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PubMed2001

Na(+)/I(-) symporter and Pendred syndrome gene and protein expressions in human extra-thyroidal tissues.

OBJECTIVE: The expression of two recently identified iodide transporters, namely the sodium/iodide symporter (NIS) and pendrin, the product of the gene responsible for the Pendred syndrome (PDS), was studied in a series of various extra-thyroidal human tissues, and especially in those known to concentrate iodide. METHODS: To this end, we used real-time kinetic quantitative PCR to detect NIS and PDS transcripts and immunohistochemistry for the analysis of their protein products. RESULTS: NIS gene and protein expression was detected in most tissues known to concentrate iodine, and particularly in salivary glands and stomach. In contrast, PDS gene expression was restricted to a few tissues, such as kidney and Sertoli cells. Interestingly, in kidney, pendrin immunostaining was detected at the apical pole of epithelial cells of the thick ascending limb of the Henle's loop and of the distal convoluted tubule. CONCLUSION: This study provides new insights on the localization and expression of two genes involved in iodide transport and emphasizes the interest of combining real-time quantitative PCR and immunohistochemistry for the comparison of gene and protein expression in tissues.

Biological Transport↗
PubMed2004

Characterization and prevalence of PITX2 microdeletions and mutations in Axenfeld-Rieger malformations.

PURPOSE: Mutations of the homeodomain protein PITX2 produce Axenfeld-Rieger (AR) malformations of the anterior chamber, an autosomal dominant disorder accompanied by a 50% risk of glaucoma. Twenty-nine mutations of PITX2 have been described, with a mutational prevalence estimated between 10% and 60% in AR. In the current study, the possible role of altered PITX2 gene dosage in the etiology of AR was investigated. Gross gene deletions and duplications should alter PITX2 activity analogously to hypomorphic and hypermorphic mutations, respectively. METHODS: Sixty-four patients with AR, iridogoniodysgenesis (IGD), iris hypoplasia (IH), or anterior segment dysgenesis (ASD) were screened for PITX2 mutations by sequencing. PITX2 gene dosage was concurrently examined in these patients by real-time quantitative PCR. Microsatellite markers were used to map 4q25 microdeletions at a contig scale, as well as for haplotype analysis in an extended AR kindred. An additional 27 patients with other assorted ocular phenotypes were evaluated by similar methods, amounting to a total of 91 cases analyzed. RESULTS: Three novel mutations of PITX2 (4.7%) were identified among 64 patients with AR, IGD, IH, or ASD. Deletions of PITX2 were as frequent as mutations in our sample. Chromosome 4q25 microdeletions were physically mapped relative to several microsatellite markers in each patient. Cosegregation of AR and a PITX2 deletion was demonstrated in an extended kindred. CONCLUSIONS: Point mutations and gross deletions of PITX2 appear to produce an equivalent haploinsufficiency phenotype. Quantitative PCR is an efficient means of detecting causative PITX2 deletions in patients with AR and may increase the detection rate at this locus.

Anterior Eye Segment↗
PubMed2003

Quantitative analysis of circulating mitochondrial DNA in plasma.

BACKGROUND: Recent studies have demonstrated the existence of circulating mitochondrial DNA in plasma and serum, but the concentrations and physical characteristics of circulating mitochondrial DNA are unknown. The aim of this study was to develop an assay to quantify mitochondrial DNA in the plasma of healthy individuals. METHODS: We adopted a real-time quantitative PCR approach and evaluated the specificity of the assay for detecting mitochondrial DNA with a cell line (rho(0)) devoid of mitochondria. The concentrations and physical characteristics of circulating mitochondrial DNA were investigated by experiments conducted in three modules. In module 1, we evaluated the concentrations of mitochondrial DNA in plasma aliquots derived from four blood-processing protocols. In module 2, we investigated the existence of both particle-associated and free forms of mitochondrial DNA in plasma by subjecting plasma to filtration and ultracentrifugation. In module 3, we used filters with different pore sizes to investigate the size characteristics of the particle-associated fraction of circulating mitochondrial DNA. RESULTS: The mitochondrial DNA-specific, real-time quantitative PCR had a dynamic range of five orders of magnitude and a sensitivity that enabled detection of one copy of mitochondrial DNA in plasma. In module 1, we found significant differences in the amounts of circulating mitochondrial DNA among plasma aliquots processed by different methods. Data from module 2 revealed that a significant fraction of mitochondrial DNA in plasma was filterable or pelletable by ultracentrifugation. Module 3 demonstrated that filters with different pore sizes removed mitochondrial DNA from plasma to different degrees. CONCLUSIONS: Both particle-associated and free mitochondrial DNA are present in plasma, and their respective concentrations are affected by the process used to harvest plasma from whole blood. These results may have implications in the design of future studies on circulating mitochondrial DNA measured in different disease conditions.

Blood Circulation↗
PubMed2006

[Bezafibrate up-regulate endothelial nitric oxide gene expressions via peroxisome proliferator-activated receptors alpha-dependent and independent pathways in cultured Bovine endothelial cells].

OBJECTIVE: To investigate the effects and related mechanisms of bezafibrate, a ligand of peroxisome proliferator-activated receptors alpha (PPARalpha), on endothelial nitric oxide synthase (eNOS). METHODS: Firstly, in cultured bovine aorta endothelial cells (BAEC), the effects of bezafibrate on eNOS mRNA and protein levels were investigated by Northern blot and Western blot. The half-life of eNOS mRNA and NO production determined from BAECs treated with bezafibrate were performed by real-time quantitative PCR and NO assay. Next, the effects of bezafibrate on signal pathways in BAEC, through inhibitors of PPARalpha, PI3 kinase and MAPK, were investigated by Western blot. Then luciferase reporter gene driven by human eNOS promoter were cloned and transfected endothelial cells to see the effects of bezafibrate on eNOS promoter-driven luciferase activity. RESULTS: In cultured BAEC, bezafibrate significantly upregulated eNOS expressions at protein and mRNA levels in a concentration-dependent fashion (50 - 200 micromol/L) (P < 0.05), and increased nitric oxide (NO) production, respectively[control (14.97 +/- 1.29) micromol/L, different concentration groups (25.12 +/- 1.25) micromol/L, (30.12 +/- 1.85) micromol/L, (33.47 +/- 1.22) micromol/L], and enhanced phosphorylation of eNOS-ser-1179 site (P < 0.05), but not eNOS-thr-497 site. Through real-time quantitative PCR, bezafibrate prolonged eNOS mRNA half-life from 3.1 hour to 6.1 hour were demonstrated. Further studies showed that bezafibrate treatments significantly enhanced dose-dependently luciferase activity (relative luciferase activity in 100 micromol/L and 200 micromol/L groups 4429.43 +/- 391.41 and 6187.5 +/- 307.53), compared with untreated luciferase reporter gene group (3361.81 +/- 316.85) (P < 0.05 and P < 0.01, respectively), and induced MAPK phosphorylation expression (P < 0.05 and P < 0.01, respectively). Then these effects of bezafibrate upregulated eNOS expressions could be blocked by PPARalpha antagonist, MAPK and PI3K inhibitor while not affected by PKC and MEK inhibitor (P < 0.01). CONCLUSIONS: Bezafibrate can upregulate eNOS expression, enhance eNOS-ser-1179 site phosphorylation, increase NO production and stabilize eNOS mRNA through PPARalpha dependent pathway and PPARalpha independent MAPK and PI3K pathways. This mechanism provides additional anti-atherosclerotic and anti-hypertension benefits of bezafibrate in addition of lipid-lowering effects.

Animals↗
PubMed1999

Reproducibility of detection of tyrosinase and MART-1 transcripts in the peripheral blood of melanoma patients: a quality control study using real-time quantitative RT-PCR.

In recent years, large discrepancies were described in the success rate of the tyrosinase reverse transcription polymerase chain reaction (RT-PCR) for detecting melanoma cells in the peripheral blood of melanoma patients. We present a quality control study in which we analysed the reproducibility of detection of tyrosinase and MART-1 transcripts in 106 blood samples from 68 melanoma patients (mainly stages III and IV). With this study, we aimed to improve insight in the reproducibility of a RT-PCR for the detection of (minimal) amounts of circulating melanoma cells. We performed two reverse transcriptions on each mRNA sample and performed tyrosinase and MART-1 nested PCRs in duplicate per cDNA sample. Thus, four tyrosinase and four MART-1 measurements were performed per blood sample. In our study, the majority of blood samples was negative for tyrosinase (80%) or MART-1 (66%). Only four samples were positive in all four determinations for tyrosinase and seven for MART-1. Variable results (1-3 times positive results) were obtained for tyrosinase and MART-1 in 16% and 27% respectively. MART-1 PCR had a better performance than tyrosinase PCR. Sensitivity increased when both markers were used. We reasoned that the low number of melanoma marker PCR-positive blood samples can be explained by differences in mRNA quality. By using real-time quantitative PCR, we found that this was not the case: amplification of porphobilinogen deaminase (PBGD), a low copy household gene, was not different in blood samples in which a melanoma marker was not detected from groups in which this marker was detected more or less consistently (1-4 times). When applying real-time quantitative PCR for tyrosinase and MART-1, we found that a low amount of SK-MEL-28 cell equivalents was present in the blood of melanoma patients, with a higher number of equivalents in the group with a consistently positive result. We conclude that low reproducibility of a repeated assay for the detection of circulating melanoma cells is not caused by differences in mRNA quality between the samples, but due to low numbers of amplifiable target mRNA molecules in the mRNA sample. Use of more than one marker and repetition of the assay will increase the probability of finding positive PCR results.

Adolescent↗
PubMed1998

[Ischemia and reperfusion reduce the mRNA expression of endogenous basic fibroblast growth factor (bFGF) in rat skeletal muscles].

OBJECTIVE: To explore the expression of bFGF gene in normal and wounded rat skeletal muscles. METHODS: In situ hybridization and quantitative PCR methods were used to evaluate the location of basic fibroblast growth factor (bFGF) mRNA and the amount of bFGF mRNA expression in normal, ischemic or ischemic and reperfused rat skeletal muscles. RESULTS: According to the intensity of the stain in situ hybridization, four main classes of fibers could be identified: strong, moderate, weak and negative stained fibers. bFGF mRNA was found in the cytoplasm in both normal and wounded muscles; however, the signal intensity was much stronger in normal muscles. In the normal muscles, bFGF mRNA was distributed irregularly and sparsely, and 82% of fibers had positive bFGF mRNA staining, and only 52% and 22% of fibers in ischemic or ischemic and reperfused muscles were positive for bFGF mRNA staining in situ hybridization. The results in quantitative PCR study supported the concept of the reduced bFGF mRNA expression in the wounded muscles. CONCLUSIONS: The loss of stored bFGF in wounded skeletal muscles as we showed previously is also caused by its increased damage and reduced secretion.

Animals↗
PubMed2004

Comparison of PCR-southern hybridization and quantitative real-time PCR for the detection of JC and BK viral nucleotide sequences in urine and cerebrospinal fluid.

The presence of the human polyomaviruses JCV and BKV in immunocompromised patients can lead to lethal diseases and conditions including progressive multifocal leukoencephalopathy (PML), interstitial nephritis, hemorrhagic cystitis, and kidney allograft rejection. Typically, detection of JCV and BKV in clinical samples has employed standard PCR amplification for viral nucleotide sequences, with subsequent confirmation for viral genome specificity of PCR products by southern blot hybridization. Here, we directly tested a validated PCR-southern protocol with a TaqMan real-time PCR protocol (Applied Biosystems) to assay clinical samples of urine and cerebrospinal fluid. We found equal specificity and sensitivity with both methods. However, real-time allowed for absolute viral-genome quantitation without the use of radionucleotides and was performed more rapidly, in as little as 24 h. Such advantages are important to consider in the effort to establish international standardization of controls for the detection of JCV and BKV, which would aid in screening confidence and the reliable assessment of anti-viral therapies.

BK Virus↗
PubMed2001

Modulation of intestinal gene expression by dietary zinc status: effectiveness of cDNA arrays for expression profiling of a single nutrient deficiency.

Mammalian nutritional status affects the homeostatic balance of multiple physiological processes and their associated gene expression. Although DNA array analysis can monitor large numbers of genes, there are no reports of expression profiling of a micronutrient deficiency in an intact animal system. In this report, we have tested the feasibility of using cDNA arrays to compare the global changes in expression of genes of known function that occur in the early stages of rodent zinc deficiency. The gene-modulating effects of this deficiency were demonstrated by real-time quantitative PCR measurements of altered mRNA levels for metallothionein 1, zinc transporter 2, and uroguanylin, all of which have been previously documented as zinc-regulated genes. As a result of the low level of inherent noise within this model system and application of a recently reported statistical tool for statistical analysis of microarrays [Tusher, V.G., Tibshirani, R. & Chu, G. (2001) Proc. Natl. Acad. Sci. USA 98, 5116-5121], we demonstrate the ability to reproducibly identify the modest changes in mRNA abundance produced by this single micronutrient deficiency. Among the genes identified by this array profile are intestinal genes that influence signaling pathways, growth, transcription, redox, and energy utilization. Additionally, the influence of dietary zinc supply on the expression of some of these genes was confirmed by real-time quantitative PCR. Overall, these data support the effectiveness of cDNA array expression profiling to investigate the pleiotropic effects of specific nutrients and may provide an approach to establishing markers for assessment of nutritional status.

Animals↗
PubMedDate not supplied

Molecular characterization of Encephalitozoon intestinalis (Microspora) replication kinetics in a murine intestinal cell line.

Microsporidia are obligate intracellular pathogens of invertebrate and vertebrate animals. Most human infections are caused by Enterocytozoon bieneusi or Encephalitozoon intestinalis, and result in chronic diarrhea. In order to determine the signals involved in microsporidial spore activation and invasion, kinetics of in vitro E. intestinalis replication were defined using real-time quantitative PCR. Segments of small subunit ribosomal RNA and polar tube protein 2 genes of E. intestinalis were used to quantify parasite gene copy number following infection in murine colon carcinoma cells. Parasite DNA was detectable in small but significant amounts within host cells as early as 4 h postinoculation, genome replication was completed by 36 h, and parasite progeny were released into the supernatant beginning 72 h postinoculation. Heat-treating spores did not prevent transfer of parasite DNA into cells, but did inhibit parasite replication. Treating cell cultures with albendazole suppressed but did not completely inhibit parasite replication. These results confirm observations that E. intestinalis completes its life cycle within the turnover time of its target host cells; invasion into susceptible host cells occurs independently of spore viability; and real-time quantitative PCR is a sensitive and reproducible method with which to monitor microsporidial infection under varying treatments or conditions.

Albendazole↗
PubMed2026

A Novel Complete F8 Tandem Duplication Causing Elevated Factor VIII Activity and Associated with Venous Thromboembolism.

Background Coagulation factor VIII (FVIII) is a critical component of the intrinsic coagulation pathway. While elevated FVIII levels are an established risk factor for venous thromboembolism (VTE), genetic variants in the F8 gene directly causing such elevations remain scarce. Here, we report a novel complete F8 tandem duplication identified in a female patient with splanchnic venous thrombosis (SVT). Methods We performed genetic testing using a thrombophilia panel targeting 35 genes involved in thrombosis and haemostasis to detect both point variants and copy number variations (CNVs). Family co-segregation analysis and phenotypic assays for FVIII and von Willebrand factor (VWF) were conducted. The structural basis of the identified F8 copy number gain was elucidated using optical genome mapping (OGM). Full-length F8 mRNA amplification, quantitative PCR, plasma FVIII Western blotting, and X-chromosome inactivation analysis were performed to assess the functional consequences of the duplication. Thrombin generation test (TGT) was employed to assess the hypercoagulable state. Results Genetic testing identified three copies of all 26 exons of the F8 gene in the proband, which was also detected in her mother (CNVs = 3) and son (CNVs = 2). One-stage clotting and chromogenic assays confirmed persistently elevated FVIII activity in the proband and her mother, accompanied by increased FVIII antigen levels. The OGM analysis confirmed a 229 kb tandem duplication including the F8 gene on one of the proband's X chromosomes. The junction regions exhibited high sequence homology and were rich in repetitive sequences, which precluded precise breakpoint mapping. Full-length F8 mRNA amplification revealed no aberrant transcripts, whereas quantitative PCR showed increased F8 mRNA expression in all carriers. Plasma FVIII Western blotting indicated FVIII heavy and light chains of expected molecular weights with increased band intensity in carriers. X-chromosome inactivation analysis in female carriers showed no significant skewing. TGT in two available carriers showed increased thrombin generation compared with a normal control at both low (1 pM) and high (5 pM) tissue factor concentrations. Conclusion We identified a novel complete F8 tandem duplication associated with increased FVIII expression and a hypercoagulable phenotype in a female patient with SVT. These findings support F8 gene dosage gain as a rare gain-of-function mechanism contributing to elevated FVIII levels and thrombophilia, while variation in VWF levels and acquired risk factors may modify thrombotic penetrance.

coagulation factor VIII↗
PubMed2002

Internal spatiotemporal population dynamics of infection with three Wolbachia strains in the adzuki bean beetle, Callosobruchus chinensis (Coleoptera: Bruchidae).

The adzuki bean beetle, Callosobruchus chinensis, is infected with three distinct lineages of endosymbiotic bacteria belonging to the genus Wolbachia, which were designated wBruCon, wBruOri, and wBruAus. In an attempt to understand the mechanisms underlying the infection with these three organisms, the spatiotemporal infection dynamics of the three Wolbachia strains was investigated in detail by using a quantitative PCR technique. During the development of C. chinensis, the wBruCon, wBruOri, and wBruAus infection levels consistently increased but the growth patterns were different. The levels of infection plateaued at the pupal stage at approximately 3 x 10(8), 2 x 10(8), and 5 x 10(7) wsp copy equivalents per insect for wBruCon, wBruOri, and wBruAus, respectively. At the whole-insect level, the population densities of the three Wolbachia types did not show remarkable differences between adult males and females. At the tissue level, however, the total densities and relative levels of the three Wolbachia types varied significantly when different tissues and organs were compared and when the same tissues derived from males and females were compared. The histological data obtained by in situ hybridization and electron microscopy were concordant with the results of quantitative PCR analyses. Based on the histological data and the peculiar Wolbachia composition commonly found in nurse tissues and oocytes, we suggest that the Wolbachia strains are vertically transmitted to oocytes not directly, but by way of nurse tissue. On the basis of our results, we discuss interactions among the three coinfecting Wolbachia types, reproductive strategies of Wolbachia, and factors involved in the different cytoplasmic incompatibility phenotypes.

Animals↗
PubMed2004

[Quantitative detection of SARS-CoV RNA in excreta and oropharyngeal washing fluid from convalescence patients with severe acute respiratory syndrome].

OBJECTIVE: To detect the severe acute respiratory syndrome coronavirus (SARS-CoV) RNA in excreta and oropharyngeal washing fluid (OWF) from the convalescence SARS patients, and to determine whether convalescence patients carry the SARS-CoV, and whether having infectivity. METHODS: Totally 531 samples (including urine, stool, and OWF) were collected from 177 convalescence patients with positive SARS antibody which were confirmed by ELISA method. Real-time quantitative PCR was performed to detect the RNA of the SARS-CoV, and results were analyzed by SPSS analysis software. RESULTS: There were 49 (27.7%) cases of SARS-CoV RNA detection positive in 177 patients, including 31 (17.5%) cases with one sample positive, 14 (7.9%) cases with two samples positive, and 4 (2.3%) cases with three samples positive. The positive rates of urine, stool, and OWF were 14.7% (26/177), 11.9% (21/177), and 13.6% (24/177), respectively. The quantity of SARS-CoV RNA in samples was 100-47 000 copies/ml. No significant difference was found among urine, stool, and OWF on the difference grade quantity of SARS-CoV RNA quantity. CONCLUSIONS: About 10% convalescence SARS patients might still carry the SARS-CoV in hospitalization. The detection of SARS-CoV RNA by real-time quantitative PCR may become a laboratory examination warranty for convalescence SARS patients to discharge hospital and relieve separation.

Adolescent↗
PubMed2003

Differential expression of type XII collagen in developing chicken metatarsal tendons.

Type XII collagen is a fibril-associated collagen with multiple functional domains. The purpose of this work was to determine its role in regulating tendon matrix assembly. The temporal and spatial expression patterns of both collagen and mRNA were analysed in developing chicken metatarsal tendons using immunofluorescence microscopy, in situ hybridization and real-time quantitative PCR. Temporally, type XII collagen was present during all stages of development (day 14-hatch). However, spatially, type XII collagen expression shifted from the entire tendon at day 14, when the tendon is immature and fascicles are not well developed, to the interfacial matrix (endotendinium) associated with developing fascicles. This shift was obvious beginning at day 17, becoming prominent at day 19. Associated with this shift was a gradual decrease in type XII collagen reactivity in the tendon proper (non-sheath). By hatching, the reactivity was sequestered almost exclusively to the sheaths with some reactivity remaining at the fibroblast-matrix interface within the fascicle. In situ hybridization indicated that fibroblasts in the tendon expressed type XII collagen mRNA homogeneously at day 14. However, by hatching, when the tendon matures, type XII collagen is restricted primarily to the sheath cells. Quantitative PCR analyses, of NC3 splice variants, demonstrated highest expression levels for the short splice variant mRNA at days 14-17, followed by a significant decrease at day 19 with levels remaining constant to adult. Long variant mRNA expression was highest at day 14 then decreased and was constant from day 17 to adult. These changing patterns may be related to the spatial shift in type XII collagen expression to the sheaths. Differential temporal and spatial expression patterns indicate that type XII collagen functions to integrate the developing tendon matrices and fascicles into a functional unit.

Animals↗
PubMed2004

Inflammatory malignant fibrous histiocytomas and dedifferentiated liposarcomas: histological review, genomic profile, and MDM2 and CDK4 status favour a single entity.

Inflammatory malignant fibrous histiocytoma (inflammatory MFH) is a very rare tumour that occurs most often in the retroperitoneum. So far, it has been considered to be a special subtype of MFH. As it is now widely accepted that most retroperitoneal pleomorphic MFHs are dedifferentiated liposarcomas, the present study compared histological features, genomic profile (CGH analysis), and MDM2 and CDK4 status (immunohistochemistry, FISH, and quantitative PCR) in inflammatory MFHs from 12 patients and dedifferentiated liposarcomas that had an inflammatory MFH component from eight patients. Metaphase cytogenetic and FISH analyses were also performed on one inflammatory MFH. Histological review showed areas of well-differentiated liposarcoma in nine inflammatory MFHs. CGH analysis showed 12q13-15 amplification or gain in six of seven inflammatory MFHs and in seven of seven dedifferentiated liposarcomas. Immunohistochemistry showed positivity of tumour cells for MDM2 in every tumour in both groups and for CDK4 in ten and seven inflammatory MFHs and dedifferentiated liposarcomas, respectively. Metaphase cytogenetic and FISH analysis performed on one inflammatory MFH showed the presence of a supernumerary large marker chromosome and ring chromosome with high-level amplification of both MDM2 and CDK4 genes. FISH analysis on paraffin wax-embedded sections showed amplifications of MDM2 and CDK4 in seven of seven inflammatory MFHs and in seven of seven dedifferentiated liposarcomas. Quantitative PCR showed amplification of MDM2 in six and of CDK4 in seven of nine inflammatory MFHs. In conclusion, this study strongly suggests that most so-called inflammatory MFHs are dedifferentiated liposarcomas.

Adult↗
PubMed2002

Diagnostic and prognostic implications of circulating cell-free Epstein-Barr virus DNA in natural killer/T-cell lymphoma.

PURPOSE: Natural killer/T-cell (NK/T-cell) lymphoma is a highly aggressive tumor for which no serological tumor marker has yet been established to be useful clinically. We investigated the potential of circulating EBV DNA as a tumor marker for this malignancy. EXPERIMENTAL DESIGN: A real-time quantitative PCR assay was used to measure circulating EBV DNA. RESULTS: Plasma EBV DNA levels were measured in 18 patients with NK/T-cell lymphoma at presentation and during therapy. Plasma EBV DNA was detected in 17 of the 18 patients (median, 659 copies/ml; interquartile range, 181-17,042 copies/ml) but in none of 35 control subjects (p < 0.0001). Serial measurements of plasma EBV DNA levels during therapy showed a close correlation between clinical response and changes in plasma EBV DNA levels. Clinically responding patients showed a fall of plasma EBV DNA levels to low or undetectable levels, whereas those who failed therapy showed a rapid increase in plasma EBV DNA levels. Most importantly, patients with high baseline plasma EBV DNA levels (> or = 600 copies/ml) demonstrated a significantly inferior survival than those with low baseline plasma EBV DNA (< 600 copies/ml; 21% versus 78%; P = 0.024). CONCLUSIONS: Plasma EBV DNA, as measured by real-time quantitative PCR, is a useful tumor marker for diagnosis, disease monitoring, and prediction of outcome in patients with NK/T-cell lymphoma.

Adult↗
PubMed2006

In vivo characterization of a group II nucleopolyhedrovirus isolated from Mamestra brassicae (Lepidoptera: Noctuidae) in Japan.

A Japanese isolate of Mamestra brassicae nucleopolyhedrovirus (MabrNPV) was identified phylogenetically as a group II nucleopolyhedrovirus (NPV) that is related closely to other NPVs isolated from Mamestra spp. based on nucleotide sequence data of its polh, egt and lef-3 genes. The multiplication of MabrNPV in M. brassicae larvae was characterized following inoculation at various doses and in combination with the fluorescent brightener Tinopal by measuring temporal changes in the concentrations of its viral DNA using real-time quantitative PCR. The growth curves of budded-virus replication were analysed by fitting the data of viral DNA concentration in the host haemolymph to a modified Gompertz model. When fifth-instar larvae were inoculated with an LD95 equivalent dose of MabrNPV and Tinopal, the time lag between the onset of primary and secondary infection was estimated to be 25 h. Another 65 h was required to reach a plateau titre equivalent to a level of 10(9) virions ml(-1) in the haemolymph. All larvae died during the sixth instar following this inoculation regime. In contrast, following inoculation with a 1000-fold higher dose of MabrNPV and Tinopal, the time lag between the onset of primary and secondary infection was only 20 h. Subsequently, the same plateau titre was reached after a further 20 h. Following this inoculation regime, most larvae died during the fifth instar. Quantification of viral DNA by real-time quantitative PCR and application of the Gompertz model are valuable for the characterization of baculovirus replication in vivo.

Amino Acid Sequence↗
PubMed2002

Influence of age, sex, and strength training on human muscle gene expression determined by microarray.

The purpose of this study was to determine the influence of age, sex, and strength training (ST) on large-scale gene expression patterns in vastus lateralis muscle biopsies using high-density cDNA microarrays and quantitative PCR. Muscle samples from sedentary young (20-30 yr) and older (65-75 yr) men and women (5 per group) were obtained before and after a 9-wk unilateral heavy resistance ST program. RNA was hybridized to cDNA filter microarrays representing approximately 4,000 known human genes and comparisons were made among arrays to determine differential gene expression as a result of age and sex differences, and/or response to ST. Sex had the strongest influence on muscle gene expression, with differential expression (>1.7-fold) observed for approximately 200 genes between men and women (approximately 75% with higher expression in men). Age contributed to differential expression as well, as approximately 50 genes were identified as differentially expressed (>1.7-fold) in relation to age, representing structural, metabolic, and regulatory gene classes. Sixty-nine genes were identified as being differentially expressed (>1.7-fold) in all groups in response to ST, and the majority of these were downregulated. Quantitative PCR was employed to validate expression levels for caldesmon, SWI/SNF (BAF60b), and four-and-a-half LIM domains 1. These significant differences suggest that in the analysis of skeletal muscle gene expression issues of sex, age, and habitual physical activity must be addressed, with sex being the most critical variable.

Adult↗
PubMed2001

Laboratory diagnosis of cytomegalovirus (CMV) infections in immunodepressed patients, mainly in patients with AIDS.

There is a high prevalence of cytomegalovirus (CMV) infection in the general population. An important proportion of immunodepressed patients are latent carriers of the virus, and under these conditions, the lack of cellular immunity predisposes the patient to an active infection in which the virus is replicating. Consequences for the immunodepressed patients are widely variable, ranging from completely asymptomatic infections to life-threatening situations. An important characteristic of these infections is that they have no specific clinical symptoms and are often indistinguishable from other types of infection. As such, clinical diagnosis should be supported by laboratory tests. There are many diagnostic tests available, the choice of which one to use being made on the basis of clinician-defined objectives. The CMV immunological state of the patient should be ascertained in order to indicate the possibility of reactivation or the risk of primary infection. To this end, serological tests have proven to be the most useful. Laboratory tests also allow the rapid diagnosis of active CMV infections, for which rapid culture techniques (shell-vial) or viral antigen tests (antigenemia pp65) are more appropriate. Given that not all active infections present with symptoms, the most typical problem in clinical practice is to distinguish between symptoms and illnesses caused by CMV (CMV disease: CMVD), and other infectious or non-infectious entities. To clarify this situation, diagnostic histopathology and methods able to quantify the viral load (pp65 antigenemia and quantitative PCR) are of great use. The identification of patients who, during active infection, are at high risk of contracting CMVD in the near future, is a more complex challenge for the laboratory. Once again, tests that are able to quantify the viral load are the most appropriate for identifying those patients who should receive preventive antiviral treatment. In HIV-positive patients, CMVD tends to manifest itself as retinitis and involvement of the gastrointestinal tract. The principal risk factor in the development of CMVD in these patients is the degree of immunodepression, which appears almost exclusively in patients with a CD4+ lymphocyte count lower than 50/mm3 and when other opportunistic infections have occurred. The advent of oral ganciclovir has raised the possibility of long-term prophylactic treatment of CMVD, making the search for virological and immunological markers that identify these high-risk patients a priority. Techniques allowing the quantification of CMV viremia, namely pp65 antigenemia and quantitative PCR, have been most useful to this end.

AIDS-Related Opportunistic Infections↗