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PubMed1990

Detection of avian infectious bronchitis viral infection using in situ hybridization and recombinant DNA.

A recombinant DNA probe with specificity for the 3' end of genomic RNA from the Ark 99 strain of infectious bronchitis virus (IBV) was found to hybridize with extracted RNA of three strains with the Ark serotype, as well as the Mass41, Holl52, Gray, JMK, Conn, Fla and SE17 strains of IBV. Viral infection was detected in the cytoplasm of chicken embryo kidney cells inoculated with Mass41, Ark99, SE17 or two recent field isolates of IBV using in situ cytohybridization and a biotinylated probe. In vivo infections were detected in individual cells of tracheas and lungs 2,4, and 6 days after inoculation of chicks with Mass41 and Ark99. In situ hybridization of Ark99 infected tissue sections using 32P-dATP labelled probe indicated that more viral replication was present in the trachea on day 4 than either days 2 or 6; whereas more viral RNA was found in the lungs on day 6 than days 2 or 4 after inoculation.

Animals↗
PubMed2005

Detection of dengue viruses in field-caught Aedes aegypti (Diptera: Culicidae) in Maracay, Aragua state, Venezuela by type-specific polymerase chain reaction.

Virological surveillance of dengue viruses in Aedes aegypti populations constitutes a powerful tool for early prediction of dengue outbreaks. We have standardized a protocol for viral RNA extraction from individual and pools of mosquitoes that permits a sensitive detection of dengue virus without RNA degradation or PCR inhibition when we apply a semi-nested RT-PCR. The limit of detection for each dengue serotype was 0.1 PFU. In a prospective field study conducted from November 2000 to December 2001, adult female A. aegypti mosquitoes from several municipalities with high dengue transmission in Maracay, Aragua State, Venezuela were collected and screened for dengue viruses using RT-PCR. We analyzed a total of 296 A. aegypti pools (1,632 mosquitoes); of these, 154 pools (469 mosquitoes) were collected from houses with persons with clinical diagnosis of dengue (dengue houses), and 142 pools (1,163 mosquitoes) from adjacent residences (neighbour houses). From the dengue houses, eight mosquito pools (5.2%) were positive for DENV-1 (0.7%), DENV-3 (3.2%) and DENV-4 (1.3%) viruses. From the neighbour houses, 18 mosquito pools (12.7%) were positive for DENV-3 (12%) and DENV-4 (0.7%) viruses. From these 26 RT-PCR positive mosquito pools (containing 1-25 mosquitoes each), 22 pools (84.6%) were positive for DENV-3. The most prevalent serotype in the 2001 dengue outbreak was also DENV-3. The minimum infection rate in both A. aegypti collections, from dengue houses and neighbour houses was 17 and 15 per 1,000, respectively. The relevance of these results for dengue surveillance is discussed.

Aedes↗
PubMed1976

Dual labeling in standard DNA-RNA hybridization studies using 125 I-labeled nuclear RNA and 3H-labeled DNA.

Standard DNA-RNA hybridization studies, using nucleic acids isolated from mammalian tissues, are frequently hindered by relatively low levels of radioactivity in pulse-labeled RNA and in an inability to reliably estimate the amount of DNA present in the hybrid. In the method described here nuclear RNA is labeled in vitro with 125I to 400 000- 800 000 cpm/mug and DNA is obtained from a rat glial tumor line grown in culture and labeled to specific activities of 42 000-79 000 cpm/mug. DNA-RNA hybridization is conducted in an all solution system at RNA:DNA ratios of 3.5:1 to 18:1. Assay background is controlled by pretreatment of the hybrid and free RNA at the conclusion of the annealing study with RNase, then isolation of the hybrid together with a small fraction of free RNA oligonucleotides on hydroxyapatite. The partially purified hybrids are then trapped on Millipore filters. Assay background id 0.004% of total counts present in the annealing reaction. Comparison of the annealing reactions of pulse-labeled liver nuclear RNA and in vitro 125I-labeled nuclear RNA in saturation, kinetic, and competitive hybridization studies shows them to be essentially the same. Nuclear RNA labeled by either tritium or iodine shows a 10-20-fold greater concentration of the annealing sequences over that found in the microsomal RNA. Minor differences are noted between the nuclear RNAs in the initial rates of reaction and in the magnitude of the decrease in percent hybridization at low levels of unlabeled competitor RNA. This may be due to preferential labeling in pulse-labeled RNA of molecules which are present in lower concentrations or are transcribed from more frequently repeated DNA sequences than the average population of annealing RNA molecules. The technique has application in systems where the amount of tissue for RNA extraction is small or where the system does not permit the obtaining of pulse-labeled RNA, as in experimental rodent skin carcinogenesis or in dealing with RNA from the tissues of large mammals or humans.

Animals↗
PubMed1982

Identification of storage-protein messenger RNA of the fleshfly Sarcophaga peregrina.

Storage-protein mRNA was found to be abundant in poly(A)-containing RNA extracted from the fat-body of third-instar larvae of Sarcophaga peregrina (fleshfly). This RNA sedimented at the position of 19S on sucrose-density-gradient centrifugation and the product of its translation in vitro was 75K protein (protein of mol.wt. 75 000), which was precipitated specifically with antibody against storage protein. This product was suggested to contain a signal sequence that is missing in mature storage protein. The poly(A)-containing RNA was also found to contain much of another mRNA coding for 25K protein (protein of mol.wt. 25 000), but the function of this protein is unknown.

Animals↗
PubMed1996

Changes in integrin and E-cadherin expression in neoplastic versus normal thyroid tissue.

UNLABELLED: BACK: The functional organization of polarized epithelia depends mostly on adhesion molecules belonging to the integrin and cadherin families. These molecules either recognize basement membrane components, such as laminins, or form intercellular junctions via homotypic interactions. Such tissue organization is often disrupted upon neoplastic transformation, and the resulting loss of functional polarization and cell cohesion might be a prerequisite for the invasive and metastatic behavior of carcinomas. PURPOSE: We studied modifications on thyroid adhesive mechanisms at various stages of neoplastic progression in terms of adhesion molecule expression, topography, and functional regulation by tyrosine kinases. Starting from this working hypothesis, we sought to identify one or more biological markers that would be suggestive of malignant transformation and poorer prognosis and that could be developed as a reliable indicator(s) in early diagnostic steps. METHODS: The study was carried out on both surgical samples and the corresponding fine-needle aspiration biopsy smears (numbers of specimens collected: 19 adenomas, seven follicular carcinomas, 13 papilary carcinomas, and 39 normal tissues). Immunohistochemistry of tissue sections and smears and immuno-precipitation and western blot analysis of protein extracts were done with a battery of monoclonal and polyclonal antibodies. Northern blotting was performed on RNA extracts from frozen tissue samples and use of an integrin subunit beta4 complementary DNA probe. RESULTS: Our findings can be summarized as follows: 1) In normal thyroid cells, the cooperative role of integrin alpha6beta4 and laminin 5/kalinin in hemidesmosome-mediated adhesion adhesion is missing, and recognition of the basal lamina occurs via integrin alpha3beta1 and laminin 1 and/or 2 (this pattern being maintained in adenomas but altered in carcinomas regardless of their histotype or differentiation grade); 2) only in carcinomas with clinical and/or histologic aggressiveness do neoexpression of integrin subunit beta4 and loss of laminin 2/merosin occur, indicating de novo assembly of integrin alpha6beta4; 3) pericellular redistribution and cytoskeletal disconnection of the E-cadherin-catenin complex occur; and 4) basal E-cadherin tyrosine phosphorylation decreases in carcinomas as compared with that in normal and adenomatous tissue. CONCLUSION: The malignant progression of thyroid tumors involves marked rearrangement of cell-basement membrane and cell-cell adhesion molecules and changes in their cytoskeleton linkage. These rearrangements are also easily and reproducibly detected on fine-needle aspiration biopsy smears. IMPLICATIONS: Immunodetection of adhesion molecules in sections and/or fine-needle smears may complement the toolbox of thyroid surgical pathologists; it may expand the possibilities of achieving a correct early diagnosis of thyroid tumors and of gaining some prognostic information on thyroid tumors.

Adenocarcinoma, Follicular↗
PubMed2002

Inhibition of NF-kappa B binding correlates with increased nuclear glucocorticoid receptor levels in acute alcohol-treated human monocytes.

BACKGROUND: Acute alcohol treatment blocks inflammatory cytokines via inhibition of NF-kappaB in monocytes in the presence of ongoing IkappaBalpha degradation, suggesting regulation of NF-kappaB activation downstream of IkappaBalpha degradation. DNA binding of NF-kappaB has been suggested to be regulated by other nuclear regulatory factors, including the glucocorticoid receptor (GR). Here, we show for the first time that acute alcohol (25 mM) exposure modulates GR activation in monocytes. METHODS: Human peripheral blood monocytes were treated with lipopolysaccharide (LPS) in the presence or absence of alcohol (25 mM) for 1 hour. Nuclear GR levels were estimated by Western blotting and NFkappaB activation was studied in the same extracts by gel shift analysis (EMSA). Cells were stimulated with 1 microM of Dex to be used as positive control for GR activation. GR/GRE binding was also determined in nuclear extracts by EMSA. IkappaBalpha mRNA known to be induced by GR/GRE activation was studied in total RNA extracts by the SuperArray method (SuperArray Inc., Bethesda, MD). RESULTS: LPS is a potent inducer of GR nuclear translocation and GR binding to the glucocorticoid response element (GRE). Acute alcohol treatment both induced (p < 0.05) and augmented (p < 0.05) LPS-stimulated GR nuclear levels. However, alcohol inhibits LPS-induced (nonligand bound) GR/GRE binding activity in monocytes. This inhibition of GR transactivation by alcohol was further confirmed by decreased expression (40%) of a target gene, IkappaBalpha. Thus, alcohol treatment increases nonligand-bound nuclear GR, but inhibits its transactivation function. Ligand-induced GR/GRE binding was decreased in alcohol-treated monocytes. Inhibition of ligand-induced GR/GRE binding by alcohol exposure is likely due to cytoplasmic retention of the GR. CONCLUSIONS: Our results show that acute alcohol exposure inhibits GR in monocytes by differently affecting ligand- and nonligand-induced GR nuclear translocation. These data also suggest that acute alcohol regulates GR activation in monocytes concomitant to inhibition of NF-kappaB activation.

Adolescent↗
PubMed2006

Gene expression profiling of whole-blood samples from women exposed to hormone replacement therapy.

The American Women's Health Initiative study published in July 2002 caused considerable concern among hormone replacement therapy (HRT) users and prescribers in many countries. This study is an exploratory research comparing the genome-wide expression profile in whole-blood samples according to HRT use. Within the Norwegian Women and Cancer study, 100 postmenopausal women (50 HRT users and 50 non-HRT users) born between 1943 and 1949 with normal to high body mass index and no other medication use were selected. After total RNA extraction, amplification, and labeling, the samples were hybridized together with a common reference (Universal human reference RNA, Stratagen) to Agilent Human 1A oligoarrays (G4110b, Agilent Technologies) containing 20,173 unique genes. Differentially expressed genes were used to build a classifier using the nearest shrunken centroid method (PAM). Then, we tested the significant changes in single genes by different methods like t test, Significance Analysis of Microarrays, and Bayesian ANOVA analysis. Results did not reveal any distinct gene list which predicted accurately HRT exposure (error rate, 0.40). Classifier performance slightly improved (error rate, 0.26) including only women who were using continuous combined HRT treatment. According to the small amplitude of expression alterations observed in whole blood, more quantitative technique and larger sample sizes will be needed to be able to investigate whether significant single genes are differentially expressed in HRT versus non-HRT users. Taken cautiously, significant enrichments in biological process of genes with small changes after HRT use were observed (e.g., receptor and transporter activities, immune response, frizzled signaling pathway, actin filament organization, and glycogen metabolism).

Adult↗
PubMed2006

Application of in situ ductal perfusion to facilitate isolation of high-quality RNA from mouse pancreas.

A technique to isolate high-quality intact RNA from murine pancreas is described. This technique involves in situ ductal perfusion of the pancreas with an RNase inhibitor prior to removal of the organ for RNA extraction. In this way, the pancreatic RNases are inhibited in situ allowing good yields of intact RNA, suitable for studies on pancreatic gene transcription by real-time PCR or microarray analysis, to be obtained in a reliable way.

Animals↗
PubMed2000

Development of a semi-quantitative assay to detect full-length CYP2C19 RNA.

Cytochrome P450 2C19 (CYP2C19) is the enzyme responsible for the metabolism of a number of drugs, including anticonvulsants and antidepressants. We have developed a semi-quantitative competitive RT-PCR assay to estimate the degree of expression of the full-length CYP2C19 message. This assay used a known quantity of internally deleted CYP2C19 RNA to quantitate the RT-PCR products of the CYP2C19 transcript in the RNA extracted from tissues. We determined that this method is sensitive and reproducible in assaying for CYP2C19 RNA in human livers. The lowest detectable amount of competitor RNA was 0.166 fg or 270 copies of CYP2C19 competitor RNA. Using human liver samples containing 3-23 x 10(5) copies of CYP2C19 RNA, we found the assay to be reproducible with a coefficient of intra- and interday variation of 11% and 20%, respectively. Using this assay, we measured full-length CYP2C19 RNA in 10 human livers. We found the CYP2C19 transcripts range from 0.1-23 x 10(5) copies/microgram liver total RNA. The analysis of CYP2C19 transcripts for liver of *1 and *2 genotypes, based on restriction enzyme digest analysis of RT-PCR products, suggests that only normal (*1), not the variant (*2) copy of full-length CYP2C19 RNA, was detectable in these livers. We report for the first time the quantification of full-length CYP2C19 RNA in livers.

Alleles↗
PubMed2001

Clinical and molecular responses in high-grade intraepithelial neoplasia treated with topical imiquimod 5%.

OBJECTIVE: To assess the clinical and molecular response of patients with recurrent high-grade vulvar, vaginal, or cervical intraepithelial neoplasia treated with topical 1-2(2-methylpropyl)-1H-imidazo [4,5-c] quinolin-4-amine (imiquimod) cream 5%, an immune response modifier with known efficacy in the treatment of external genital warts. METHODS: This is the first case series in the peer-reviewed literature reporting the use of imiquimod in high-grade intraepithelial neoplasia of the lower genital tract. Eight patients with high-grade intraepithelial neoplasia were treated with imiquimod in the gynecological oncology clinic and the HIV gynecology clinic at The University of Texas Medical Branch at Galveston. Frozen biopsies were available for RNA extraction on four patients before and after therapy. Using semiquantitative reverse transcription-PCR, we measured RNA levels of IFNs alpha and gamma, 2',5'-oligoadenylate synthetase, as well as CD4 and CD8 lymphocyte markers. RESULTS: Of the patients treated, four had complete responses, two had partial responses, one progressed, and one did not tolerate the therapy. Of the four complete responders, two remained disease-free (mean follow-up, 33 months). 2',5'-Oligoadenylate synthetase RNA expression showed an increased trend after therapy. CONCLUSIONS: These results obtained in this small and heterogeneous group merit further study in the use of topical 5% imiquimod use in the treatment of intraepithelial neoplasia. An important mechanism of action of imiquimod may involve 2',5'-oligoadenylate synthetase antiviral activity.

2',5'-Oligoadenylate Synthetase↗
PubMed2002

Presence of hepatitis C virus RNA in the salivary glands of patients with Sjögren's syndrome and hepatitis C virus infection.

OBJECTIVE: To determine whether hepatitis C virus (HCV) RNA could be detected in the salivary glands of patients with both a diagnosis of Sjögren's syndrome (SS) and HCV infection. METHODS: Five patients with primary SS (European criteria) and chronically infected by HCV and 3 controls (one with primary SS without HCV infection, another with HCV infection without sicca syndrome, and a third without SS and HCV infection) were tested for the presence of HCV-RNA (using reverse transcriptase-polymerase chain reaction) in their saliva, serum, and salivary glands. RESULTS: In the patient group, HCV-RNA was detected in the serum and saliva of all cases and RNA extracted from salivary gland specimens tested positive in 3 cases. In the control group, HCV-RNA was not detected in the serum, saliva, or salivary glands from subjects without HCV infection. Only the control subject with HCV but without sicca syndrome tested positive for the presence of HCV-RNA in the serum, saliva, and salivary gland tissue. CONCLUSION: Our results showed that HCV may propagate and reside within salivary gland tissue, leading to HCV associated sialadenitis or Sjögren's-like syndrome in some cases, a phenomenon that does not seem specific. However, a direct role for HCV in the physiopathology of certain cases of primary SS is suggested.

Adult↗
PubMed1988

IDDM in BB rats. Enhanced MHC class I heavy-chain gene expression in pancreatic islets.

Modulation in major histocompatibility complex (MHC) gene expression correlates with the inflammatory reactions that occur during graft rejection and autoimmune disease. We analyzed the expression of class I and II MHC genes in the pancreatic islets of prediabetic and newly diabetic BB rats by immunohistochemistry of tissue sections and Northern blotting of RNA extracted from isolated islets. We show that enhanced levels of MHC class I heavy-chain RNA are present in pancreatic islets before overt inflammation and the onset of insulin-dependent diabetes mellitus (IDDM) in the spontaneously diabetic BB rat. Immunohistochemical analysis revealed enhanced class I antigen expression throughout the pancreatic islets of newly diabetic animals but no induction of class II antigen on endocrine cells within the islet. Varying degrees of inflammatory infiltrate were observed in the sections exhibiting enhanced class I antigen expression or in nearby serial sections. Southern blot analysis revealed no restriction-fragment-length polymorphism or amplification of the endogenous class I heavy-chain genes compared with those of seroidentical disease-resistant Wistar-Furth rats. I-A alpha and I-E alpha hybridizing RNA appeared de novo before overt diabetes, although concomitantly with T-lymphocyte-receptor beta-chain and interferon-gamma gene hybridizing RNA and after MHC class I heavy-chain RNA enhancement was observed. These data indicate the possibility that enhanced class I heavy-chain gene expression plays a role in the progression of IDDM.

Animals↗
PubMed2006

The dual role of chorion peroxidase in Bactrocera oleae chorion assembly.

In the present study, we reveal for the first time that the Bactrocera oleae chorion peroxidase (bPxd) participates essentially in B. oleae chorion formation and clearly represents the homologous member of Drosophila melanogaster chorion peroxidase (Pxd). Comparative sequence analysis disclosed that the bPxd cDNA semi-central region, which encodes for the putative catalytic domain of the enzyme, exhibits great homology (98%) with its Pxd counterpart. Thus, it is very likely that bPxd is highly responsible for the chorion hardening process, through protein cross-linking mediated by the formation of di- and tri-tyrosine bonds. Distinct molecular weight bPxd RNA transcripts were detected in Northern blotting analysis of total RNA extracts of adult flies (2.9 and 1.7 kb) and ovaries (2.2 kb). The ovarian-specific bPxd RNA transcript is selectively expressed in the follicle cell layer during the late stages of oogenesis 12-14, as revealed by in situ hybridization. Moreover, reverse transcription reactions confirmed the stage-specific developmental regulation of the bPxd gene, which is maximally expressed during stage 13. Western blotting with the rabbit anti-rAePO polyclonal antibody revealed three immunoreactive bands of 76, 66 and 54 kDa in crude protein extracts from adult flies, while in larva and purified chorion preparations, a unique 54 kDa band was clearly detected. Immunolocalization experiments revealed that bPxd peroxidase constitutes an essential structural chorionic component, being abundantly localized in all the successive chorionic layers and vitelline membrane as well.

Animals↗
PubMed1999

Detection of tumor messenger RNA in the serum of patients with malignant melanoma.

Serum RNases are known to be elevated in patients with cancer. Consequently, it is not clear whether human mRNA with sufficient integrity as to permit reverse transcription-PCR (RT-PCR) amplification is detectable in serum. We examined serum from six patients with malignant melanoma for human tyrosinase mRNA using RT-PCR. Serum from 20 normal volunteers served as controls. Tyrosinase mRNA could be demonstrated in serum from four of the six melanoma patients with detection by gel electrophoresis and confirmation by blotting amplified product to a tyrosinase-specific probe. The serum remained tyrosinase mRNA positive, even if passed through a 0.45 microm filter prior to RNA extraction, indicating that the mRNA was extracellular at the time of extraction. Tyrosinase mRNA could not be detected in any control serum (0 of 20 individuals). The presence and integrity of amplifiable RNA was confirmed in all serum specimens (patients and controls) by RT-PCR amplification of c-abl mRNA. Amplifiable RNA could be demonstrated regardless of whether serum was freshly drawn or stored frozen for several years. We conclude that human mRNA can be extracted and amplified from serum. The ability to amplify tumor mRNA from serum may have important utility in cancer diagnostics and monitoring.

Blotting, Southern↗
PubMed1989

RNA metabolism in nuclei: adenovirus and heat shock alter intranuclear RNA compartmentalization.

Nuclear RNA compartmentalization has received little attention as an area where regulation of gene expression could occur. RNA transcription and processing occur in association with the nuclear matrix, a salt-insoluble proteinaceous network that fills the nuclear space and is contiguous with the peripheral lamina and pore complexes. Described here are experiments that determine the fate of nuclear RNA after it has completed these matrix-associated maturation steps. Continuous label experiments indicate that after nuclear RNA is processed it changes its state of attachment in the nucleus so that it is now removed from the nucleus in the high salt extraction step of matrix isolation. It is this salt-extractable RNA that will be transported to the cytoplasm. Late in adenovirus infection and following heat shock, when transport of cellular RNA is decreased, cellular transcripts do not make the transition from the matrix-associated to the salt-extractable nuclear pool. The implication of these data for the regulation of gene expression is discussed.

Adenoviruses, Human↗
PubMed1992

RNA isolation from cartilage using density gradient centrifugation in cesium trifluoroacetate: an RNA preparation technique effective in the presence of high proteoglycan content.

An efficient method for the isolation of RNA from cartilage is described. The difficulties in obtaining RNA from cartilage, a tissue of low cell density and high proteoglycan content, were overcome by making several modifications to the guanidine thiocyanate/cesium chloride method of RNA extraction. Cartilage tissue is frozen, crushed, and homogenized in a 4 M guanidine thiocyanate lysis buffer. The RNA is then pelleted by ultracentrifugation through a cesium trifluoroacetate density gradient. The use of cesium trifluoroacetate, rather than cesium chloride, for density gradient centrifugation improves both the yield and purity of total RNA isolated from cartilage. The ultracentrifugation has been adapted to the Beckman TL100 tabletop centrifuge and is complete in 3 h. This fast, simple method produces high quality RNA, suitable for use in RNase protection assays, polymerase chain reaction analysis, and Northern analysis. This purification procedure may be applicable to other sources, from which RNA isolation is complicated by the presence of abundant cell wall or matrix components.

Animals↗
PubMed1983

The formation of arenaviruses that are genetically diploid.

Analyses of RNA extracted from preparations of arenaviruses indicate that the relative molar proportions of the genomic L and S RNA species are frequently far from equal. In order to investigate the genetic significance of this observation temperature-sensitive (ts) mutants of two lymphocytic choriomeningitis (LCM) virus strains (ARM and WE) have been recovered and categorized into recombination groups (Groups I and II). Fingerprint analyses of wild-type progeny viruses obtained from dual infections with ARM Group II and WE Group I ts viruses indicate that they have L/S RNA genotypes of WE/ARM. It is concluded that the ARM Group II ts viruses have mutations in their L RNA species and that the WE Group I ts viruses have mutations in their S RNA species. Correspondingly it is deduced that the ARM Group I ts viruses have S RNA mutations and the WE Group II ts viruses mutations in their L RNA species. Cells coinfected with certain WE Group I mutants, or an ARM Group I and certain WE Group I ts mutants, have also yielded wild-type viruses. Fingerprint analyses have shown that the wild-type viruses obtained from the latter crosses are diploid with respect to their S RNA species. On subsequent passage these wild-type viruses shed high proportions of ts mutants. We interpret the data to indicate that the original Group I ts mutants that yielded the diploid viruses have mutations in different S RNA gene products so that the progeny produce plaques at the nonpermissive temperature by gene product complementation. No wild-type recombinant viruses have been obtained from crosses involving Pichinde and LCM ts mutants.

Arenaviruses, New World↗
PubMed1994

Detection of hepatitis C virus RNA by reverse-transcriptase and polymerase chain reaction: clinical applications of quantitative analysis.

Polymerase chain reaction (PCR) applications to diagnostics allowed the detection of viral nucleic acids in expected and unexpected clinical circumstances. This has raised some scepticism on the practical usefulness of PCR in the routine laboratory and emphasized the need for quantitative analysis. We addressed this question detecting HCV-RNA by a single step RT-PCR in serum samples from 50 patients with chronic non-A, non-B hepatitis included in clinical trials for recombinant alpha-interferon therapy. We obtained at least 5 serum specimens from each patient (baseline, during and after therapy samples) during an 18-month mean follow-up (range 12-45 months). RT-PCR was performed on total RNA extracted from 100 microl serum aliquots using primers for the highly conserved 5'NCR of HCV-RNA and 35 amplification cycles. PCR products were analyzed by agarose gel electrophoresis and Southern blot hybridization against a P(32)-oligonucleotide probe. Sensitivity was evaluated in separate experiments on tenfold dilutions of a reference Chimp serum containing 10(6) CID(50)/ml. The overall sensitivity of our assay ranged between 10(2) and 10(3) genome Eq./ml. We establish a semiquantitative score system to evaluate viremia levels: 2 = HCV-RNA levels >10(4) genome Eq./ml; 1 = levels between 10(3) and 10(4) g.Eq./ml; 0 = levels less than 10(2) g.Eq/ml. The reproducibility of this scoring system was confirmed testing repeatedly in duplicate end-point dilutions of positive serum samples. A statistically significant relation was observed between elevated HCV-RNA and ALT values (83.8%, chi-square 159.963 P < 0.001). Response to IFN therapy was significantly better in patients with lower baseline HCV-RNA levels. A time relation was found between flare-ups of serum HCV-RNA levels and ALT elevations higher than 3 x normal values viremia elevations coincident or occurring about 1 month earlier than ALT elevations. This finding suggests that immuno pathogenesis might be responsible of HCV-induced liver damage as in chronic hepatitis B where identical relations were observed between viremia and ALT serum levels. In conclusion, single-step HCV-RNA RT-PCR can be a specific and reproducible semiquantitative assay and provides useful diagnostic informations for therapeutic decision making and monitoring of HCV-infected patients.

Journal Article↗