Saliva, diagnostics, and dentistry.

Saliva, a scientific and clinical entity familiar to every oral health researcher and dental practitioner, has emerged as a translational and clinical commodity that has reached national visibility at the National Institutes of Health and the President's Office of Science and Technology. "Detecting dozens of diseases in a sample of saliva" was issued by President Obama as one of the 14 Grand Challenges for biomedical research in the 21(st) Century (National Economic Council, 2010). In addition, NIH's 2011 Government Performance Report Act (GPRA) listed 10 initiatives in the high-risk long-term category (Collins, 2011). The mandate is to determine the efficacy of using salivary diagnostics to monitor health and diagnose at least one systemic disease by 2013. The stage is set for the scientific community to capture these national and global opportunities to advance and substantiate the scientific foundation of salivary diagnostics to meet these goals. A specific calling is to the oral, dental, and craniofacial health community. Three areas will be highlighted in this paper: the concept of high-impact diagnostics, the role of dentists in diagnostics, and, finally, an infrastructure currently being developed in the United Kingdom--The UK Biobank--which will have an impact on the translational and clinical utilizations of saliva.

Standardized hepatitis C virus RNA panels for nucleic acid testing assays.

BACKGROUND: Methods for the quantification of hepatitis C virus (HCV) RNA are useful in the clinical management of infected patients. However, the introduction of assays based upon various nucleic acid testing (NAT) technologies, each utilizing a different set of standards, creates the potential for misinterpretation of patient results. OBJECTIVE: In order to address the need for worldwide standardization of these assays, a HCV RNA quantification panel (NAP HCV-RNA) calibrated against the World Health Organization (WHO) First International Standard for HCV RNA was prepared. The eight-member HCV RNA quantification panel was evaluated utilizing a variety of commercially available and in-house NAT technologies. STUDY DESIGN: NAP HCV-RNA panels were tested and analyzed using the methods and data reduction protocols specific to each NAT technology employed in this study (bDNA, TMA and two PCR methods). Proprietary units of measure from each assay were compared to WHO International Units (IU) for each panel member (0, 50, 500, 5000, 50000, 200000, 500000 and 2000000 IU/ml). RESULTS: Evaluation of the NAP HCV-RNA in a variety of NAT procedures demonstrated linearity across the range of target concentrations detected in each assay and R(2) values ranged from 0.9929 to 0.9995 across the four technologies. As expected, the correspondence of assay specific proprietary units to IU differed depending upon the technology utilized. CONCLUSIONS: NAP HCV-RNA provides a consistent, standardized method for comparing results across laboratories and technologies and is useful in ensuring the quality of NAT testing for HCV RNA, independent of the methodology used.

Detection of hepatitis C after liver transplantation. Four serologic tests compared.

To determine the best method for detecting HCV infection in immunosuppressed patients, stored frozen serum from 101 liver transplant recipients was tested for hepatitis C virus. Each sample was tested by four assays. HCV RNA was detected by both polymerase chain reaction (PCR) and branched DNA signal amplification. Antibody to HCV was determined using second-generation enzyme-linked immunoassay (EIA) and recombinant immunoblot assay. Forty one transplant recipients met the working definition for true positives of HCV infection. Of these "true positives," 98% were positive by HCV RNA PCR assay, 88% by b-DNA signal amplification assay, 88% by anti-HCV EIA, and 63% demonstrated two or more reactive bands on recombinant immunoblot. Five of 57 (9%) HCV-antibody negative recipients had HCV RNA detected by both methods. Of 44 HCV enzyme-linked immunoassay (EIA) repeatedly reactive samples, the recombinant immunoblot was negative in 2 and indeterminate in 13. HCV RNA was present in 9 of 13 recombinant immunoblot indeterminate sera. Nine EIA repeatedly reactive sera were negative by both tests for HCV RNA. In liver transplant recipients, HCV infection is best determined by measurement of HCV RNA. Antibody formation may be delayed or suppressed in a minority of patients despite > 10(9) equivalents/L (> 10(6)/mL) of HCV RNA in serum. Recombinant immunoblots with a single reactive band pattern often indicate HCV infection in immunosuppressed patients.

Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay.

The aim of this study was to establish the performance characteristics of a nonradioisotopic branched DNA (bDNA) signal amplification assay for quantitation of hepatitis B virus (HBV) DNA in human serum. Quantitation was determined from a standard curve and expressed as HBV DNA equivalents/mL (Eq/mL; 285,000 Eq = 1 pg of double stranded HBV DNA). The bDNA assay exhibited a nearly four log dynamic range of quantitation and an analytical detection limit of approximately 100,000 Eq/mL. To ensure a specificity of 99.7%, the quantitation limit was set at 700,000 Eq/mL. The interassay percent coefficient of variance for quantification values ranged from 10% to 15% when performed by novice users with different sets of reagents. Using the bDNA assay, HBV DNA was detected in 94% to 100% of hepatitis B e antigen-positive specimens and 27% to 31% of hepatitis B e antigen-negative specimens from chronic HBV-infected patients. The bDNA assay may be useful as a prognostic and therapy monitoring tool for the management of HBV-infected patients undergoing antiviral treatment.

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

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. In testing a standardized, coded panel, bDNA showed 100% specificity and detected five of six sera proven to transmit hepatitis C to the chimpanzee; PCR detected all six infectious sera. Serial samples were measured in two acute and five chronic cases of transfusion-associated hepatitis and in three commercial seroconversion panels. In acute cases, 10(7)-10(8) molecular equivalents per ml (eq per ml) of HCV RNA were detected prior to peak alanine aminotransferase (ALT) activity and then rapidly declined to non-detectable levels. Similar levels of HCV RNA were observed early in the course of two patients who progressed to chronic hepatitis; the chronic course was characterized by diminished, fluctuating and sometimes non-detectable levels of HCV RNA. In two chronic cases, HCV RNA was not detected, or only transiently detected by bDNA, but was present when assayed by PCR. In one chronic case, the periodicity of HCV RNA levels closely paralleled the fluctuations of ALT suggesting a relationship between viral replication and subsequent hepatocellular injury. In testing 50 blood donors whose anti-HCV reactivity was confirmed by a recombinant immunoblot assay (RIBA), HCV RNA was detected by bDNA in 41 (81%), while PCR was positive in 45 (90%); the overall concordance between bDNA and PCR in 100 anti-HCV enzyme immunoassays (EIA) reactive donor samples was 96%.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of quantitative cDNA-PCR with the branched DNA hybridization assay for monitoring plasma hepatitis C virus RNA levels in haemophilia patients participating in a controlled interferon trial.

The branched DNA (bDNA) assay was compared with a semi-quantitative cDNA-polymerase chain reaction (cDNA-PCR) assay for monitoring HCV RNA levels in plasma in 17 haemophilia patients participating in a controlled alpha-interferon trial. Good correlation between the HCV RNA levels as detected by the two assays was observed, with a correlation co-efficient of 0.83 (P < 0.0001) and 0.90 (P < 0.0001) at week 0 and 24, respectively. Hepatitis C virus RNA (HCV RNA) levels could be assessed with the bDNA assay in 14/17 (82 percent) HCV cDNA-PCR positive pretreatment samples. The bDNA assay apparently failed to detect low viral titres. Interferon treated patients (n = 11) showed either a complete response, being a large reduction in HCV RNA level to below the detection limit of the HCV cDNA-PCR assay (6/11) or no significant reduction in HCV RNA level (5/11). A "partial" virological response was not observed. The changes in HCV RNA plasma levels in non-responders during interferon (IFN) treatment were similar to the (small) natural fluctuations in viral load observed in controls (untreated patients). Although the bDNA assay was not as sensitive as cDNA-PCR, given its user friendliness and quantitative results, it is concluded that it is a useful test for monitoring HCV RNA levels in patients treated with interferon. However, patients who are non-reactive in the bDNA assay have to be retested by cDNA-PCR because low viral titres are not detected by the bDNA assay.

Quantitative detection of hepatitis C virus RNA with a solid-phase signal amplification method: definition of optimal conditions for specimen collection and clinical application in interferon-treated patients.

To determine the optimal conditions for preparation of serum specimens for quantitative hepatitis C virus RNA determination, patient samples were processed such that differences in time from clot formation to centrifugation, centrifugation to separation of serum and collection of serum until freezing could be independently assessed. The effects of multiple cycles of freezing and thawing were also determined. There was progressive and significant loss of hepatitis C virus RNA activity when the time from the formation of the clot until centrifugation was longer than 2 hr. This reduction reached 32% after 6 hr and 49% after 24 hr. If centrifugation was performed immediately after formation of the clot, loss of hepatitis C virus RNA activity was reduced to less than 10% even though the serum remained unseparated from the clot for up to 6 hr. Centrifugation of blood through a paraffin plug (serum separator tube) prevented loss of hepatitis C virus RNA activity for up to 24 hr. There was no loss of hepatitis C virus RNA activity with up to three freeze-thaw cycles. When patient specimens were prepared under these optimal conditions, the sensitivity of the quantitative branched DNA signal amplification assay in patients with hepatitis C virus infection was 83% and the specificity in patients with liver disease was 100%. Fluctuations in hepatitis C virus RNA levels were shown to correlate with biochemical changes observed in patients treated with recombinant interferon-alpha 2b. These data demonstrate that improper or inconsistent methods of serum preparation may result in falsely low and unreliable levels of hepatitis C virus RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitation of hepatitis C virus RNA in liver transplant recipients.

BACKGROUND/AIMS: Hepatitis C virus (HCV) infection is common in liver transplant recipients, yet the effects of immunosuppression on HCV RNA levels and the relationship of HCV RNA levels to hepatic damage have not been studied. METHODS: To explore these issues, we measured HCV RNA in serum by polymerase chain reaction amplification and branched DNA assay from 100 HCV-infected patients undergoing liver transplantation. RESULTS: Mean posttransplant levels were 16-fold higher than pretransplant values (7,935,000 and 496,000 Eq/mL, respectively; n = 65; P < 0.0001). Patients with high pretransplant levels had higher mean posttransplant levels than those with low pretransplant levels (17,119,000 and 6,504,000 Eq/mL, respectively; P = 0.064). Posttransplant levels were similar in patients with recurrent and acquired infection and were independent of time of sampling. Fifty percent of patients with HCV infection had normal liver biopsy specimens, and there was no strong relationship between level of viremia and degree of hepatic damage. CONCLUSIONS: HCV RNA levels increase markedly following liver transplantation. The frequent finding of viremia in the absence of histological hepatitis suggests that a "carrier state" is common. Absence of allograft damage in some (despite high levels of viral RNA) suggests that in immunosuppressed patients, HCV infection may be tolerated without direct hepatic damage.

Quantitative evaluation of hepatitis C virus RNA in patients with concurrent human immunodeficiency virus infections.

Quantitation of the hepatitis C virus (HCV) provides a powerful epidemiologic and therapeutic method for the evaluation of infected patients. In this study semiquantitative reverse transcriptase polymerase chain reaction (PCR) is compared with a new branched DNA signal amplification methodology. Samples from HCV-infected patients as well as from human immunodeficiency virus-infected patients were evaluated. Reverse transcriptase PCR correlated well with the branched DNA assay (r = 0.7036, P < 0.05). HCV RNA was found to occur at significantly higher titers (P < 0.05) in patients coinfected with the human immunodeficiency virus compared with titers in those infected with HCV alone. Immune status as defined by the CD4+ count was not associated with the observed difference in viral titer.

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.

Altered form of placental alkaline phosphatase produced by JAR choriocarcinoma cells in culture.

The alkaline phosphatase activity expressed by JAR choriocarcinoma cells was compared to the placental isoenzyme of human alkaline phosphatase by several criteria. JAR cell alkaline phosphatase was similar to the placental isoenzyme with respect to heat and urea stability and sensitivity to most inhibitors, but it differed significantly from placental alkaline phosphatase in its sensitivity to L-phenylalanylglycylglycine. In contrast to the serum form of placental alkaline phosphatase, the JAR cell enzyme was highly hydrophobic as determined by octyl agarose chromatography and appeared to be larger than the placental isoenzyme based on gel filtration and polyacrylamide gel electrophoresis. The slowly migrating hydrophobic JAR cell activity could be converted into a fast-migrating hydrophilic form similar to the serum placental isoenzyme by treatment with trypsin-like proteases. Conversion to the hydrophilic form was accompanied by a decrease in subunit molecular weight from 68,000 to 66,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 33P-labeled enzyme. Peptide mapping of the 33P-labeled molecules by limited proteolysis in the presence of sodium dodecyl sulfate showed JAR cell alkaline phosphatase to be closely related to the placental isoenzyme with respect to primary structure. However, placental alkaline phosphatase appeared to be richer in sialic acid residues than was the JAR cell enzyme. We conclude that JAR cells produce a form of placental alkaline phosphatase that contains a hydrophobic region not associated with the free serum enzyme and that the tumor cell enzyme is modified or processed differently than is the enzyme found in the term placenta.

Expression of oncodevelopmental gene products by human tumor cells in culture.

Sixty-seven human tumor cell lines and 15 lines derived from normal tissue were examined for the production of the oncodevelopmental markers carcinoembryonic antigen, alpha and beta subunits of chorionic gonadotropin, placental and nonplacental forms of alkaline phosphatase, gamma-glutamyltransferase, cystyl aminopeptidase, and calcitonin. Both intracellular and extracellular levels of these markers were determined at three phases during the growth of each culture. Sixty-eight percent of the cell lines produced elevated levels (greater than or equal to 90th percentile) of at least one marker. Of those, 46% produced elevated levels of one marked only, 29% produced two, 22% produced three, and 4% produced four markers. No cell line produced more than four markers at elevated levels. In most instances, however, the expression of any two particular markers was discordant. For approximately 50% of the possible marker pairs, Spearman rank-ordered correlation analyses showed significant negative correlations, indicating that when one marker was produced at elevated levels by a given cell line, other markers were usually absent ot produced at relatively low levels. In no instance was a significant positive correlation found between two markers. These data indicated that, although most human malignant cells examined produced one or more oncodevelopmental gene markers at elevated levels, no predictable coexpression of any two of the markers was seen.

Content of gonadotropins in cultured human malignant cells and effects of sodium butyrate treatment on gonadotropin secretion by HeLa cells.

Twenty-one of 82 human cell lines examined for production of human chorionic gonadotropin and its subunits (HCG-alpha and HCG-beta) produced either one or both subunits at some phase in their growth. Of these, 14 produced an excess amount of free alpha subunit, and seven produced HCG-beta or complete HCG without evidence for free alpha subunit synthesis. Five of the HCG-producing cell lines also contained or secreted the beta subunit of human luteinizing hormone. CBT cells derived from a glioblastoma multiforme and JAR choriocarcinoma cells secreted significant amounts of the beta subunit of human luteinizing hormone, while three other cell lines (breast carcinoma MCF-7, HeLa S3, and melanoma A375) produced small amounts of the beta subunit of human luteinizing hormone but did not appear to secrete it. Two cell lines (the melanoma line A375 and the SV40-transformed line SV80) appeared to contain small amounts of human follicle-stimulating hormone. Sodium butyrate caused a 40-fold induction in the secretion of both HCG-alpha and HCG-beta by HeLa S3 cells, but the total amount of HCG-alpha secretion induced was 800-fold greater than that of HCG-beta. Induction was blocked by actinomycin D (1 microgram/ml) and cycloheximide (5 microgram/ml) but was not affected by 1-beta-D-arabinofuranosylcytosine at a concentration (5 microgram/ml) that blocked DNA synthesis 99%. These results indicate that a number of malignant human cell lines produce the subunits of both placental and pituitary gonadotropins and that there is frequently an excess secretion of the free alpha subunit common to these hormones.

Electron microscopy of late adenovirus type 2 mRNA hybridized to double-stranded viral DNA.

R loops were generated with late adenovirus type 2 (Ad2) mRNA in double-stranded viral DNA, and visualized by electron microscopy. Unpaired DNA sequences in Ad2:Ad2+ND4 heteroduplex DNA served as a visual marker for the orientation of R loops with respect to the conventional DNA map. The most abundant classes of late Ad2 mRNA observed by this technique hybridized, in order of R-loop frequency, with midpoints near posit1ons 0.57, 0.88, 0.77, and 0.40 to 0.50 of the DNA map. The R loop at position 0.57, 0.88, 0.77, and 0.40 containing the hexon gene; the one at position 0.88 corresponded to a region containing the fiber gene. The relative frequencies of these two R loops paralleled those of the encoded gene products. The mRNA sizes, calculated from those of the respective R loops, were slightly larger than needed to code for these polypeptides. Using the R-loop technique, two locations at which adjacent mRNA's hybridized to different strands were accurately mapped at positions 0.61 and 0.91 of the DNA. The map positions of late Ad2 mRNA correlated well to published RNA and protein maps.

Early gene expression of adenovirus type 2: R-loop mapping of mRNA and time course of viral DNA, mRNA, and protein synthesis.

Adenovirus type 2 DNA was hybridized to early mRNA isolated from the cytoplasm of infected cells prior to the initiation of viral DNA synthesis. Resulting R loops were visualized in the electron microscope, and their positions were oriented with the help of DNA fragments generated by digestion with the restriction endonuclease BamHI. Early RNA was found to map (in order of relative R-loop frequency) with midpoints near positions 0.95, 0.80, 0.03, 0.65, and 0.09 on the conventional adenovirus map. The time of appearance of individual viral mRNA's was compared to the time course of viral protein and DNA synthesis. We present a refined map of adenovirus gene functions which is based on results documented in this and the accompanying study by Meyer et al. (1977), as well as on data published by other laboratories.

In Vitro system for the study of bacteriophage phi chi 174 adsorption and eclipse.

An in vitro system was developed for the study of the initial stages of bacteriophage phi chi 174 infection. Escherichia coli C cells were incubated with 20% sucrose and then subjected to cold osmotic shock in 5 mM MgSO4. The concentrated supernatant shock fluid inactivated phi chi 174 with the same kinetics and requirements as for normal infection. Shock fluids prepared from phi chi 174-resistant strains of E. coli did not show this effect. The 114S phage were initially converted into 70S particles, the process termed "eclipse". These structurally altered phages then attached to a component of the shock fluid, producing fast-sedimenting complexes, and eventually released at least a part of their DNA into the medium. The fast-sedimenting complex could be radioactively labeled with oleic acid. Radioactivity was found to co-chromatograph with both biological activity and the majority of the high-molecular-weight carbohydrates present in the shock fluid. It is concluded that E. coli C osmotic shock fluid contains isolated phi chi 174-specific receptor sites composed of lipopolysaccharides. This system conveniently separates the early stages of phage phi chi 174 infection from the intracellular events.