Home About us Contact
|
Home About us Contact | ||
|
|
||
Reverse-transcriptase Polymerase Chain Reaction (reverse-transcriptase + polymerase_chain_reaction)
Kinds of Reverse-transcriptase Polymerase Chain Reaction Selected AbstractsMLL/GRAF fusion in an infant acute monocytic leukemia (AML M5b) with a cytogenetically cryptic ins(5;11)(q31;q23q23)GENES, CHROMOSOMES AND CANCER, Issue 4 2004Ioannis Panagopoulos More than 30 fusions involving the MLL gene at 11q23 have been reported in acute myeloid leukemia (AML). Some of these chimeras are rather common, such as MLL/MLLT3(AF9), but many are quite rare, with some, for example, MLL/GRAF, described only in a single case. The MLL/GRAF fusion, in which the reciprocal hybrid was not expressed, suggesting that the former transcript was the leukemogenic one, was detected in a juvenile myelomonocytic leukemia with a t(5;11)(q31;q23). Here, we report a second case,an infant acute monocytic leukemia (AML M5b),with an MLL/GRAF fusion. By conventional G-banding, the karyotype was normal. However, Southern blot and fluorescence in situ hybridization analyses revealed that MLL was rearranged and that the 5, part of the MLL gene was inserted into 5q in the vicinity of 5q31, which harbors GRAF. Reverse-transcriptase polymerase chain reaction (PCR) showed that exon 9 of MLL was fused in-frame with exon 19 of GRAF. Extralong genomic PCR with subsequent sequence analysis demonstrated that the breakpoints occurred in intron 9 of MLL, nine base pairs (bp) downstream from exon 9, and in intron 18 of GRAF, 117 bp downstream from exon 18. A 6-bp insertion (ACACTC) of unknown origin was present at the junction. The putative MLL/GRAF fusion protein would retain the AT-hook DNA-binding domain, the DNA methyl transferase motif, the transcription repression domain of MLL, and the SH3 domain of GRAF. As expected, the reciprocal GRAF/MLL was neither expressed nor generated at the genomic level as a consequence of the ins(5;11)(q31;q23q23). On the basis of the now-reported two cases with MLL/GRAF, we conclude that this transcript,but not the reciprocal one,characterizes a rare genetic subgroup of infant AML. © 2004 Wiley-Liss, Inc. [source] GlcNAc6ST-1-mediated decoration of MAdCAM-1 protein with L-selectin ligand carbohydrates directs disease activity of ulcerative colitisINFLAMMATORY BOWEL DISEASES, Issue 5 2009Motohiro Kobayashi MD Abstract Background: A diffuse lymphocyte infiltrate is 1 of the characteristic features of ulcerative colitis (UC). Such lymphocyte recruitment requires lymphocyte rolling mediated by L-selectin ligand carbohydrates (6-sulfo sialyl Lewis X-capped O -glycans) and/or mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expressed on high endothelial venule (HEV)-like vessels. The present study was undertaken to elucidate the role of MAdCAM-1 posttranslationally modified ("decorated") with L-selectin ligand carbohydrates in UC pathogenesis and consequent clinical outcomes. Methods: Biopsy specimens composed of active and remission phases of UC as well as normal colonic mucosa were immunostained for CD34, MAdCAM-1, and MECA-79, and the immunostained sections were quantitatively analyzed. Reverse-transcriptase polymerase chain reaction (RT-PCR) was carried out to evaluate transcripts of MAdCAM-1 and N -acetylglucosamine-6- O -sulfotransferases (GlcNAc6STs). CHO and Lec2 cells transfected with CD34 and MAdCAM-1 together with enzymes involved in L-selectin ligand carbohydrate biosynthesis were analyzed by immunofluorescence, FACS, and Western blotting to characterize the biochemical properties of GlcNAc6STs. Results: The number of MAdCAM-1+ vessels was increased in UC, with no significant difference between active and remission phases. An increased ratio of MECA-79+ to MAdCAM-1+ vessels with preferential GlcNAc6ST-1 transcripts was observed in the active phase of UC compared to the remission phase. MAdCAM-1 protein was colocalized with L-selectin ligand carbohydrates at the luminal surface of HEV-like vessels in situ. GlcNAc6ST-1 preferentially utilizes MAdCAM-1 as a scaffold protein for GlcNAc-6- O -sulfation in L-selectin ligand carbohydrate biosynthesis. Conclusions: UC disease activity is not regulated by expression of MAdCAM-1 protein itself, but rather by GlcNAc6ST-1-mediated decoration of MAdCAM-1 protein with L-selectin ligand carbohydrates. (Inflamm Bowel Dis 2008) [source] Role of functional polymorphisms of NRAMP1 gene for the development of Crohn's diseaseINFLAMMATORY BOWEL DISEASES, Issue 10 2008Maria Gazouli PhD Abstract Background: Crohn's disease (CD) is characterized by chronic activation of macrophages. Natural resistance-associated macrophage protein 1 (NRAMP1) gene exerts many pleiotropic effects on macrophage functions. Hence, NRAMP1 may be also involved in the resistance to intracellular pathogens, and this effector of the innate immunity might be involved in CD pathogenesis. Polymorphic alleles at the NRAMP1 locus have been previously associated with susceptibility both to the putative infectious agents and to autoimmune disorders. Based on these indications, in the present study we investigate its candidacy as a genetic determinant for CD in a Greek population in an association-based study, comparing frequencies of 274 CD patients to these of 200 healthy control subjects. Methods: The 5,(GT)n promoter polymorphism and 9 either single nucleotide (SNPs) or insertion/deletion type polymorphisms were genotyped across the NRAMP1 gene. Reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry were performed in order to investigate the NRAMP1 mRNA levels in RNA isolated from biopsies of CD patients as well as protein expression in tissues. Results: Three NRAMP1 polymorphisms [5,(GT)n, D543N, and INT4G/C] were significantly associated with CD. Consistent with previous autoimmune disease studies, allele 3 at the functional 5,(GT)n promoter region repeat polymorphism, was significantly associated with CD when compared to healthy controls (odds ratio 1.50; 95% confidence interval [CI]: 1.16,1.95; P = 0.002). Interestingly, we observed that CD patients homozygous for allele 3 expressed higher NRAMP1 mRNA levels compared to carriers of allele 2. Furthermore, the protein levels of allele 3 carriers in tissues were also elevated compared to those of allele 2 carriers. Based on these data we can speculate that overrepresentation of allele 3 in CD patients could lead to hyperactivation of bowel-wall macrophages that are chronically exposed to lipopolysaccharide and this could subsequently cause the autoimmune-like phenotype characteristic of CD. Conclusions: Collectively, our data indicate that genetic polymorphisms of NRAMP1 might be associated with susceptibility to CD. (Inflamm Bowel Dis 2008) [source] E-cadherin synergistically induces hepatospecific phenotype and maturation of embryonic stem cells in conjunction with hepatotrophic factorsBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2005Anouska Dasgupta Abstract Since effective cell sourcing is a major challenge for the therapeutic management of liver disease and liver failure, embryonic stem (ES) cells are being widely investigated as a promising source of hepatic-like cells with their proliferative and pluripotent capacities. Cell,cell interactions are crucial in embryonic development modulating adhesive and signaling functions; specifically, the cell,cell adhesion ligand, cadherin is instrumental in gastrulation and hepatic morphogenesis. Inspired by the role of cadherins in development, we investigated the role of expression of E-cadherin in cultured murine ES cells on the induction of hepatospecific phenotype and maturation. The cadherin-expressing embryonic stem (CE-ES) cells intrinsically formed pronounced cell aggregates and cuboidal morphology whereas cadherin-deficient cadherin-expressing embryonic stem (CD-ES) cells remained more spread out and corded in morphology. Through controlled stimulation with single or combined forms of hepatotrophic growth factors; hepatocyte growth factor (HGF), dexamethasone (DEX) and oncostatin M (OSM), we investigated the progressive maturation of CE-ES cells, in relation to the control, CD-ES cells. Upon growth factor treatment, the CE-ES cells adopted a more compacted morphology, which exhibited a significant hepatocyte-like cuboidal appearance in the presence of DEX-OSM-HGF. In contrast, the CD-ES cells exhibited a mixed morphology and appeared to be more elongated in the presence of DEX-OSM-HGF. Reverse-transcriptase polymerase chain reaction was used to delineate the most differentiating condition in terms of early (alpha-fetoprotein (AFP)), mid (albumin), and late-hepatic (glucose-6-phosphatase) markers in relation to growth factor presentation for both CE-ES and CD-ES cells. We report that following the most differentiating condition of DEX-OSM-HGF stimulation, CE-ES cells expressed increased levels of albumin and glucose-6-phosphatase, whereas the CD-ES cells showed low levels of AFP and marginal levels of albumin and glucose-6-phosphatase. These trends suggest that the membrane expression of E-cadherin in ES cells can elicit a marked response to growth factor stimulation and lead to the induction of later stages of hepatocytic maturation. Thus, cadherin-engineered ES cells could be used to harness the cross-talk between the hepatotrophic and cadherin-based signaling pathways for controlled acceleration of ES hepatodifferentiation. © 2005 Wiley Periodicals, Inc. [source] Semiquantitative determination of Alicyclobacillus acidoterrestris in orange juice by reverse- transcriptase polymerase chain reaction and capillary electrophoresis , laser induced fluorescence using microchip technologyELECTROPHORESIS, Issue 21-22 2004Maribel Funes-Huacca Abstract The semiquantitative detection of Alicyclobacillus acidoterrrestris in orange juice by reverse-transcriptase polymerase chain reaction (RT-PCR) with a linear dynamic range of 2×105, 2 colony forming units (CFU)/mL in terms of cell count is described. Separation, detection, and quantification of the RT-PCR products were accomplished using the Agilent 2100 bioanalyzer in conjunction with the DNA 1000 LabChip kit. After 0 and 12 h of enrichment, it was possible to generate a linear standard curve between the amount of cells and amplicon concentration of RT-PCR and PCR products. Using this method, cell diminution was verified in samples of orange juice treated with a natural inhibitor (Sapindus saponaria), determining the persistence of viable cells. Semiquantitative RT-PCR using the Agilent 2100 bioanalyzer is a potentially useful approach for rapid in vitro determination of A. acidoterrestris and monitoring of inhibitor susceptibility for the orange juice-producing industry. [source] Gene expression in caged fish as a first-tier indicator of contaminant exposure in streamsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 12 2005Aaron P. Roberts Abstract The development of sensitive, biologically based indicators of contaminant exposure (i.e., biomarkers) is an ongoing topic of research. These indicators have been proposed as a first-tier method of identifying contaminant exposure. The primary objective of this research was to implement a biomarker-based method of exposure assessment using caged fish and real-time reverse-transcriptase polymerase chain reaction (rtRT-PCR) measurements of gene expression. Primers were developed for the CYPIA, metallothionein, and vitellogenin genes in rainbow trout (Oncorhynchyus mykiss), cutbow trout (Oncorhynchyus clarkii × mykiss), and Atlantic salmon (Salmo salar). Each of these genes has been shown to respond specifically to planar aromatic compounds, heavy metals, and environmental estrogens, respectively. Juvenile fish were placed in cages and exposed in situ at reference and contaminated sites on the Cache la Poudre River (CO, USA), the Arkansas River (CO, USA), the St. John River (NB, Canada), and two urban creeks near Dayton (OH, USA). Quantitative gene expression was determined using rtRT-PCR. Biomarker expression profiles were obtained that demonstrated differences in CYP1A, metallothionein, and vitellogenin mRNA production unique to each site, indicating that specific types of compounds were bioavailable and present in sufficient concentrations to elicit transcriptional responses in the organism. These findings support the use of a biomarker-based approach to exposure identification and assessment. [source] EVOLUTION OF INSECT METAMORPHOSIS: A MICROARRAY-BASED STUDY OF LARVAL AND ADULT GENE EXPRESSION IN THE ANT CAMPONOTUS FESTINATUSEVOLUTION, Issue 4 2005Michael A. D. Goodisman Abstract Holometabolous insects inhabit almost every terrestrial ecosystem. The evolutionary success of holometabolous insects stems partly from their developmental program, which includes discrete larval and adult stages. To gain an understanding of how development differs among holometabolous insect taxa, we used cDNA microarray technology to examine differences in gene expression between larval and adult Camponotus festinatus ants. We then compared expression patterns obtained from our study to those observed in the fruitfly Drosophila melanogaster. We found that many genes showed distinct patterns of expression between the larval and adult ant life stages, a result that was confirmed through quantitative reverse-transcriptase polymerase chain reaction. Genes involved in protein metabolism and possessing structural activity tended to be more highly expressed in larval than adult ants. In contrast, genes relatively upregulated in adults possessed a greater diversity of functions and activities. We also discovered that patterns of expression observed for homologous genes in D. melanogaster differed substantially from those observed in C. festinatus. Our results suggest that the specific molecular mechanisms involved in metamorphosis will differ substantially between insect taxa. Systematic investigation of gene expression during development of other taxa will provide additional information on how developmental pathways evolve. [source] Enhanced expression of vascular endothelial growth factor-A in ground glass hepatocytes and its implication in hepatitis B virus hepatocarcinogenesis,HEPATOLOGY, Issue 6 2009Jui-Chu Yang Ground glass hepatocytes (GGH) in chronic hepatitis B virus (HBV) infection harbor HBV pre-S deletion mutants in endoplasmic reticulum (ER) and exhibit complex biologic features such as ER stress, DNA damage, and growth advantage. The presence of pre-S mutants in serum has been shown to predict the development of hepatocellular carcinoma (HCC) in HBV carriers. GGHs hence represent a potentially preneoplastic lesion. Whether a specific growth factor is overexpressed and activated in GGHs remains to be clarified. In this study, growth factor(s) up-regulated by pre-S mutants was identified using a growth factor array in HuH-7 cells. Immunohistochemistry, reverse-transcriptase polymerase chain reaction, and Western blot analysis were performed to study the participation of these genes and their signal pathways in HuH-7 cells and liver tissues. We demonstrate that vascular endothelial growth factor-A (VEGF-A) was up-regulated by pre-S mutants in HuH-7 cells and further confirmed in GGHs by immunostaining. The VEGF-A up-regulation by pre-S mutants could be suppressed by vomitoxin, an ER stress inhibitor. Furthermore, pre-S mutants-expressed HuH-7 cells exhibited activation of Akt/mTOR (mammalian target of rapamycin) signaling and increased growth advantage, which could be inhibited by VEGF-A neutralization. Consistent with this notion, enhanced expression of VEGF-A and activation of Akt/mTOR signaling, comparable to the levels of paired HCC tissues, were also detected in HBV-related nontumorous livers. Conclusion: The enhanced expression of VEGF-A in GGHs provides potential mechanism to explain the progression from preneoplastic GGHs to HCC in chronic HBV infection. (HEPATOLOGY 2009;49:1962,1971.) [source] Kupffer cell,derived interleukin 10 is responsible for impaired bacterial clearance in bile duct,ligated miceHEPATOLOGY, Issue 2 2004Tetsuya Abe Extrahepatic cholestasis often evokes liver injury with hepatocyte apoptosis, aberrant cytokine production, and,most importantly,postoperative septic complications. To clarify the involvement of aberrant cytokine production and hepatocyte apoptosis in impaired resistance to bacterial infection in obstructive cholestasis, C57BL/6 mice or Fas-mutated lpr mice were inoculated intraperitoneally with 107 colony-forming units of Escherichia coli 5 days after bile duct ligation (BDL) or sham celiotomy. Cytokine levels in sera, liver, and immune cells were assessed via enzyme-linked immunosorbent assay or real-time reverse-transcriptase polymerase chain reaction. BDL mice showed delayed clearance of E. coli in peritoneal cavity, liver, and spleen. Significantly higher levels of serum interleukin (IL) 10 with lower levels of IL-12p40 were observed in BDL mice following E. coli infection. Interferon , production from liver lymphocytes in BDL mice was not increased after E. coli infection either at the transcriptional or protein level. Kupffer cells from BDL mice produced low levels of IL-12p40 and high levels of IL-10 in vitro in response to lipopolysaccharide derived from E. coli. In vivo administration of anti,IL-10 monoclonal antibody ameliorated the course of E. coli infection in BDL mice. Furthermore, BDL- lpr mice did not exhibit impairment in E. coli killing in association with little hepatic injury and a small amount of IL-10 production. In conclusion, increased IL-10 and reciprocally suppressed IL-12 production by Kupffer cells are responsible for deteriorated resistance to bacterial infection in BDL mice. Fas-mediated hepatocyte apoptosis in cholestasis may be involved in the predominant IL-10 production by Kupffer cells. (HEPATOLOGY 2004;40:414,423.) [source] The Impact of Interferon Gamma Receptor Expression on the Mechanism of Escape From Host Immune Surveillance in Hepatocellular CarcinomaHEPATOLOGY, Issue 3 2000Mitsuo Nagao M.D. Interferon gamma (IFN-,) plays an important role in host defense mechanism and participates in the progression of chronic liver disease. IFN-, exerts its pleiotrophic effects by transcriptional regulation of expression of numerous genes, such as major histocompatibility complex (MHC) class I and Fas, through interaction with IFN-, receptor (IFN-,-R). Although hepatocytes in normal liver express weak or no IFN-,-R, those in acute and chronic liver disease up-regulate its expression. A study using IFN-,-R ,-chain knock-out mice revealed the actions of IFN-, on tumor cells as an extrinsic tumor-suppressor mechanism. However, it is unclear whether or how hepatocellular carcinoma (HCC) blocks the signal transduction of IFN-, to evade host immune surveillance. We examined the expression of IFN-,-R and IFN-,,inducible genes in 44 cases with HCC using real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. In noncancerous liver tissues (n = 38), IFN-,-R expression on the cell surface was up-regulated in 27 cases. In IFN-,-R,negative cases (n = 15), tumor size was larger (P = .032), serum ,-fetoprotein (AFP) level was higher (P = .001), intrahepatic and extrahepatic metastasis was more common (P = .044 and .013, respectively), and Ki-67 labeling index (LI) was higher (P = .041), compared with IFN-,-R,positive cases. Accordingly, the evasion mechanism may play an important role in progression, especially metastasis, in HCC. The significant correlation between the status of IFN-,-R and the expression of Fas and MHC implies that the loss of IFN-,-R might contribute to the mechanism of escape from host immune rejection in HCC. [source] Activation of the cannabinoid 2 receptor (CB2) protects against experimental colitisINFLAMMATORY BOWEL DISEASES, Issue 11 2009Martin A. Storr MD Abstract Background: Activation of cannabinoid (CB)1 receptors results in attenuation of experimental colitis. Our aim was to examine the role of CB2 receptors in experimental colitis using agonists (JWH133, AM1241) and an antagonist (AM630) in trinitrobenzene sulfonic acid (TNBS)-induced colitis in wildtype and CB2 receptor-deficient (CB mice. Methods: Mice were treated with TNBS to induce colitis and then given intraperitoneal injections of the CB2 receptor agonists JWH133, AM1241, or the CB2 receptor antagonist AM630. Additionally, CB mice were treated with TNBS and injected with JWH133 or AM1241. Animals were examined 3 days after the induction of colitis. The colons were removed for macroscopic and microscopic evaluation, as well as the determination of myeloperoxidase activity. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) for CB2 receptor was also performed in animals with TNBS and dextran sodium sulfate colitis. Results: Intracolonic installation of TNBS caused severe colitis. CB2 mRNA expression was significantly increased during the course of experimental colitis. Three-day treatment with JWH133 or AM1241 significantly reduced colitis; AM630 exacerbated colitis. The effect of JWH133 was abolished when animals were pretreated with AM630. Neither JWH133 nor AM1241 had effects in CB mice. Conclusions: We show that activation of the CB2 receptor protects against experimental colitis in mice. Increased expression of CB2 receptor mRNA and aggravation of colitis by AM630 suggests a role for this receptor in normally limiting the development of colitis. These results support the idea that the CB2 receptor may be a possible novel therapeutic target in inflammatory bowel disease. (Inflamm Bowel Dis 2009) [source] Tetomilast suppressed production of proinflammatory cytokines from human monocytes and ameliorated chronic colitis in IL-10-deficient miceINFLAMMATORY BOWEL DISEASES, Issue 11 2008Hitoshi Ichikawa MD Abstract Background: Tetomilast (OPC-6535) was originally developed as a compound inhibiting superoxide production in neutrophils. Although its mechanism of action is not completely understood, phosphodiesterase type 4 inhibitory function has been postulated. The therapeutic effect of PDE4 inhibitors has been reported for chronic inflammatory disorders such as chronic obstructive pulmonary diseases. In this study we aimed to examine whether tetomilast could be a novel drug for inflammatory bowel diseases by further clarifying its antiinflammatory effects. Methods: Cytokines from human peripheral blood mononuclear cells were measured by enzyme-linked immunosorbent assay (ELISA) and Cytokine Beads Array. The transcripts were quantified by reverse-transcriptase polymerase chain reaction (RT-PCR). Phosphorylation of transcription factors was examined by phosflow. To examine its in vivo effect, a once-daily oral dose of tetomilast was tested in murine IL-10,/, chronic colitis. Results: Tetomilast suppressed TNF-, and IL-12 but not IL-10 production from lipopolysaccharide (LPS)-stimulated human monocytes. It suppressed TNF-,, IFN-,, and IL-10 from CD4 lymphocytes. Tetomilast suppressed cytokine production at the transcriptional level but did not alter phosphorylation of p65, ERK, p38, and STAT3. HT-89, a protein kinase A inhibitor, did not abolish the effect of tetomilast, suggesting that it was independent from the classical cAMP/PKA pathway. IL-10 was not essential to the inhibitory effect of tetomilast on TNF-, and IL-12. Tetomilast ameliorated IL-10,/, chronic colitis with reduced clinical symptoms, serum amyloid A, and histological scores with decreased TNF-, mRNA expression. Conclusions: Tetomilast exerts its antiinflammatory effects on human monocytes and CD4 cells. Combined with in vivo data these findings support the feasibility of tetomilast as a novel drug for inflammatory bowel diseases. (Inflamm Bowel Dis 2008) [source] Mucosal NOD2 expression and NF-,B activation in pediatric Crohn's diseaseINFLAMMATORY BOWEL DISEASES, Issue 3 2008Laura Stronati PhD Abstract Background: Recent advances in the pathogenesis of Crohn's disease (CD) have suggested that an aberrant innate immune response initiates the cascade of events leading to T-cell activation and to disease development. NOD2 protein, which is mainly expressed by innate immunity cells, appears to play a key role against bacteria by triggering a host defense response through the activation of the transcriptor factor NF-,B and a consequent proinflammatory cytokine production. The present study was aimed at investigating the expression and activity of NOD2, NF-,B, and of 2 proinflammatory cytokines, TNF, and IL-1,, in mucosal biopsies of CD affected children compared to healthy controls. Methods: In all, 22 children with active CD and 10 matched controls were entered in the study. mRNA and protein expressions were detected using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot; NF-,B binding activity was assessed by electromobility gel shift assay (EMSA). Results: NOD2 and IL-1, mRNAs were upregulated in CD children. Protein levels of NOD2, TNF,, and nuclear NF-,B, as well as the binding activity of NF-,B to a consensus DNA sequence, were significantly increased in inflamed mucosa of patients as compared to controls. Moreover, NF-,B activity was strongly upregulated in patients also when bound to the NOD2 promoter site. No difference was seen between patients and controls when NF-,B binding activity was determined in the uninflamed tissue. Conclusions: This study suggests that altered mechanisms regulating NOD2 induction, NF-,B activation and cytokine production may contribute to dysregulate the innate immune response underlying pediatric CD. (Inflamm Bowel Dis 2007) [source] Characterization of cecal gene expression in a differentially susceptible mouse model of bacterial-induced inflammatory bowel diseaseINFLAMMATORY BOWEL DISEASES, Issue 7 2007Matthew H. Myles DVM Abstract Background: A/JCr mice develop typhlitis in response to Helicobacter hepaticus infection, whereas C57BL/6 mice coexist with this bacterium in a "commensal" relationship and do not develop disease even during prolonged colonization. Methods: To determine mechanisms that control this balance between responsiveness and nonresponsiveness, the mucosal response of A/JCr and C57BL/6 mice to acute H. hepaticus colonization was evaluated using genome-wide profiling. Transcription levels for a subset of gene discoveries were then evaluated longitudinally by semiquantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) to identify changes in gene expression that occur during progression from the acute to chronic phase of colonization. To determine whether chronic mucosal inflammation in A/JCr mice was mediated through a Th1 mechanism, as was inferred from the gene expression data, mice with typhlitis were treated with neutralizing antibody targeting IL-12/23p40 or IFN-gamma and the response to treatment was determined by cecal lesion severity and transcription of disease-related genes. Results: A/JCr mice had a biphasic expression of proinflammatory genes that corresponded with the acute and chronic phases of disease. In contrast, C57BL/6 mice exhibited a less robust acute transcriptional response that waned by day 30 postinoculation. Sustained upregulation of proinflammatory signals and responsiveness to anti-IL-12/23p40 and anti-IFN-, antibody suggests that inflammation in A/JCr mice was mediated through a Th1 mechanism. Prolonged upregulation of SOCS3 during the acute response to colonization suggests that C57BL/6 mice maintain mucosal homeostasis, at least in part by attenuating responsiveness to cytokine signaling. Conclusions: Collectively, these findings provide a foundation for understanding the immunological mechanisms that confer resistance or susceptibility to H. hepaticus -induced typhlitis. (Inflamm Bowel Dis 2007) [source] Expression and functional characterization of FOXP3+CD4+ regulatory T cells in ulcerative colitis,INFLAMMATORY BOWEL DISEASES, Issue 2 2007Qi T. Yu BS Abstract Background: CD4+CD25+ regulatory T cells (TR) can prevent or treat experimental murine colitis but little is known about their potential role in human inflammatory bowel disease (IBD). FOXP3 is a transcription factor that plays a critical role in the development and function of CD4+CD25+ TR. The aim of this study was to examine the presence and functional characteristics of TR cells in colonic lymphoid tissues in patients with ulcerative colitis (UC). Methods: FOXP3 expression was assessed by flow cytometry, immunohistochemistry, and reverse-transcriptase polymerase chain reaction (RT-PCR). Functional characterization of CD4+CD25+ cells was analyzed by suppression of proliferation and secretion of cytokines by cocultured effector CD4+CD25, T cells. Results: FOXP3+CD4+ T cells are increased in the lamina propria (LP) of inflamed and noninflamed areas of UC colon compared to normal colon. CD4+CD25+ T cells in UC mesenteric lymph nodes (MLN) express FOXP3 mRNA and protein and suppress the proliferation of autologous MLN CD4+CD25, T cells. The suppressor activity of MLN CD4+CD25+ T cells is cell contact-dependent but cytokine-independent. In addition, CD4+CD25+ T cells potently suppress the production of both Th1 (IFN-,, IL-2) and Th2 (IL-5, IL-13) cytokines by cocultured CD4+CD25, T cells. FOXP3+ cells localized in the T-cell-rich areas of MLN and occasionally present in the follicles. Conclusions: There is an expansion of FOXP3+CD4+ T cells in mucosal lymphoid tissues in UC. CD4+CD25+ isolated from UC MLN express FOXP3 and display features of TR cells in spite of active mucosal inflammation. These data suggest that their suppressor activity may be abrogated in vivo or they are unable to counterbalance the chronic mucosal inflammation in UC. (Inflamm Bowel Dis 2007) [source] Overexpression of MLH-1 and psoriasin genes in cutaneous angiofibromas from tuberous sclerosis complex patientsJOURNAL OF CUTANEOUS PATHOLOGY, Issue 9 2006Michelangelo La Placa Background:, Tuberous sclerosis complex (TSC) is associated with mutations in two likely tumor-suppressor genes (TSC1 and TSC2) and characterized by the development of tumor-like growths (angiofibromas) in a variety of tissues and organs, particularly brain and skin. Methods:, Employing a DNA-microarray assay, able to detect mRNA production from 1176 different basic genes, we analyzed the gene-expression levels in a cutaneous hamartoma sample from a TSC patient. Altered gene expressions detected by microarray technology were further checked by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) in the same material and in cutaneous hamartoma samples obtained from five other TSC patients. Results:, The results obtained by the microarray technology in one hamartoma specimen, confirmed by the RT-PCR results obtained in the same material and in five other hamartoma specimens, demonstrated that TSC-related angiofibromas exhibit significant mRNA overexpression of two genes, represented by MLH-1 and psoriasin. Conclusions:, The overexpression of MLH-1, which codes for a DNA mismatch repair protein, and psoriasin, which codes for a specific chemoattractant factor for CD4+ T cells, implicated in the pathogenesis of inflammatory skin disease, and expressed in excess during abnormal pathways of cell growth, may shed light on the pathogenesis of the proliferative skin lesion. [source] Impact of human metapneumovirus in childhood: Comparison with respiratory syncytial virus and influenza viruses,JOURNAL OF MEDICAL VIROLOGY, Issue 1 2005Samantha Bosis Abstract This study evaluated the overall impact of human metapneumovirus (hMPV) infection in 1,505 children and their households, and compared it with infections due to respiratory syncytial virus (RSV) and influenza viruses. Nasopharyngeal swabs were used at enrollment to collect specimens for the detection of hMPV, RSV, and influenza virus RNA by reverse-transcriptase polymerase chain reaction (RT-PCR). hMPV was detected in 42 children (2.8%), RSV in 143 (9.5%; P,<,0.0001 vs. hMPV), and influenza viruses in 230 (15.3%; P,<,0.0001 vs. hMPV). Of the 42 hMPV-positive samples, one was also positive for RSV and six for influenza viruses, for a co-infection rate of 16.7%. Clinically, hMPV was identified only in patients with acute respiratory infection, whereas RSV and influenza viruses were also detected in patients with different clinical manifestations. Symptoms with statistically significant different proportions at presentation were fever (more frequent in the hMPV- and influenza-positive children) and wheezing with bronchiolitis or asthma exacerbation (more frequent among hMPV- and RSV-positive cases). The households of the hMPV- and the influenza-positive children had significantly more illnesses, needed significantly more medical visits, received more antipyretics, and missed significantly more work or school days than those of the RSV-positive children. Results show that hMPV is an emerging cause of acute respiratory infection in childhood, and may have a significant clinical and socioeconomic impact on children and their families. J. Med. Virol. 75:101,104, 2005. © 2005 Wiley-Liss, Inc. [source] Decreased Detectability of Grapevine Leafroll-associated virus 3 in Sakasly Grapevines Cultivated Under the Sahara ConditionsJOURNAL OF PHYTOPATHOLOGY, Issue 9 2006A. Ben salem-Fnayou Abstract The detectability of grapevine-leafroll-associated virus 3 (GLRaV-3) was investigated by enzyme-linked immunosorbent assay (ELISA) and reverse-transcriptase polymerase chain reaction (RT,PCR) in the domestic grapevine cultivar Sakasly, grown over two successive years in the Sahara, at Rjim-Mâatoug in Tunisia. Self-rooted cuttings, infected with GLRaV-3 were cultivated and the presence of the virus was checked over 2 years and compared with controls. During the first year, 80% of the originally infected vines were negative for GLRaV-3 using ELISA. After the second year, 93% and 95% of these plants were negative for GLRaV-3 using ELISA and RT,PCR, respectively. Furthermore, rooted cuttings derived from GLRaV-3-negative plants and grown under controlled conditions in a greenhouse (at 16,20°C) were ELISA-negative in most cases (84%). In addition, biological indexing on Vitis vinifera cv. Gamay Rouge de la Loire showed no leafroll symptoms on this indicator in 92% of the grafted vines. These results suggest a naturally occurring heat therapy in the Tunisian Sahara, which could be of practical importance for the production of GLRaV-3-free grapevine cuttings, especially as scale insect and mealybug vectors have not been observed in this area. [source] Cross-species hybridization of a Borrelia burgdorferi DNA array reveals infection- and culture-associated genes of the unsequenced genome of the relapsing fever agent Borrelia hermsiiMOLECULAR MICROBIOLOGY, Issue 3 2004Jianmin Zhong Summary The known genome sequence of Borrelia burgdorferi, an agent of Lyme borreliosis, was used to study the genetic content and gene expression in B. hermsii, another spirochete pathogen and a cause of relapsing fever. Cross-species hybridization of a DNA array representing 1628 open reading frames (ORF) of B. burgdorferi with genomic DNA of B. hermsii indicated that the latter organism has at least 81% of the chromosomal genes and 43% of the plasmid genes of B. burgdorferi. We then carried out quantitative hybridization of the arrays with multiple replicates of cDNA produced from B. hermsii cells growing in the blood of infected mice or in culture medium that was adjusted to the same pH, temperature and a spirochete density as infected blood. Of 642 B. burgdorferi ORFs hybridized by all replicates under both conditions, 12 (1.9%) demonstrated differential expression by a regularized t -test and stringent criteria. BBP07 and BBG30, two plasmid-borne ORFs with the greatest measurable difference in expression between in vivo and in vitro conditions, putatively encode proteins of unknown function. Orthologues of BBP07 in B. hermsii were identified, and increased expression in infected mice was demonstrated by quantitative reverse-transcriptase polymerase chain reaction. [source] Overexpression of EgROP1, a Eucalyptus vascular-expressed Rac-like small GTPase, affects secondary xylem formation in Arabidopsis thalianaNEW PHYTOLOGIST, Issue 4 2009Camille Foucart Summary ,,To better understand the genetic control of secondary xylem formation in trees we analysed genes expressed during Eucalyptus xylem development. ,Using eucalyptus xylem cDNA libraries, we identified EgROP1, a member of the plant ROP family of Rho-like GTPases. These signalling proteins are central regulators of many important processes in plants, but information on their role in xylogenesis is scarce. ,,Quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) confirmed that EgROP1 was preferentially expressed in the cambial zone and differentiating xylem in eucalyptus. Genetic mapping performed in a eucalyptus breeding population established a link between EgROP1 sequence polymorphisms and quantitative trait loci (QTLs) related to lignin profiles and fibre morphology. Overexpression of various forms of EgROP1 in Arabidopsis thaliana altered anisotropic cell growth in transgenic leaves, but most importantly affected vessel element and fibre growth in secondary xylem. Patches of fibre-like cells in the secondary xylem of transgenic plants showed changes in secondary cell wall thickness, lignin and xylan composition. ,,These results suggest a role for EgROP1 in fibre cell morphology and secondary cell wall formation making it a good candidate gene for marker-based selection of eucalyptus trees. [source] Blood,brain barrier impairment with enhanced SP, NK-1R, GFAP and Claudin-5 expressions in experimental cerebral toxocariasisPARASITE IMMUNOLOGY, Issue 10 2008C.-W. LIAO SUMMARY Infection by Toxocara canis in humans may cause cerebral toxocariasis (CT). Appreciable numbers of T. canis larvae cross the blood,brain barrier (BBB) to invade the brain thus causing CT. In the present studies, we evaluated the BBB permeability and BBB injury as assessed by the cerebral Evans blue (EB) concentration as well as by pathological changes and glial fibrillary acidic protein (GFAP) expression in T. canis -infected mice monitored from 3 days (dpi) to 8 weeks post-infection (wpi). The vasodilation neuropeptides, the expressions of substance P (SP) and its preferred binding neurokinin-1 receptor (NK-1R) as well as claudin-5 of tight-junction proteins associated with BBB impairment were also assessed by Western blotting and reverse-transcriptase polymerase chain reaction. Results revealed that BBB permeability increased as evidenced by a significantly elevated EB concentration in brains of infected mice. BBB injury appeared due to enhanced GFAP protein and mRNA expressions from 4 to 8 wpi. Leukocytes might have been unrelated to BBB impairment because there was no inflammatory cell infiltration despite T. canis larvae having invaded the brain; whereas markedly elevated SP protein and NK-1R mRNA expressions concomitant with enhanced claudin-5 expression seemed to be associated with persistent BBB impairment in this experimental CT model. [source] Research note: Characterization of a cDNA encoding glutamine synthetase II from Gelidium crinale (Rhodophyta)PHYCOLOGICAL RESEARCH, Issue 1 2002D. Wilson Freshwater SUMMARY A cDNA encoding glutamine synthetase (GS) was characterized from the red alga, Gelidium crinale (Turner) Gaillon, using reverse-transcriptase polymerase chain reaction and the 5,- and 3,-rapid amplification of cDNA ends. Sequence analysis of a 1231-bp GS cDNA transcript included both 5, and 3, untranslated regions and a 1056-bp open reading frame encoding a 352 amino acid polypeptide. Comparison with GS sequences from other organisms revealed that the G. crinale cDNA encodes a type-II GS, and the absence of a N-terminal plastid signal sequence suggests that it is a cytosolic isoenzyme. Phylogenetic analyses of GSII amino acid sequences supports the multiple origin of cytosolic and plastid isoenzymes during eukaryotic evolution. [source] Functional characterization of AP3, SOC1 and WUS homologues from citrus (Citrus sinensis)PHYSIOLOGIA PLANTARUM, Issue 3 2007Fui-Ching Tan Flowering and flower formation are defining features of angiosperms and the control of these developmental processes involves a common repertoire of genes which are shared among different species of flowering plants. These genes were first identified using various homeotic and flowering time mutants of Arabidopsis and snapdragon, and homologous genes have subsequently been isolated from a wide range of different plant species based on the conservation of protein sequence and function. Using degenerate reverse-transcriptase polymerase chain reaction, we have isolated one APETALA3 -like (CitMADS8) and two SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1)-like (CsSL1 and CsSL2) homologues from sweet orange (Citrus sinensis L.). Although the translated amino acid sequence of CitMADS8 shares many similarities with other higher plant APETALA3 proteins, CitMADS8 fails to complement the floral organ identity defects of the Arabidopsis ap3-3 mutant. By contrast, the two citrus SOC1 -like genes, particularly CsSL1, are able to shorten the time taken to flower in the Arabidopsis wild-type ecotypes Columbia and C24, and functionally complement the late flowering phenotype of the soc1 mutant, essentially performing the endogenous function of Arabidopsis SOC1. Once flowering has commenced, interactions between specific flowering genes and a gene required for meristem maintenance, WUSCHEL, ensure that the Arabidopsis flower is a determinate structure with four whorls. We have isolated a citrus WUSCHEL homologue (CsWUS) that is capable of restoring most of the meristem function to the shoots and flowers of the Arabidopsis wus-1 mutant, implying that CsWUS is the functional equivalent of Arabidopsis WUSCHEL. [source] Temporal and spatial regulation of ,6 integrin expression during the development of the cochlear-vestibular ganglionTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 5 2007Dawn Davies Abstract The neurons of the cochlear-vestibular ganglion (CVG) that innervate the sensory hair cells of the inner ear are derived from the otic epithelium early in development. Neuroblasts detach from neighboring cells, migrate into the mesenchyme where they coalesce to form the ganglion complex, then send processes back into the epithelium. Cell migration and neuronal process formation involve changes in cellular interactions with other cells and proteins in the extracellular matrix that are orchestrated by cell surface-expressed adhesion molecules, including the integrins. I studied the expression pattern of the ,6 integrin subunit during the early development of the CVG using immunohistochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR) in murine tissue sections, otocyst, and ganglion explants. At embryonic day (E)10.5 ,6 integrin was expressed in the otic epithelium but not in migrating neuroblasts. Importantly, the loss of ,6 was associated with exit from the epithelium, not neuronal determination, revealing differentiation cues acutely associated with the cellular environment. Markers of glial and neuronal phenotype showed that ,6-expressing cells present in the CVG at this stage were glia of neural crest origin. By E12.5 ,6 expression in the ganglion increased alongside the elaboration of neuronal processes. Immunohistochemistry applied to otocyst cultures in the absence of glia revealed that neuronal processes remained ,6-negative at this developmental stage and confirmed that ,6 was expressed by closely apposed glia. The spatiotemporal modulation of ,6 expression suggests changing roles for this integrin during the early development of inner ear innervation. J. Comp. Neurol. 502:673,682, 2007. © 2007 Wiley-Liss, Inc. [source] Two Subgroups of Stapes Fixation: Otosclerosis and Pseudo-Otosclerosis,THE LARYNGOSCOPE, Issue 11 2005Tamás Karosi MD Abstract Hypothesis: Stapes ankylosis is a disease with variable histopathology and can be caused by otosclerosis or pseudo-otosclerosis. Viral pathogenesis of otosclerosis could be established only by correlative analysis: histologic examination of the stapes footplate and reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of the viral RNA. Background: Presence of the RNA genome of measles virus was demonstrated in the footplates of clinically otosclerotic patients by RT-PCR, and also viral proteins were detected by immunohistochemistry. Methods: Nucleic acids were extracted from ankylotic stapes footplates of clinically stapes fixation patients (n = 104). Measles virus genomic nucleoprotein (NP) RNA was amplified by seminested RT-PCR. Amplification results were correlated to postoperative histologic and audiologic findings. Results: Measles virus RNA was detectable only in histologically otosclerotic stapes footplates (n = 67). Histology for virus negative footplates (n = 37) excluded otosclerosis. Virus negative stapes footplates showed nonotosclerotic, degenerative disorders. Conclusions: Stapes ankylosis is a heterogeneous disease causing conductive hearing loss with different etiologies. Nonotosclerotic stapes fixations could be established as pseudo-otosclerosis and may belong to nonspecific, degenerative disorders with variable and noncharacteristic histopathology. Otosclerosis is an inflammatory disease caused by persisting measles virus infection of the otic cap-sule. [source] FGF17 is an autocrine prostatic epithelial growth factor and is upregulated in benign prostatic hyperplasiaTHE PROSTATE, Issue 1 2004Nathaniel Polnaszek Abstract BACKGROUND Fibroblast growth factors (FGFs) are known to play an important role in the growth of prostatic epithelial cells. Benign prostatic hyperplasia (BPH) is characterized by increased epithelial and stromal proliferation within the transition zone of the prostate. FGF2, FGF7, and FGF9 are expressed in BPH tissue but expression of FGF17 has not been previously characterized in human prostate tissue. METHODS Expression of FGF17 in human prostate tissue and primary cultures of prostatic epithelial and stromal cells was determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Growth response to FGF17 was assessed by addition of recombinant FGF17 to immortalized normal and neoplastic epithelial cell lines and primary cultures of prostatic stromal cells in the presence of insulin. Quantitative analysis of expression of FGF17 relative to keratin 18 and/or ,-actin in normal and hyperplastic prostate and prostate carcinoma was carried out by real-time quantitative RT-PCR. RESULTS FGF17 is expressed by prostatic epithelial cells and can act as an autocrine growth factor for immortalized and neoplastic prostatic epithelial cells. It can also promote stromal proliferation, although only at higher concentrations. Expression of FGF17 per epithelial cell was increased 2-fold in BPH. CONCLUSIONS FGF17 is expressed by normal, hyperplastic, and neoplastic prostatic epithelial cells and can promote epithelial proliferation in an autocrine manner. FGF17 expression is increased 2-fold in BPH and may contribute to the increased epithelial proliferation seen in this disease. © 2004 Wiley-Liss, Inc. [source] Immunohistolocalization and Gene Expression of the Carbonic Anhydrase Isoenzymes (CA-II and CA-VI) in Glands Associated with the Canine Lacrimal ApparatusANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 1 2010Y. Sugiura Summary Cytosolic and secretory carbonic anhydrase isoenzymes (CA-II and CA-VI, respectively) were detected by immunohistolocalization using specific canine CA-II and CA-VI antisera. CA-II and CA-VI were identified in glands associated with the canine lacrimal apparatus, such as lacrimal gland, superficial gland of the third eyelid (third eyelid gland) and tarsal gland. CA-II and CA-VI mRNA signals were also detected by reverse-transcriptase polymerase chain reaction in the same tissues. Some serous acinar cells and duct segments in the lacrimal gland and serous acinar cells in the third eyelid gland were immunopositive for anti-CA-II and CA-VI antisera. In particular, some immunopositive acini to CA-II and CA-VI on the edge of the third eyelid gland are histologically similar to sebaceous gland cells. Sebaceous gland cells in the tarsal and ciliary glands also showed immunopositivity to both CA antisera. CA-II and CA-VI gene transcripts were detected in the same regions. These results suggest that secreted CA-VI may form together with cytosolic CA-II, a high-activity isozyme mostly considered as a bicarbonate producer, in a mutually complementary system for the maintenance of bicarbonate levels to regulate pH in tear fluid and protect the corneal epithelia against injuries. In sebaceous gland cells in the lacrimal apparatus, CA-VI may be related to lipogenesis in an unknown function. [source] Immunohistolocalization and Gene Expression of the Secretory Carbonic Anhydrase Isozymes (CA-VI) in Canine Oral Mucosa, Salivary Glands and OesophagusANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 1 2007T. Kasuya Summary The immunohistolocalization of secretory carbonic anhydrase isoenzymes (CA-VI) in canine salivary glands, parotid, submandibular, sublingual and zygomatic glands, oral and oesophageal mucosa was studied using a specific antiserum against a canine CA-VI. In addition, the gene expression of CA-VI from the same tissue was studied using a real-time reverse-transcriptase polymerase chain reaction. In all salivary glands and oesophageal gland, immunostaining intensely localized CA-VI antiserum throughout the cytoplasm of serous acinar cells, including serous demilune and ductal epithelial cells. In contrast, no immunoreaction localized CA-VI in the mucous acinar cells of the gland. CA-VI gene transcripts were also detected in the same areas. The physiological significance of secretory CA-VI in the oral and oesophageal cavity is thought to play a highly specialized role in the maintenance of bicarbonate level in saliva and to protect mucosa from acid injury. It is shown that the major sites of the CA-VI secretion in dogs were in serous (demilune) secretory cells in all four major salivary glands and oesophageal glands in particular. [source] Association between parasite infection and immune responses in multiple sclerosisANNALS OF NEUROLOGY, Issue 2 2007Jorge Correale MD Objective To assess whether parasite infection is correlated with a reduced number of exacerbations and altered immune reactivity in multiple sclerosis (MS). Methods A prospective, double-cohort study was performed to assess the clinical course and radiological findings in 12 MS patients presenting associated eosinophilia. All patients presented parasitic infections with positive stool specimens. In all parasite-infected MS patients, the eosinophilia was not present during the 2 previous years. Eosinophil counts were monitored at 3- to 6-month intervals. When counts became elevated, patients were enrolled in the study. Interleukin (IL)-4, IL-10, IL-12, transforming growth factor (TGF)-,, and interferon-, production by myelin basic protein,specific peripheral blood mononuclear cells were studied using enzyme-linked immunospot (ELISPOT). FoxP3 and Smad7 expression were studied by reverse-transcriptase polymerase chain reaction. Results During a 4.6-year follow-up period, parasite-infected MS patients showed a significantly lower number of exacerbations, minimal variation in disability scores, as well as fewer magnetic resonance imaging changes when compared with uninfected MS patients. Furthermore, myelin basic protein,specific responses in peripheral blood showed a significant increase in IL-10 and TGF-, and a decrease in IL-12 and interferon-,,secreting cells in infected MS patients compared with noninfected patients. Myelin basic protein,specific T cells cloned from infected subjects were characterized by the absence of IL-2 and IL-4 production, but high IL-10 and/or TGF-, secretion, showing a cytokine profile similar to the T-cell subsets Tr1 and Th3. Moreover, cloning frequency of CD4+CD25+ FoxP3+ T cells was substantially increased in infected patients compared with uninfected MS subjects. Finally, Smad7 messenger RNA was not detected in T cells from infected MS patients secreting TGF-,. Interpretation Increased production of IL-10 and TGF-,, together with induction of CD25+CD4+ FoxP3+ T cells, suggests that regulatory T cells induced during parasite infections can alter the course of MS. Ann Neurol 2007 [source] Significance of micrometastases in pelvic lymph nodes detected by real-time reverse transcriptase polymerase chain reaction in patients with clinically localized prostate cancer undergoing radical prostatectomy after neoadjuvant hormonal therapyBJU INTERNATIONAL, Issue 2 2007Hideaki Miyake OBJECTIVE To clarify the significance of micrometastases in pelvic lymph nodes in patients treated by radical prostatectomy (RP) for prostate cancer after neoadjuvant hormonal therapy (NHT). PATIENTS AND METHODS The study included 52 patients with clinically localized prostate cancer who received NHT followed by RP. The expression of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) in 989 lymph nodes isolated from the 52 patients were assessed by a fully quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). We regarded specimens in which either PSA or PSMA mRNA were positive as showing the ,presence of micrometastasis'. Lymph node specimens were also stained immunohistochemically with an antibody against PSA. RESULTS Pathological examinations detected tumour cells in 11 lymph nodes from four patients, and real-time RT-PCR further identified micrometastasis in 40 lymph nodes from 19 patients with no pathological evidence of nodal involvement. The presence of micrometastatic cancer cells was confirmed by immunohistochemical staining in 19 lymph nodes from 11 patients with pathologically negative nodes. The presence of micrometastases was significantly associated with other conventional prognostic variables, including the pretreatment serum PSA level, biopsy Gleason score and surgical margin status. The biochemical recurrence-free survival rate in patients with no micrometastasis was significantly higher than that in those with micrometastasis. Furthermore, multivariate analysis identified the presence of micrometastasis as an independent factor predicting biochemical recurrence. CONCLUSIONS Although residual foci of atrophic prostate cancer cells in resected lymph nodes after NHT can be difficult to diagnose by routine pathological examination, the present results show the usefulness of quantitative real-time RT-PCR targeting PSA and PSMA genes for detecting micrometastatic tumour foci in pelvic lymph nodes from patients with localized prostate cancer treated by NHT followed by RP. Furthermore, the present findings suggest that micrometastases in pelvic lymph nodes might be, at least partly, important in the development of biochemical recurrence in some patients undergoing RP after NHT. [source] | ||||||||||||||||||||||