Reverse Transcriptase Assay Research Articles

Molecular and biological characterization of simian-human immunodeficiency virus-like particles produced by recombinant fowlpox viruses

Virus-like particles (VLPs) mimicking the simian-human immunodeficiency virus SHIV 89.6P (VLP SHIV) were produced by co-infection of Vero cells with fowlpox SIV gag/ pol (FP gag/ pol SIV) and fowlpox HIV-1 env 89.6P (FP env 89.6P) recombinant viruses. As a necessary prerequisite for a more efficient vaccine approach, ultrastructural, functional and molecular characterizations of VLP SHIV were performed in the SHIV-macaque model to verify the similarity of these particles to SHIV 89.6P. Here we show that VLP SHIV can infect T cells by fusion without replication, as demonstrated by the absence of new viral progeny in VLP SHIV-infected C8166 cells. Biochemical characterization showed identical protein profiles of VLP SHIV and SHIV 89.6P, and ultrastructural analysis of Vero cells releasing VLP SHIV also confirmed the morphological similarity of these pseudovirions to SHIV 89.6P particles. Viral mRNAs were also found packaged inside the core of VLP SHIV by RT-PCR and reverse transcriptase assays.

825. Product Enhanced Reverse Transcriptase (PERT) Assay for RCL/RCR Detection

Product Enhanced Reverse Transcriptase (PERT) assay has been used extensively to detect reverse transcriptase (RT) activity associated with retroviruses. We have assessed the usefulness of the PERT assay for RCL and RCR testing and compared it with existing assays for RCL (p24gag ELISA/gag PCR) and RCR (S+/L|[minus]|) detection. As a first step towards evaluating the usefulness of PERT for RCL detection, we determined the sensitivity of detection of purified and virus associated HIV-RT by PERT. The PERT assay was able to detect |[sim]| 100 molecules of purified HIV-1 RT and 1-0.1 IU of a replication competent HIV-1 virus, R7-GFP, in two independent experiments. In a RCL detection assay comprising of a 3-week amplification phase and a 1-week indicator phase, 1 IU of R7-GFP was detected by all three assays (p24 ELISA, gag PCR and PERT) in spite of higher backgrounds associated with the PERT assay. To provide additional support for the use of PERT for RCL testing, we also examined the effect of competing vector particles on RCL detection by p24 ELISA and PERT. Both assays were able to detect 1 IU of R7-GFP mixed with varying concentrations (100, 1000, 5000 and 10,000 ng of p24) of a HIV-1 vector, CS-CGW (provided by Philip Zoltick, Philadelphia, PA), in the RCL detection assay. These results suggest that the PERT assay is as sensitive as p24gag ELISA and gag PCR for detection of replication competent HIV-1 in a RCL detection assay. For evaluating the ability of the PERT assay to detect RCR, we determined the sensitivity of detection of purified M-MuLV-RT and compared the detection of replication competent retroviruses (GALV-SEATO, RD114, and 4070A) by PERT and S+/L- assays. The PERT assay detected approximately 100 molecules of purified MuLV-RT in three independent experiments. Both assays detected 1 IU of RD114 and 10 IU of 4070A; detection of the GALV-SEATO virus was more sensitive by the S+/L- assay (1-10 IU) than the PERT assay (10-100 IU). These studies indicate that the sensitivity of detection of retroviruses is equivalent by PERT and S+/L- assays. To evaluate the sensitivity of PERT in a RCR detection assay, GALV-SEATO and RD114 viruses were amplified for 3-weeks and serial dilutions of amplified material were tested by S+/L- and PERT assays. Both assays detect 1 IU of RD114 and GALV after the amplification phase suggesting that they have similar sensitivities in an extended RCR assay. In conclusion, the PERT assay can be used for RCL and RCR testing of a variety of retroviral vectors regardless of the structure, sequence, and envelope of the vectors.

Evaluation of a Low Cost Reverse Transcriptase Assay for Plasma HIV-1 Viral Load Monitoring

We evaluated a low cost manual reverse transcriptase assay (ExaVir Load V.1 and V.2; Cavidi Tech AB) against commercially available HIV RNA assays that quantify viral load to assess its suitability for use in resource-constrained settings. Frozen plasma samples previously tested for RNA by RT-PCR (Roche Diagnostics) and bDNA (Bayer Diagnostics) were retested for RT activity. Text sequence obtained from HIV genotype analysis was submitted to the Stanford HIV Resistance Database V.3.9 and were examined for resistant virus. Detectable RT was present in 98% of samples (V.1; n=127) and in 95% of samples (V.2; n=69) with RNA >10,000 and >1,000 copies/ml respectively. Positive association was found between the log10 RNA copies/ml and log10 RT copies/ml equivalents variables using Pearson's correlation (V.1: r=0.89, n=189; V.2: r=0.89, n=85). The RT activity over time closely followed the trend for RNA levels in samples from 10 HIV seropositive patients with progressive disease. A strong association between RT and RNA was also found with paired samples from 19 patients taken at initiation or change of antiretroviral therapy and again within 2 months. Current (n=40) or no (n=119) exposure to efavirenz therapy had no effect on RT assay performance despite efavirenz binding tightly to the RT enzyme. Samples that demonstrated resistance to the non-nucleoside RT inhibitors (n=112) had a decrease in RT of 0.20 log10 indicating a possible decrease in RT fitness. The RT assay showed good association with current molecular assays, and V.2 is sufficiently sensitive for monitoring HIV viral load in resource-constrained settings.

CDNA Microarray Analysis of Nerve Growth Factor-Regulated Gene Expression Profile in Rat PC12 Cells

Nerve growth factor (NGF)-driven differentiation of PC12 cells into neuronal-like cells provides a representative model system for studying neuronal differentiation processes. Despite of extensive research, gene regulation associated with the differentiation program in PC12 cells still needs to be elucidated. We used cDNA microarray analysis to characterize the response of PC12 cells to NGF at mRNA expression. Forty-six genes were reproducibly influenced by 2-fold or more after NGF treatment for 5 days. Twenty-five of the regulated transcripts were matched to genes which have known functions. Among the microarray results confirmed with real-time reverse transcriptase assay, several genes have not previously known to be modulated by NGF. The results mostly reflected changes in molecules regulating neural plasticity, cytoskeletal organization, and lipid metabolism, which include neuritin, PDZ protein Mrt1, lipoprotein lipase, tropomodulin 1 and rhoB. These observed genetic changes may provide new information about molecular mechanisms underlying NGF-promoted differentiation of PC12 cells.

Detection of serum reverse transcriptase activity in patients with ALS and unaffected blood relatives

Retroviral involvement in the etiology of sporadic ALS has been suspected for several years since the recognition that both murine and human retroviruses can cause motor neuron disease-like syndromes. In a pilot study, an increased prevalence of a retroviral marker (reverse transcriptase [RT] activity) was demonstrated in the serum of British patients with ALS. The current investigation was designed to confirm and extend these findings in a geographically distinct patient cohort under blinded testing conditions. A highly sensitive product-enhanced RT assay was employed to test coded sera obtained from 30 American patients with sporadic ALS and from 14 of their blood relatives, 16 of their spouses, and 28 nonrelated, nonspousal control subjects. Serum RT activity was detected in a higher proportion of ALS patients (47%) than in non-blood-related controls (18%; p = 0.008). The prevalence of RT activity in the serum of spousal controls (13%) was similar to that in other non-blood-related controls. Unexpectedly, the prevalence of serum RT activity in blood relatives of ALS patients (43%) approached that in the ALS patients themselves. These results confirm that patients with ALS have a significantly higher prevalence of serum reverse transcriptase (RT) activity than that seen in unrelated control subjects. The finding of a similarly increased prevalence in blood relatives of ALS patients raises the possibility that the observed RT activity might be due to an inherited endogenous retrovirus.

Detection of reverse transcriptase activity in human melanoma cell lines and identification of a murine leukemia virus contaminant

Stimulated by earlier reports on the presence of retroviruses in mouse and hamster melanoma cell lines, we addressed the question as to whether human melanoma cell lines might also harbour a retrovirus. The melanoma cell lines SK-MEL-25, SK-MEL-28, MEL-JUSO, MML-I, MeWo, A-375, Colo-38, BS-780 were confirmed to be human by human leucocyte antigen (HLA) typing, and supernatants were tested by the product-enhanced reverse transcriptase (PERT) assay for reverse transcriptase (RT) activity. Cell lines SK-MEL-25, SK-MEL-28, MEL-JUSO and MML-I were positive, whereas cell lines MeWo, A-375, Colo-38 and BS-780 were negative. The RT activity peaked at a buoyant density in sucrose typical for retroviruses. From this peak fraction an R-U5 sequence indistinguishable from murine leukemia virus (MLV) was identified by particle-associated retrovirus RNA amplification (PARRA). Semiquantitative MLV-specific RNA-PCR demonstrated colocalization of the MLV-like RNA and RT activity on the sucrose gradient of SK-Mel-25. MLV RNA and DNA were also detectable in culture supernatants of SK-MEL-28, MEL-JUSO and MML-I, but not of MeWo, A-375, Colo-38 and BS-780 by semiquantitative polymerase chain reaction (PCR). Sequence comparison revealed highest homology with the RET sequence previously identified in mouse myeloma SP2/0-AG14 cells. Taken together, our data strongly suggest that certain human melanoma cell lines are productively infected by a MLV which was probably introduced during tumour passage in mice or by laboratory contamination many years ago and subsequently spread to other lines. We recommend mandatory testing of melanoma and other human cell lines for contamination with infectious MLV or other animal retroviruses, similar to mycoplasma screening, in order to avoid artificial experimental data.

Anti-HIV-1 activities in extracts from some medicinal plants as assessed in anin vitro biochemical HIV-1 reverse transcriptase assay

An in vitro HIV-1 reverse transcriptase (RT) assay was used for screening of anti-HIV activity of extracts obtained from some Kenyan medicinal plants. The assay utilises [3H]-methyl thymidine triphosphate (dTTP) as the enzyme substrate and polyadenylic acid.oligodeoxythymidylic acid [poly(rA).p(dT)(12-18)] as the template-primer dimmer. This assay was optimised and standardised with respect to the various experimental parameters in a microtiter plate methodology. The assay was then applied to test for potential antiviral activities of several Kenyan medicinal plant extracts and the concentrations producing 50% inhibition (IC50) of the HIV-1 RT were determined. This assay is described in this report and results obtained with some of the extracts are presented.

Improved HIV-1 viral load determination based on reverse transcriptase activity recovered from human plasma

A more sensitive version of ExaVir Load, a test that utilizes reverse transcriptase (RT) activity from virions in plasma to determine HIV-1 viral load, is described. The virions were immobilized on a gel that was washed, followed by lysis of the virions, elution of purified RT, and finally RT activity determination. The changes made to the original test were: (1) improved washing of the immobilized virions by addition of a non-lytic detergent to the wash buffer, (2) improved virion lysis procedure, including changes in salt, detergent and pH, (3) the use of larger sample volumes in the RT assay, and (4) prolonged RT reaction time. The alterations gave a tenfold increased sensitivity compared to the original version. The correlation between RT load by the current test and RNA PCR was the same as previously (r=0.90). Using colorimetric product detection, the average detection limit in a panel of 262 patient plasma from Stockholm was 0.5 fg RT/ml, corresponding to approximately 170 RNA copies/ml. None of 54 HIV-1 RNA negative samples exhibited RT. The amount of RT load positive samples were 19% for samples containing 50-400 RNA, 71% for samples with 400-1,500, and 100% among samples with >8,000 copies/ml (according to Roche Amplicor). The sensitivity could be increased further using fluorimetric detection. In conclusion, the modifications of the test described result in an important increase in sensitivity. It can now be regarded as a competitive alternative method for HIV viral load determinations.

Reverse transcriptase activity for quantitation of HIV-1 subtype C in plasma: Relation to RNA copy number and CD4 T-cell count

The present study monitored the changes in human immunodeficiency virus (HIV) viral load using a reverse transcriptase (RT) assay and an HIV-1 RNA based assay, and relates these data to the dynamics of CD4 cell counts. The samples examined originate from a prospective study of HIV-1 subtype C infected, untreated Ethiopians followed twice yearly over a period of up to 5 years. The ExaVir Load test, version 1, was used for isolation and quantitation of HIV-1 RT in plasma. The RT activities recovered were compared to the HIV-1 RNA copy numbers, which had been determined previously by the NucliSens HIV-1 QT Test. There was a significant correlation between the data obtained in the two tests (r = 0.65, P < 0.0001). During follow-up, the median RT and RNA levels increased more or less in parallel up to approximately four times the values at admittance. CD4 cell counts, which had also been determined previously, decreased slowly but continuously from approximately 310 to 190 CD4 cells/ml. In the majority of individual patients, there was an inverse correlation between CD4 T-cell counts and RT activity, and with the RNA copy number, and the data obtained by either test could be used to predict CD4 T-cell counts. The ExaVir Load test thus provides data equivalent to the estimation of the number of HIV-1 RNA copies for the prediction of CD4 T-cell counts. It is based on a simple technique, can be run in any routine diagnostic laboratory, and is a competitive alternative for use in resource limited settings.

Anti-HIV activities of organic and aqueous extracts of Sutherlandia frutescens and Lobostemon trigonus

A screening process was applied to extracts made from Sutherlandia frutescens (L.) R. Br (Fabaceae) and Lobostemon trigonus (Boraginaceae) as identified by the Botany Department, University of Port Elizabeth to detect if any of the extracts inhibited the human immunodeficiency virus (HIV). For purposes of dereplication, sulphated polysaccharides were removed and bovine serum albumin (BSA) was included in the assays to adsorb non-specific tannins potentially present. In the reverse transcriptase (RT) assay, an aqueous extract of the Lobostemon leaves inhibited HIV-1 RT with an IC 50 value of 49 μg/ml, while in the protease assay no inhibition was seen. In the α- and β-glucosidase assays, no significant inhibition was seen with the inclusion of BSA, indicating tannin-based inhibitory effects on these two enzymes. The β-glucuronidase inhibitory activity, however, was retained in the presence of BSA. The study shows that Sutherlandia extracts contain inhibitory compounds active against HIV target enzymes, while aqueous Lobostemon leaf extracts contain a potent HIV-1 RT inhibitor, thus showing a potential mechanistic action of these plants in aiding HIV-positive patients.

A novel partial modification at C2501 in Escherichia coli 23S ribosomal RNA.

Escherichia coli is the best-characterized organism with respect to posttranscriptional modifications of its ribosomal RNA (rRNA). It is presently believed that all the modified nucleotides have been identified, primarily on the basis of two detection methods; modification-induced inhibition of the enzyme reverse transcriptase or analysis by combined HPLC and electrospray ionization mass spectrometry. Comparison of data from these different approaches reveals a disagreement regarding modification of C2501 in E. coli 23S rRNA. A. Bakin and J. Ofengand previously reported the detection of a modification at this site based on a reverse transcriptase assay. J.A. McCloskey and coworkers could not confirm the existence of such a modification using an electrospray ionization mass spectrometry approach. C2501 is therefore generally considered unmodified. We have used a strategy involving isolation of a specific rRNA fragment from E. coli 23S rRNA followed by Matrix Assisted Laser Desorption/Ionization mass spectrometry and tandem mass spectrometry to investigate this controversy. Our data reveal a novel 16-Da partial modification at C2501. We believe that the data reported here clarify the above discrepancy, because a minor partial modification detected in a reverse transcriptase assay would not necessarily be detected by the original mass spectrometry approach. The level of modification was furthermore monitored in different growth situations, and we found a significant positive regulation in stationary phase cells. C2501 is universally conserved and implicated in structure folds very close to the catalytic center of the ribosome. Moreover, several antibiotics bind to nucleotides in this region, which altogether make a modification at this site interesting.

84. Enhanced Detection Method for Retrovirus in Vaccine

Detecting any presence of retroviruses in biological material is important to ensure safety and prevent retroviral infection, which has been associated with various disorders including malignancies, immunodeficiencies, and neurological disorders. Highly specific and sensitive PCR techniques are used to detect specifically the presence of a known retrovirus. In contrast, the presence of an unknown retrovirus can only be examined by non-specific methods such as transmission electron microscopy and growth of virus on susceptible cells. However, detection methodologies involving cell culture techniques may be ineffective because certain retroviruses are not cytopathic in vitro. Retroviruses possess RNA-dependent DNA polymerase known as reverse transcriptase (RT), which is inherent in all kinds of retrovirus. Taking advantage of its ubiquitous nature in retroviruses, enzymatic activity of RT may be measured to detect retroviral contamination in biological products. On the basis of the polymerase enhanced reverse transcriptase (PERT) assay, two different assay systems, PCR-based RT assay (PERT-PCR) and ELISA-based RT assay (PERT-ELISA), were developed. Respective optimum conditions were determined. Sensitivity, linearity and reproducibility of the methods were studied on purified RT. Both PERT-PCR and PERT-ELISA could detect as little as 10-10 unit/ml of RT. Nonetheless, no additional increase in sensitivity was observed by performing ELISA after PCR amplification. Treatment with RNase after cDNA synthesis increased sensitivity over 100 times, and the addition of synthetic DNA competitors, which suppress RT-like activities of cellular DNA-dependent DNA polymerases, greatly increased specificity of the assay. A modified form of PERT-PCR assay was used and subsequently RT activities were detected in vaccines and culture supernatant of retrovirus-producing cells. This enhanced form of PERT-PCR assay is easy to perform and is able to detect up to 10-10 unit/ml of purified RT, as well as RT in vaccines and cell culture supernatant. Thus, this study presents an enhanced assay, applicable to screening of all replication-competent retrovirus, quantification of the viral load, drug susceptibility testing of RT, and control of virus inactivation in biological products.

Anti-HIV-1 Cycloartanes from Leaves and Twigs ofGardenia thailandica

Thailandiol ( 1), gardenolic acid A ( 2), quadrangularic acid E ( 3) and 3beta-hydroxy-5alpha-cycloart-24(31)-en-28-oic acid ( 4) have been isolated from the leaves and twigs of Gardenia thailandica Tirveng (order: Rubiales; family: Rubiaceae). In addition, 5-hydroxy-7,2',3',4',5',6'-hexamethoxyflavone ( 5), 5,7-dihydroxy-2',3', 4',5',6'-pentamethoxyflavone ( 6), 5-hydroxy-7,2',3',4',5'-pentamethoxyflavone ( 7) and 5,7-dihydroxy-2',3',4',5'-tetramethoxyflavone ( 8) were also isolated from the same source. The structures were elucidated by spectroscopic methods. Crude extracts and compounds 1 - 4 displayed anti-HIV-1 activities as determined by using the (Delta)(Tat/Rev)MC99 virus and 1A2 cell line system. The EC (50) values determined by the syncytium assay ranged from < 7.8 to 110 microg/mL. They also exhibited moderate to high activities in reverse transcriptase (RT) assay; the IC (50) values of compounds 1 - 4, ranged from < 22.5 to 156.8 microg/mL.

Activation of primary porcine endothelial cells induces release of porcine endogenous retroviruses.

Endothelial cells form the interface between the porcine graft and the recipient and frequently become activated after xenotransplantation. To evaluate the safety of xenotransplantation further, we assessed the effect of cellular activation on the expression and release of porcine endogenous retroviruses from primary endothelial cells isolated from transgenic and nontransgenic pigs. Primary porcine endothelial cells, cultured from pigs transgenic for human decay accelerating factor, were treated with human tumor necrosis factor-alpha, porcine interferon-gamma, or lipopolysaccharide. The release of porcine endogenous retroviruses into the supernatant was monitored at 24-hr intervals (up to 72 hr) by polymerase chain reaction-based reverse transcriptase (PBRT) assay. Activated and unactivated endothelial cells were co-cultured with human cells to investigate the capacity of any virus released from the porcine cells to infect human cells. Virus was not detected in supernatants from quiescent cells by PBRT analysis. The number of viral particles released from endothelial cells was 10 to 5 x 10 viral particles/mL after cellular activation with tumor necrosis factor-alpha, interferon-gamma, or lipopolysaccharide, as shown by PBRT analysis. In contrast, in vitro infection of human cells was observed with unactivated endothelial cells only and was not observed in co-cultures with the activated porcine cells. Cytokine treatment of primary porcine endothelial cells results in an increase in the release of virus into the supernatant, but the observed increase in viral titer was not mirrored by an increase in infectivity toward human cells.

Human DNA polymerase-eta, an A-T mutator in somatic hypermutation of rearranged immunoglobulin genes, is a reverse transcriptase.

We have proposed previously that error-prone reverse transcription using pre-mRNA of rearranged immunoglobulin variable (IgV) regions as templates is involved in the antibody diversifying mechanism of somatic hypermutation (SHM). As patients deficient in DNA polymerase-eta exhibit an abnormal spectrum of SHM, we postulated that this recently discovered Y-family polymerase is a reverse transcriptase (RT). This possibility was tested using a product-enhanced RT (PERT) assay that uses a real time PCR step with a fluorescent probe to detect cDNA products of at least 27-37 nucleotides. Human pol-eta and two other Y-family enzymes that are dispensable for SHM, human pols-iota and -kappa, copied a heteropolymeric DNA-primed RNA template in vitro under conditions with substantial excesses of template. Repeated experiments gave highly reproducible results. The RT activity detected using one aliquot of human pol-eta was confirmed using a second sample from an independent source. Human DNA pols-beta and -mu, and T4 DNA polymerase repeatedly demonstrated no RT activity. Pol-eta was the most efficient RT of the Y-family enzymes assayed but was much less efficient than an HIV-RT standard in vitro. It is thus feasible that pol-eta acts as both a RNA- and a DNA-dependent DNA polymerase in SHM in vivo, and that Y-family RT activity participates in other mechanisms of physiological importance.

Characterisation of endogenous retrovirus in rodent cell lines used for production of biologicals

Rodent cells are used widely to manufacture recombinant proteins for pharmaceutical use in humans and animals. However, all rodent cell lines express endogenous retroviruses that require appropriate testing regimes for identification and characterisation. In this communication we report the results of transmission electron microscopy, reverse transcriptase assay and infectious virus assays for retrovirus in 185 manufacturer cell banks of mouse, rat or hamster origin. The results indicated considerable variability of retroviral expression levels by transmission electron microscopy and reverse transcriptase assay, but nevertheless characteristic features of each cell type were observed. Infectious retrovirus was detected in mouse myeloma and hybridoma cell lines, but not in cell lines of hamster or rat origin. There was no evidence of contamination of cell banks with exogenous retrovirus. The results of retroviral characterisation of the parental mouse cell lines NS0, NS-1 and Sp2/0Ag14 by the above assays were consistent with the results of the survey. Co-cultivation of the above parental mouse cell lines with mouse and human cell lines suggested that the ability to infect human cells was related to threshold susceptibility of cell types and the levels of expression of infectious xenotropic retrovirus by mouse cells.

Monitoring of human immunodeficiency virus infection in resource-constrained countries.

The reference standards used to monitor human immunodeficiency virus (HIV) infection are flow cytometric analysis of T lymphocyte subsets to provide the CD4+ T cell count and molecular assays to quantify plasma HIV load. Few laboratories in resource-constrained countries can afford to perform these tests. A number of lower-cost assays requiring less expensive equipment may be well-suited to such countries. These include manual CD4 cell assays (Dynal, Coulter, BioRad) and ultrasensitive reverse transcriptase (Cavidi) and p24 (Perkin Elmer Life Sciences) assays to monitor virus load. Quality control and access to quality assurance programs are essential. The total lymphocyte count, although readily available and inexpensive, generally does not correlate as closely with CD4+ T cell counts. Other surrogate markers, such as beta2-microglobulin, are not suitable for routine monitoring of HIV infection. This review discusses the above assays and their role in addition to clinical monitoring in resource-constrained countries.

Detection of porcine endogenous retrovirus in cultures of freshly isolated porcine bone marrow cells.

Pigs are under consideration as possible sources of organs for xenotransplantation in humans. The induction of hematopoietic microchimerism through xenotransplantation of source animal hematopoietic cells has been suggested as a means to induce tolerance in potential recipients. Because all porcine cells contain genetic information for porcine endogenous retrovirus (PERV), coculture techniques, reverse transcriptase (RT) and reverse transcriptase-polymerase chain reaction assays were used to determine whether infectious PERV is released from fresh porcine bone marrow cells cultured in the presence or absence of porcine cytokines. Human embryonic kidney cell line, HEK-293 cells cocultured with porcine bone marrow cells were positive for PERV RNA but never became positive for viral RT activity, suggesting the PERV infection was not productive. In contrast, high levels of RT activity was detected in porcine ST-IOWA cells after coculture, demonstrating that these cells became productively infected. PERV was released from cultured porcine bone marrow cells without stimulation, and combinations of the porcine hematopoietic cytokines, interleukin-3, granulocyte macrophage-colony stimulating factor and stem cell factor had no additional effect on the infectivity or in vitro tropism of released PERV virions.

Detection of telomerase status by semiquantitative and in situ assays, and by real-time reverse transcription-polymerase chain reaction (telomerase reverse transcriptase) assay in bladder carcinomas.

To investigate whether telomerase activity could be used as a diagnostic and/or prognostic marker of bladder carcinoma, by assessing telomerase activity using semiquantitative and in situ methods, and quantifying telomerase reverse transcriptase (hTERT) mRNA expression in a series of bladder carcinomas. Telomerase activity was detected by the telomeric repeat amplification protocol (TRAP) assay using a telomerase polymerase chain reaction (PCR) enzyme-linked immunosorbent assay (ELISA) kit on a series of 29 bladder carcinomas and on three normal bladder samples. hTERT mRNA levels were quantified using real-time quantitative reverse transcription (RT)-PCR. For the in situ detection of telomerase activity, the same telomerase PCR ELISA kit was used and applied to frozen-tissue sections only for discordant cases between telomerase activity and hTERT mRNA status. Telomerase activity was positive in 15 of the 29 bladder carcinomas (52%) and negative for the three normal bladder samples. hTERT was detected and quantified in all tumour samples, with major differences in hTERT values. None of the three normal bladder samples had quantifiable hTERT mRNA, giving complete sensitivity and specificity for the method in diagnosing bladder carcinoma. Comparing the results of RT-PCR and TRAP assay showed a significant association between the enzyme activity and levels of hTERT mRNA expression, with only five discordant cases, most of them being TRAP-negative and hTERT-positive. Among these cases the in situ results of telomerase activity were concordant with hTERT mRNA levels by RT-PCR and not with TRAP assay results, as nuclear fluorescence of malignant epithelial cells. The semiquantitative evaluation of positive cell numbers showed a heterogeneity of telomerase activity within the tumour tissue. There was a significant correlation between RT-PCR and histopathological variables (grade and stage), and a significant correlation between TRAP assay results and histopathological grade. Detecting hTERT mRNA by RT-PCR seems to be a promising method, with a much better sensitivity than the TRAP assay in diagnosing bladder carcinomas, and a better correlation with histopathological variables. False-negative cases on the TRAP assay are explained by the heterogeneity of telomerase activity within tumour cells. Thus evaluating hTERT gene expression levels could be used as a marker of malignant progression, useful in the early diagnosis and follow-up of bladder carcinomas.

Problems associated with product enhancement reverse transcriptase assay using bacteriophage MS2 RNA as a template

In order to identify the reverse transcriptase activity in sera and conditioned media from peripheral blood mononuclear cells (PBMCs) of large granular lymphocyte leukemia patients product enhanced reverse transcriptase activity (PERT) assays were performed using bacteriophage MS2 RNA as a template. All samples obtained from conditioned media of virus-infected cell lines as well as PBMCs of lymphocytic leukemia patients and normal healthy individuals tested positive with this assay. Therefore the validity of the assay was questioned. Careful evaluation of the assay revealed that some of the essential reagents used, such as Taq DNA polymerase and RNase inhibitor contained indigenous amplifiable DNA. DNase I treatment of Taq DNA polymerase before PCR reduced the product significantly. Moreover, no false positive results were observed when encephalomyocarditis virus RNA was used instead of MS2 RNA as the template. These results suggest a need for caution when using bacteriophage MS2 RNA as the template in PERT assays to confirm the presence of retroviral infection or for identification of novel retroviruses.