Mickey S. Urdea

Significance of serum hepatitis C virus RNA levels in chronic hepatitis C.

Hepatitis C virus (HCV) is the main cause of parenteral non-A, non-B hepatitis and serum can be tested for the virus itself by reverse-transcription polymerase chain amplification. What of the level of this viraemia? To find out if quantitative study of HCV RNA might be useful clinically we took advantage of participation in trials of interferon-alpha in patients with chronic HCV infection and applied a new assay, branched DNA (bDNA) signal amplification. Paired serum and liver biopsy specimens from 47 patients with confirmed chronic HCV infection and evidence of HCV RNA in their serum were studied. The quantitative bDNA assay (detection limit 350,000 equivalents/mL [eq/mL]) was positive in 34 sera (sensitivity 72%). Patients who acquired HCV infection by blood transfusion had a higher viraemia (median 2,701,000 eq/mL, n = 29) than health workers and intravenous drug users (635,000 eq/mL, n = 13; p < 0.01). Patients with a sustained complete response to interferon-alpha therapy had lower pre-treatment viraemia levels (median at bDNA cut-off, n = 11) than complete responders who relapsed after the drug was stopped (1,613,000 eq/mL, n = 15; p < 0.01) and non-responders (3,066,000 eq/mL, n = 20; p < 0.01). High viraemia levels were not related to the histological diagnosis but were associated with lobular inflammation, lymphoid aggregates, and bile-duct lesions. These findings indicate that mode of acquisition is an important determinant of HCV viraemia and that patients with low HCV viraemia levels are more likely to respond to interferon in a sustained fashion.

Isolation and nucleotide sequence of a cDNA encoding the precursor of mouse nerve growth factor.

Nerve growth factor (NGF) is a polypeptide that enhances survival, nerve fibre outgrowth and neurotransmitter biosynthesis in sympathetic and sensory neurones1–3. Administration of antibodies against NGF to developing animals leads to atrophy of the sympathetic system4. NGF is not normally detectable in innervated tissues but ablation of the innervating neurones leads to the production of measurable NGF in the target tissue5. After transplantation of the denervated tissue, reinnervation occurs, then NGF decreases to undetectable levels. Thus NGF seems to act as a neurotrophic messenger and its level is regulated by innervating neurones. Because of the minute levels present it is very difficult to study NGF biosynthesis in innervated tissue. However, NGF can be isolated from male mouse submaxillary glands, where it exists in inexplicably high levels6. Its amino acid sequence has been determined7, and the synthesis of NGF and its larger precursors has been demonstrated in cultured submaxillary glands8. We report here the nucleotide sequence of a submaxillary cDNA encoding the mouse NGF precursor (preproNGF). In contrast to previous suppositions8 the NGF moiety is situated near the carboxy-terminus of the polyprotein precursor. It is flanked at the amino-terminus by 187 amino acids which may be cleaved at dibasic residues to generate three peptides; there are only two additional amino acids at the carboxy-terminus.

Isolation of the yeast calmodulin gene: Calmodulin is an essential protein

Calmodulin was purified from Saccharomyces cerevisiae based on its characteristic properties. Like other calmodulins, the yeast protein is small, heat-stable, acidic, retained by hydrophobic matrices in a Ca2+-dependent manner, exhibits a pronounced Ca2+-induced shift in electrophoretic mobility, and binds 45Ca2+. Using synthetic oligonucleotide probes designed from the sequences of two tryptic peptides derived from the purified protein, the gene encoding yeast calmodulin was isolated. The gene (designated CMD1) is a unique, single-copy locus, contains no introns, and resides on chromosome II. The amino acid sequence of yeast calmodulin shares 60% identity with other calmodulins. Disruption or deletion of the yeast calmodulin gene results in a recessive-lethal mutation; thus, calmodulin is essential for the growth of yeast cells.

Requirements for High Impact Diagnostics in the Developing World

This paper focuses on the technologies required to meet the global health diagnostics needs presented in the previous papers, highlighting how new diagnostic technologies might have the potential to change medical and public-health scenarios in the developing world.

Rapid and precise quantification of HIV-1 RNA in plasma using a branched DNA signal amplification assay.

The level of human immunodeficiency virus type 1 (HIV-1) RNA in human plasma has been quantitated directly with use of a solid-phase nucleic acid hybridization assay, based on branched DNA (bDNA) signal amplification technology with chemiluminescent detection. Signal amplification is accomplished by the incorporation of sites for 1,755 alkaline phosphatase-labeled probes per genome of HIV-1, after successive hybridization of target-specific oligonucleotides and bDNA amplifier molecules. The assay is performed in microwells, much like an immunoassay, and is amenable to routine laboratory use. Reproducibility and specificity studies indicated that the bDNA method was precise and showed no reactivity with seronegative donors. HIV-1 RNA levels were quantitated for 348 seropositive specimens, with a detection rate of 83% for those specimens from patients with < 500 CD4+ T-cell counts. Plasma RNA levels were found to change with disease stage, and in response to antiviral therapy. Quantitation of HIV-1 RNA in the plasma of HIV-1-infected patients, with use of the bDNA assay, may be a useful method for monitoring HIV-1 disease progression and therapeutic response.

Activation of tumor necrosis factor-α system in chronic hepatitis C virus infection

Tumor necrosis factor-α (TNF-α)plays a central role in the hosts immunomodulatoryresponse to infective agents. To evaluate theTNF-α system in patients with chronic hepatitis Cvirus (HCV) infection, plasma, serum, and peripheral bloodmononuclear cells (PBMC) were prospectively collectedfrom 53 patients and 33 healthy control subjects.Circulating TNF-α and TNF receptors were assayed by their respective enzyme immunoassays. Inaddition, TNF-α mRNA was quantitated in PBMC usinga branched DNA assay, and production of TNF-α byPBMC with and without lipopolysaccharide was also assessed. Patients with chronic HCV infectionhad a higher level of circulating TNF-α comparedto healthy control subjects (9.62 ± 6.01 vs 3.66± 1.23 pg/ml, P < 0.001). They also had highercirculating levels of TNF receptors compared to control(CD120a: 3323 ± 1267, pg/ml, N = 49 vs 1855± 422 pg/ml, N = 33, P < 0.001; CD120b: 1290± 650 pg/ml, N = 51, vs 863 ± 207 pg/ml,N = 33, P < 0.001). Plasma TNF-α level correlated with circulatingCD120a (r = 0.52, N = 49, P < 0.001) and weakly withCD120b (r = 0.32, N = 51, P = 0.02). Plasma TNF-αalso correlated with markers of hepatocellular injury, including ALT (r = 0.34, N = 53, P = 0.01) andα-GST (r = 0.31, N = 43, P = 0.042), but not withserum HCV RNA levels. There was no difference in theTNF-α mRNA levels in PBMC between patients with chronic HCV infection (1.4 ± 1.9units/106 cells, N = 8) and healthy control subjects(2.1 ± 1.4 units/106 cells, N = 8, P = NS). Therewas also no difference in the spontaneous production ofTNF-α by PBMC (1 × 106 cells/ml)between patients with chronic HCV infection (14.2± 36.5 pg/ml, N = 11) and healthy subjects (11.9± 14.0 pg/ml, N = 14, P = NS). However, patientswith chronic HCV infection produced more TNF-α upon stimulation withlipopolysaccharide compared to healthy control subjects(1278 ± 693 pg/ml, N = 11, vs 629 ± 689pg/ml, N = 14, P < 0.05). These data indicate thatthe TNF-α system is activated in patients with chronicHCV infection.

Development of a Type 2 Diabetes Risk Model From a Panel of Serum Biomarkers From the Inter99 Cohort

OBJECTIVE The purpose of this study was to develop a model for assessing the 5-year risk of developing type 2 diabetes from a panel of 64 circulating candidate biomarkers. RESEARCH DESIGN AND METHODS Subjects were selected from the Inter99 cohort, a longitudinal population-based study of ∼6,600 Danes in a nested case-control design with the primary outcome of 5-year conversion to type 2 diabetes. Nondiabetic subjects, aged ≥39 years, with BMI ≥25 kg/m2 at baseline were selected. Baseline fasting serum samples from 160 individuals who developed type 2 diabetes and from 472 who did not were tested. An ultrasensitive immunoassay was used to measure of 58 candidate biomarkers in multiple diabetes-associated pathways, along with six routine clinical variables. Statistical learning methods and permutation testing were used to select the most informative biomarkers. Risk model performance was estimated using a validated bootstrap bias-correction procedure. RESULTS A model using six biomarkers (adiponectin, C-reactive protein, ferritin, interleukin-2 receptor A, glucose, and insulin) was developed for assessing an individuals 5-year risk of developing type 2 diabetes. This model has a bootstrap-estimated area under the curve of 0.76, which is greater than that for A1C, fasting plasma glucose, fasting serum insulin, BMI, sex-adjusted waist circumference, a model using fasting glucose and insulin, and a noninvasive clinical model. CONCLUSIONS A model incorporating six circulating biomarkers provides an objective and quantitative estimate of the 5-year risk of developing type 2 diabetes, performs better than single risk indicators and a noninvasive clinical model, and provides better stratification than fasting plasma glucose alone.

A novel method for the rapid detection of specific nucleotide sequences in crude biological samples without blotting or radioactivity; application to the analysis of hepatitis B virus in human serum.

The detection of a little as 0.2 pg (60,000 molecules) of hepatitis B viral (HBV) DNA in human serum samples in 4 h has been demonstrated using a solution-hybridization and bead-capture method. An amplification method based on chemically crosslinked oligodeoxyribonucleotides was coupled with a horseradish peroxidase-labeling scheme for the ultimate detection of the analyte. Two sets of HBV complementary synthetic oligodeoxyribonucleotide probes containing one of two types of single-stranded (ss) overhangs were employed. These ss overhangs were used to capture the probe-analyte complex onto a bead and subsequently to label it. Detection was achieved with either a chemiluminescent or colorimetric output substrate for the enzyme. Only in the presence of the virus was label specifically bound to the support. The assay was relatively unaffected by either sample composition or by the presence of heterologous nucleic acids.

A chemical 5'-phosphorylation of oligodeoxyribonucleotides that can be monitored by trityl cation release

Abstract A new phosphoramidite-derived reagent, (2-cyanoethoxy)-2-(2′-0-4,4′- dimethoxytrityloxyethylsulfonyl)ethoxy-N,N-diisopropylaminophosphine, for the 5′- phosphorylatlon of ollgodeoxyribonucleotides has been developed. Phosphorylatlon efficiency can be determined by the release of 4,4′-dimethoxytrityl cation in acid.

Evaluation of branched DNA signal amplification for the detection of hepatitis C virus RNA

Summary. There is an increasing need for a practical assay to measure HCV RNA to assess the viral burden in chronic hepatitis C virus (HCV) infection as viral load relates to transmission and therapeutic response. This study evaluates branched DNA (bDNA) signal amplification, a technique that avoids many of the pitfalls of polymerase chain reaction (PCR). The bDNA assay uses a microtitre well format and a series of capture, target and amplification probes that bind RNA to the well and then successively bind oligonucleotides to the RNA and branched DNA molecules to the oligonucleotides. Enzyme‐labelled probes are bound to the arms of the bDNA and light output from a chemiluminescent substrate is directly proportional to the amount of starting HCV RNA. Appropriate standards provide direct quantitation. Whereas PCR amplifies the HCV genome, bDNA amplifies the hybridization signal.