasispecies in plasma and peripheral blood mononuclear cells of treatment naïve chronically infected patients.

Peripheral blood mononuclear cells (PBMCs) from 45 treatment naïve, HIV-negative, chronically hepatitis C virus (HCV)-infected patients were analyzed for the presence of HCV RNA. Viral RNA was detected in 73% of the studied patients. Single-strand conformation polymorphism assays and sequence analysis of the HCV 5'untranslated regions amplified from RNA recovered from both Plasma and PBMCs suggested virus compartmentalization in 57.6% of patients studied. In summary, our study presents evidence that HCV RNA can be found in PBMCs of treatment naïve chronically infected patients that are not immunocompromised or co-infected with the human immunodeficiency virus.

Efficient gene transfer in mouse neural precursors with a bicistronic retroviral vector.

Gene transfer into neural precursors is a powerful approach to study the function of specific gene products during nervous system development. Here we describe a retrovirus-based methodology to transduce foreign genes into mouse neural precursors. We used a high-titer bicistronic retroviral vector that encodes a marker gene, placental alkaline phosphatase (plap), and a selection gene, neomycin phosphotransferase II (neoR), under the translational control of two retroviral internal ribosome entry segments. Transduction efficiency even without selection was up to 95% for multipotential neurospheres derived from embryonic striata and grown with basic fibroblast growth factor 2. Expression of plap and neoR was sustained with time in culture and upon differentiation into neurons, astrocytes, and oligodendrocytes, as shown by double immunofluorescence labeling with cell type-specific markers, Western blotting, and neomycin resistance. However, levels of plap were decreased in differentiated oligodendrocytes. Transduction with the same vector of neonatal oligodendrocyte precursors grown in oligospheres consistently resulted in a lower proportion of plap-immunoreactive cells and enhanced cell death in the absence of neomycin. However, plap expression was maintained in some differentiated oligodendrocytes expressing galactocerebroside or myelin basic protein. In that neurospheres can be easily expanded in vitro and factors enabling their differentiation into the three main central nervous system cell types are being elucidated, this methodology could be used in the future to produce large number of transduced, differentiated neural cells.

An internal ribosome entry segment promotes translation of the simian immunodeficiency virus genomic RNA.

The retroviral genomic RNA is the messenger for the synthesis of the group-specific antigen (gag) and polymerase precursors of the major structural proteins and enzymes of the virion. The 5'-untranslated leader of the simian immunodeficiency virus (SIV) genomic RNA is formed of highly structured domains involved in key steps of the viral life cycle. Thus, the presence of stable RNA structures between the 5'-cap and the gag start codon are thought to strongly inhibit scanning of a 43 S preinitiation ribosomal complex. This prompted us to look for an alternative to the canonical ribosome scanning. By using a standard bicistronic assay in the rabbit reticulocyte lysate, we show that the SIVmac 5'-leader contains an internal ribosome entry segment (IRES) and that gene expression driven by this IRES is stimulated upon cleavage of eukaryotic initiation factor 4G. Deletion analysis revealed that the sequence between the major splice donor and the gag AUG codon is required for IRES activity. DNA transfection and viral transduction experiments in both NIH-3T3 and COS-7 cells confirmed that translation driven by the SIV leader is IRES-dependent and thus insensitive to the immunosuppressant rapamycin. Identification of an IRES in SIV is of particular interest for the understanding of lentivirus replication and also for the design of novel lentiviral vectors suitable for gene transfer.

Identification of an internal ribosome entry segment in the 5' region of the mouse VL30 retrotransposon and its use in the development of retroviral vectors.

Mouse virus-like 30S RNAs (VL30m) constitute a family of retrotransposons, present at 100 to 200 copies, dispersed in the mouse genome. They display little sequence homology to Moloney murine leukemia virus (MoMLV), do not encode virus-like proteins, and have not been implicated in retroviral carcinogenesis. However, VL30 RNAs are efficiently packaged into MLV particles that are propagated in cell culture. In this study, we addressed whether the 5' region of VL30m could replace the 5' leader of MoMLV functionally in a recombinant vector construct. Our data confirm that the putative packaging sequence of VL30 is located within the 5' region (nucleotides 362 to 1149 with respect to the cap structure) and that it can replace the packaging sequence of MoMLV. We also show that VL30m contains an internal ribosome entry segment (IRES) in the 5' region, as do MoMLV, Friend murine leukemia virus, Harvey murine sarcoma virus, and avian reticuloendotheliosis virus type A. Our data show that both the packaging and IRES functions of the 5' region of VL30m RNA can be efficiently used to develop retrotransposon-based vectors.

Retroviral vectors for the expression of two genes in human multipotent neural precursors and their differentiated neuronal and glial progeny.

Retroviral vectors allow stable integration of exogenous DNA into genomic DNA and therefore gene transmission to progeny. Multipotent neural precursors and immortal cell lines prepared from them have been demonstrated to integrate into either adult or developing brain in a nontumorigenic, functional manner, without interfering with normal neurobiological processes. These cells thus appear to provide a Trojan horse ideally adapted to directing the expression of transgenes appropriately in a host brain. Here we investigated and optimized the transduction capacity of MuLV-based retroviral vectors in which internal ribosomal entry segments (IRESs) drive coexpression of two heterologous gene products from a single bicistronic mRNA in a human multipotent neural precursor cell line, "Dev," which was prepared from a medulloblastoma. For this, two vectors containing two different combinations of three viral IRESs were used and the capacity of different pseudotyped vectors to permit an efficient and stable transduction of Dev cells was compared. Our data show that (1) the best recombinant vectors for Dev cell transduction are those pseudotyped with the 10A1 MuLV envelope (40% of transduction) and (2) the initial coexpression of neo and plap, observed in transduced undifferentiated Dev cells, is maintained in differentiated Dev cells with a neuronal or glial phenotype. Therefore, these double-IRES vectors may provide an efficient means of transducing the coexpression of two proteins in undifferentiated human neural precursors that is maintained in their differentiated progeny. These data suggest that the double-IRES strategy is well adapted to potential therapeutic situations when coexpression of two different transgenes may be required in the same cell.

Detection of the infectious hematopoietic necrosis virus directly from infected fish tissues by dot blot hybridization with a non-radioactive probe.

A method was developed for the rapid diagnosis of the infectious hematopoietic necrosis virus (IHNV) based on dot blot hybridization with a non-radioactive probe. When the assay was developed through a color reaction both biotin- and alkaline phosphatase-labeled probes were highly specific for IHNV and the sensitivity reached to 20 pg of viral RNA. When the alkaline phosphatase-labeled probe was developed by a chemiluminiscent reaction, the sensitivity showed a five-fold increase to 4 pg of viral RNA. The procedures successfully detected IHNV directly from infected symptomatic and asymptomatic fishes exhibiting higher sensitivity than the traditional approaches involving viral propagation in cell cultures. Additional advantages of the method are its simplicity and it takes only about 6 h to carry out the procedures from the initial processing of the tissues to diagnosis.

Characterization of an internal ribosomal entry segment within the 5' leader of avian reticuloendotheliosis virus type A RNA and development of novel MLV-REV-based retroviral vectors.

The murine leukemia virus (MLV)-related type C viruses constitute a major class of retroviruses that includes numerous endogenous and exogenous mammalian viruses and the related avian spleen necrosis virus (SNV). The MLV-related viruses possess a long and multifunctional 5' untranslated leader involved in key steps of the viral life cycle--splicing, translation, RNA dimerization, encapsidation, and reverse transcription. Recent studies have shown that the 5' leader of Friend murine leukemia virus and Moloney murine leukemia virus can direct cap independent translation of gag precursor proteins (Berlioz et al., 1995; Vagner et al., 1995b). These data, together with structural homology studies (Koning et al., 1992), prompted us to undertake a search for new internal ribosome entry segment (IRES) of retroviral origin. Here we describe an IRES element within the 5' leader of avian reticuloendotheliosis virus type A (REV-A) genomic RNA. Data show that the REV-A 5' IRES element maps downstream of the packaging/dimerization (E/DLS) sequence (Watanabe and Temin, 1982; Darlix et al., 1992) and the minimal IRES sequence appears to be within a 129 nt fragment (nucleotides 452-580) of the 5' leader, immediately upstream of the gag AUG codon. The REV-A IRES has been successfully utilized in the construction of novel high titer MLV-based retroviral vectors, containing one or more IRES elements of retroviral origin. These retroviral constructs, which represent a starting point for the design of novel vectors suitable for gene therapy, are also of interest as a model system of internal translation initiation and its possible regulation during development, cancer, or virus infection.

Determination of gizzerosine activity in fish meal with a homologous radioimmunoassay.

A homologous RIA for the determination of toxic gizzerosine activity in commercial fish meals has been developed. Three polyclonal antibodies (GR316, GR415, and GR418) were produced in female rabbits and extensively characterized for their potential use in individual RIA. The RIA had lower detection limits of 0.048, 0.78, and 0.39 ng/mg using the three respective antibodies. Because gizzerosine is derived from lysine and histidine, crossreactivity with these amino acids, and with histamine was examined. The antibodies crossreacted with histamine from 21 to 100%. No crossreactivity with histidine or lysine was observed for any of the three antibodies. Antibody GR415 was chosen for determination of gizzerosine in extracted fish meal samples because crossreactivity of histamine using this antibody was only present at high concentrations, and the Ka value for gizzerosine was 10-fold greater than for histamine. A mild buffer extraction procedure was used, resulting in 98% gizzerosine recovery. Displacement curves from extracted and serially diluted fish meal samples were parallel with gizzerosine standard. Inter-and intra-assay coefficients of variation were 11 and 15%, respectively. We used the RIA for determination of gizzerosine activity in a pool of 23 fish meal samples of known gizzerosine scores (determined with a chick bioassay), and histamine content. The partial correlation coefficient between gizzerosine content determined by the RIA and gizzerosine scores from the bioassay was high (0.83), and significant (P < 0.01). There were also significant correlations between gizzerosine scores and histamine content of the fish meals (0.63, P < 0.01), and between histamine content and gizzerosine levels determined by the RIA (0.59, P < 0.01). The application of the homologous RIA for the determination of gizzerosine activity in commercial fish meals could be of importance for the prevention of gizzard erosions in the poultry industry, and for studying gizzerosine-induced pathology and metabolism.

Inhibitors of infectious pancreatic necrosis virus (IPNV) replication.

In attempts to detect inhibitors of infectious pancreatic necrosis virus (IPNV) replication, we have evaluated, by an IPNV plaque inhibition assay, a group of compounds that have broad spectrum antiviral activity for both single- and double-stranded RNA viruses. The inosine monophosphate dehydrogenase (IMP dehydrogenase) inhibitors 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) and 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), and the orotidine monophosphate decarboxylase (OMP decarboxylase) inhibitor 4-hydroxy-3-beta-D-ribofuranosylpyrazole-5-carboxamide (pyrazofurin), were found to inhibit IPNV replication. For EICAR and pyrazofurin the concentrations that inhibited the IPNV plaque formation by 50% (EC50) were 0.01 micrograms/ml and 0.5 micrograms/ml, respectively. The cytotoxic concentrations required to reduce cell viability by 50% (CC50) were 50 micrograms/ml and 100 micrograms/ml, respectively, and the concentrations that reduced [methyl-3H] thymidine incorporation by 50% (IC50) were 0.5-1 and 50 micrograms/ml. Thus, for both compounds the IPNV-inhibitory concentration was 50-100 times lower than the concentration that affected DNA synthesis in growing cells. EICAR and pyrazofurin seem to be good candidates for further evaluation in an in vivo model of IPNV infection.

Polyacrylamide gel electrophoresis of viral genomic RNA as a diagnostic method for infectious pancreatic necrosis virus detection.

A rapid and simple technique for the diagnosis of IPNV from cell culture and infected fish tissues has been developed. It is based on the observation of IPNV bisegmented double-stranded RNA genome in silver stained polyacrylamide gels after electrophoresis. The method is highly specific and can detect as little as 1 ng of viral RNA, which corresponds to 1 x 10(5) pfu, making it possible to visualize the viral genome as soon as the initial cythopatic effect appears. Furthermore, the RNA viral genome could be detected directly from fish tissues when fish showed clear clinical signs of the disease. The method has the advantage of detecting any strain of IPNV. An acrylamide concentration of 6% and bisacrylamide concentration of 0.13% give a rapid and definitive result in less than 6 h.