Advances in the diagnosis of respiratory tract infections: role of the Luminex xTAG respiratory viral panel.

Clinical laboratories providing an etiological diagnosis of respiratory tract infections (RTI) have increasingly relied on nucleic acid amplification tests. Polymerase chain reaction-based methods are becoming more standardized, and several have undergone the scrutiny of regulatory agencies mandated to assess the risks and benefits of implementing pathogen-detection assays into diagnostic algorithms. Respiratory viruses lead to both upper and lower RTI and are implicated in exacerbations of chronic pulmonary conditions.

A prospective molecular surveillance study evaluating the clinical impact of community-acquired respiratory viruses in lung transplant recipients.

Background: Community-acquired respiratory viral infections (RVIs) are common in lung transplant patients and may be associated with acute rejection and bronchiolitis obliterans syndrome (BOS). The use of sensitive molecular methods that can simultaneously detect a large panel of respiratory viruses may help better define their effects.

Methods: Lung transplant recipients undergoing serial surveillance and diagnostic bronchoalveolar lavages (BALs) during a period of 3 years were enrolled.

Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead-based assay.

Virology laboratories historically have used direct fluorescent-antibody assay (DFA) and culture to detect six or seven respiratory viruses. Following the discovery of five new human respiratory viruses since 2000, there is an increasing need for diagnostic tests to detect these emerging viruses. We have developed a new test that can detect 20 different respiratory virus types/subtypes in a single 5-h test.

Technical validation of a multiplex platform to detect thirty mutations in eight genetic diseases prevalent in individuals of Ashkenazi Jewish descent.

Purpose: This study determines the analytic accuracy of a Luminex bead-based commercial analyte-specific reagent for the simultaneous analysis of 30 mutations prevalent in Ashkenazi Jews at eight genetic disease loci.

Methods: DNA from 20 samples with known abnormal genotypes were run a total of 109 times. DNA from 820 patients with unknown genotypes submitted for Ashkenazi Jewish testing panels were analyzed using our current laboratory techniques.

Analytical validation of the tag-it high-throughput microsphere-based universal array genotyping platform: application to the multiplex detection of a panel of thrombophilia-associated single-nucleotide polymorphisms.

Background: We have developed a novel, microsphere-based universal array platform referred to as the Tag-It platform. This platform is suitable for high-throughput clinical genotyping applications and was used for multiplex analysis of a panel of thrombophilia-associated single-nucleotide polymorphisms (SNPs).

Methods: Genomic DNA from 132 patients was amplified by multiplex PCR using 6 primer sets, followed by multiplex allele-specific primer extension using 12 universally tagged genotyping primers.

Novel signal amplification technology with applications in DNA and protein detection systems.

A non-enzymatic approach to signal amplification has practical advantages over conventional target amplification methods. We have designed a simple, cost-efficient signal amplification system that can be used to enhance the detection of nucleic acids or protein. The signal amplification process requires initial capture of analyte by a specific probe, which, depending on the analyte, can be an oligomer or an antibody.

Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes.

A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T(2)-biotin.dT-T(2) loop.

The cDNA sequence and the deduced amino acid sequence of human transcobalamin II show homology with rat intrinsic factor and human transcobalamin I.

The cellular uptake of cobalamin (Cbl, vitamin B12) is mediated by transcobalamin II (TCII), a plasma protein that binds Cbl and is secreted by human umbilical vein endothelial (HUVE) cells. These cells synthesize and secrete TCII and, therefore, served as the source of the complementary DNA (cDNA) library from which the TCII cDNA was isolated. This full-length cDNA consists of 1866 nucleotides that code for a leader peptide of 18 amino acids, a secreted protein of 409 amino acids, a 5'-untranslated segment of 37 nucleotides, and a 3'-untranslated region of 548 nucleotides.

A base substitution at a splice site in the COL3A1 gene causes exon skipping and generates abnormal type III procollagen in a patient with Ehlers-Danlos syndrome type IV.

The dermis of a child with Ehlers-Danlos syndrome type IV (EDS-IV) contained about 11% of the normal amount of type III collagen and cultured dermal fibroblasts produced a reduced amount of type III procollagen which was secreted poorly. Type III collagen produced by these cells contained normal and abnormal alpha-chains and cyanogen bromide peptides. The site of the structural defect in the abnormal alpha 1 (III) chains was localized to the region of Met797, which is at the junction of the two carboxyl-terminal CB5 and CB9 cyanogen bromide peptides.

Hormonal regulation of levels of the messenger RNA encoding hepatic P450 2c (IIC11), a constitutive male-specific form of cytochrome P450.

A cDNA clone for rat hepatic cytochrome P450 2c (gene product IIC11) was isolated and used to study the sex specificity, expression during development, and hormonal regulation of the mRNA encoding this protein in rat liver. P450 2c mRNA levels were about 16-fold higher in males than in females and were only slightly increased in male rats after administration of phenobarbital, a drug that dramatically raises the levels of mRNAs encoding several other members of the P450 II family. In contrast to the mRNA encoding P450 f (gene product IIC7), which increases gradually over the first 6 weeks of life, P450 2c mRNA showed a dramatic increase at puberty, between 4.

Studies on the mechanism of entry of vaccinia virus in animal cells.

In order to study the mechanism of entry of vaccinia virus into cells the fate of virion associated polypeptides was investigated during infection of african green monkey kidney (BSC-40) cells with 35 S-methionine labelled virus. Approximately 12-15 percent of the virion polypeptides were degraded to acid-soluble products by 3 hours post-infection. Proteolysis was inhibited (50 percent) by methylamine, suggesting a lysosomal site of degradation.

Isolation and characterization of neutralizing monoclonal antibodies to vaccinia virus.

Cells producing neutralizing monoclonal antibodies (mAbs) to UV-inactivated vaccinia virus strain WR were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. Three mAbs that reacted strongly with purified virus envelopes as determined by enzyme-linked immunosorbent assay were studied. The three mAbs recognized a 14,000-molecular-weight (14K) envelope protein of vaccinia virus and were shown to be immunoglobulin G2b (mAbC3 and mAbB11) and immunoglobulin M (mAbF11).

Endocytosis, proteolysis, and exocytosis of exogenous proteins by cultured myotubes.

The endocytic activity of chick myotubes in cultures was investigated. Differentiated myotubes internalized the fluid-phase marker horseradish peroxidase in membrane-bound particles which typically displayed reaction product at the inner surface of the vesicle. Pulse-chase experiments demonstrated a rapid decrease in the number of horseradish peroxidase-containing vesicles and a redistribution from a uniform to a perinuclear pattern.

Inhibition of intracellular proteolysis in muscle cultures by multiplication-stimulating activity. Comparison of effects of multiplication-stimulating activity and insulin on proteolysis, protein synthesis, amino acid uptake, and sugar transport.

The effects of the insulin-like growth factor, multiplication-stimulating activity (MSA), on chick myotube cultures were investigated. In serum-free media, MSA at levels reported to be present in fetal serum (5 ng/ml) significantly inhibited overall rates of protein degradation and stimulated protein synthesis and amino acid uptake. Half-maximal effects on protein degradation (-30%), synthesis (+25%), and amino acid uptake (+50%) occurred at approximately 0.