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PubMed2002

Real-time RT-PCR for quantitation of hepatitis C virus RNA.

A newly developed real-time RT-polymerase chain reaction assay for quantitation of hepatitis C virus (HCV) RNA in human plasma and serum was applied. A pair of primers and a probe (molecular beacon) were designed that are specific for the recognition of a highly conservative 5'-non-coding region (5'-NCR) in HCV genome. HCV real-time RT-PCR assay had a sensitivity of 1000 RNA copies per reaction, with a dynamic range of detection between 10(3) and 10(7) RNA copies. The coefficient variation of threshold cycle (Ct) values in intra- and inter-runs were less than 1.37 and 4.66%, respectively. The real-time RT-PCR assay on the HCV sero-positive samples yielded reproducible data, with less than 2.09% of the inter-assay variation. In order to determine its potential for clinical diagnosis, real-time RT-PCR was used to examine the HCV RNA levels in plasma from sero-positive and negative subjects, showing that the assay is highly sensitive and has specificity of 100%. It was demonstrated that the real-time RT-PCR was able to amplify HCV RNA in reference sera with seven genotypes (1A, 1B, 2B, 3A, 4, 5A and 6A) that include six major HCV genotypes circulated in the world. Since HCV is a major pathogen of post-transfusion and community-transmitted non-A, non-B hepatitis, this assay has a broad application for basic and clinical investigations.

5' Untranslated Regions↗
PubMed1997

Arthritis severity and spirochete burden are determined by serotype in the Borrelia turicatae-mouse model of Lyme disease.

Immunodeficient mice infected with Borrelia turicatae, a relapsing fever agent, have a disorder that resembles disseminated Lyme disease. Two serotypes, A and B, differed in their arthritogenicity in both CB-17 SCID and C3H SCID mice. In CB-17 SCID mice infected with serotype A or B, arthritis was assessed by measurement of tibiotarsal diameter, functional ability on a beam walk test, and microscopic assessment of joint inflammation. Serotype B-infected mice had greater joint swelling, functional disability, and leukocytic infiltration in the joints than serotype A-infected mice. Joint swelling and disability peaked at 2 weeks of infection and then decreased, while leukocyte infiltration in the joints persisted. To investigate the basis for the differences in arthritogenicity of serotypes A and B, spirochete burdens in infected mice were measured by quantitative PCR of spirochete DNA in joints, direct immunofluorescence of spirochetes in joints, and counts of spirochetes in the blood. At 2 weeks of infection there were seven times more spirochetes in the joints of serotype B-infected mice than in those of serotype A-infected mice, measured by both quantitative PCR and direct enumeration. Although serotypes A and B had the same infectivity and growth rate in vivo, serotype B spirochetes were eightfold more abundant in the blood than serotype A spirochetes and produced greater fatality in newborn mice. These findings indicate that differences in disease severity in mice infected with serotype A or B are attributable to differences in the spirochete burden in the joints and blood.

Animals↗
PubMed2005

Variation in pituitary expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and type-I and type-II activin receptors during the chicken ovulatory cycle.

Secretion of LH and FSH from the anterior pituitary is regulated primarily by hypothalamic GnRH and ovarian steroid hormones. More recent evidence indicates regulatory roles for certain members of the transforming growth factor beta (TGFbeta) superfamily including inhibin and activin. The aim of this study was to identify expression of mRNAs encoding key receptors and ligands of the inhibin/activin system in the hen pituitary gland and to monitor their expression throughout the 24-25-h ovulatory cycle. Hens maintained on long days (16 h light/8 h dark) were killed 20, 12, 6 and 2 h before predicted ovulation of a midsequence egg (n = 8 per group). Anterior pituitary glands were removed, RNA extracted and cDNA synthesized. Plasma concentrations of LH, FSH, progesterone and inhibin A were measured. Real-time quantitative PCR was used to quantify pituitary expression of mRNAs encoding betaglycan, activin receptor (ActR) subtypes (type I, IIA), GnRH receptor (GnRH-R), LH beta subunit, FSH beta subunit and GAPDH. Levels of mRNA for inhibin/activin betaA and betaB subunits, inhibin alpha subunit, follistatin and ActRIIB mRNA in pituitary were undetectable by quantitative PCR (<2 amol/reaction). Significant changes in expression (P<0.05) of ActRIIA and betaglycan mRNA were found, both peaking 6 h before ovulation just prior to the preovulatory LH surge and reaching a nadir 2 h before ovulation, just after the LH surge. There were no significant changes in expression of ActRI mRNA throughout the cycle although values were correlated with mRNA levels for both ActRIIA (r = 0.77; P<0.001) and beta-glycan (r = 0.45; P<0.01). Expression of GnRH-R mRNA was lowest 20 h before ovulation and highest (P<0.05) 6 h before ovulation; values were weakly correlated with betaglycan (r = 0.33; P = 0.06) and ActRIIA (r = 0.34; P = 0.06) mRNA levels. Expression of mRNAs encoding LH beta and FSH beta subunit were both lowest (P<0.05) after the LH surge, 2 h before ovulation. These results are consistent with an endocrine, but not a local intrapituitary, role of inhibin-related proteins in modulating gonadotroph function during the ovulatory cycle of the hen, potentially through interaction with betaglycan and ActRIIA. In contrast to mammals, intrapituitary expression of inhibin/activin subunits and follistatin appears to be extremely low or absent in the domestic fowl.

Activin Receptors↗
PubMed2005

Effect of granulocyte macrophage-colony stimulating factor on extracellular matrix deposition by dermal fibroblasts from patients with scleroderma.

OBJECTIVE: . To investigate the in vitro effect of granulocyte macrophage-colony stimulating factor (GM-CSF) on the deposition of extracellular matrix (ECM) in fibroblasts obtained from the skin of patients with systemic sclerosis (Ssc), compared to healthy controls. METHODS: Dermal fibroblasts obtained from 14 patients with SSc (7 with the diffuse form and 7 with CREST syndrome) and from 7 controls were studied. Both SSc and normal skin fibroblast cultures were stimulated for 4 and 8 days with 100 ng/ml GM-CFS. GM-CSF receptor (GM-CSFR) expression was determined by Western blot of cell lysates. Immunofluorescence was used to determine GM-CSFR expression and to investigate the deposition of ECM (type I collagen, fibronectin, and tenascin). Quantitative analysis of ECM was performed by ELISA. Expression of type I collagen and metalloproteinase 1 (MMP-1) mRNA was determined by real-time quantitative PCR. RESULTS: Deposition of ECM by normal fibroblasts appeared not to be influenced by stimulation with GM-CSF; in contrast, after stimulation with GM-CSF SSc fibroblasts showed increased deposition of fibronectin and tenascin, while type I collagen production was decreased; these results were found with both immunofluorescence and ELISA. Quantitative PCR revealed that GM-CSF inhibited the expression of mRNA type I collagen in SSc fibroblasts but not in normal fibroblasts, whereas levels of the main collagenolytic enzyme, MMP-1, were not affected. CONCLUSION: These results suggest that in SSc fibroblasts GM-CSF exerts a blocking effect on the deposition of type I collagen, through an inhibitory action on mRNA, while the production of other components of ECM such as fibronectin and tenascin is increased by stimulation with this cytokine.

Adult↗
PubMed2002

Experimental model of congenital toxoplasmosis in guinea-pigs: use of quantitative and qualitative PCR for the study of maternofetal transmission.

Maternofetal transmission of Toxoplasma gondii was assessed in pregnant guinea-pigs, with a gestational period of 65 +/- 5 days. A total of 56 female guinea pigs was infected by the intraperitoneal route (RH strain), by the oral or the intraperitoneal route (Prugniaud strain; PRU) or by the oral route (76K strain). Inoculation was performed 90 +/- 18 days or 30 +/- 9 days before the onset of gestation or 20 +/- 6 days or 40 +/- 6 days after. Gestational age was determined by a progesterone assay. Parasite loads (fetal brain and liver) were assessed by nested PCR and real-time PCR quantification on Light Cycler was performed with a SYBR Green I technique. The 76K strain appeared to be the most virulent in the model: the neonatal survival rate was 31%, in contrast to 53% and 68% for the PRU and RH strains, respectively. The percentage of survival of neonates for all strains taken together was lower after inoculation at 40 days' gestation (25%) than at 20 days' gestation (77%). Whatever the strain, maternofetal transmission determination was greater with nested PCR (54% for RH, 84% for PRU and 86% for 76K strains) than with real-time quantitative PCR (31% for RH, 66% for PRU and 76% for 76K strains). However, real-time quantitative PCR showed that neonatal parasite load was greater with the cystogenic strains (76K, PRU) and that high hepatic load (> 10000 parasites/g) was often associated with disease severity (11 of 12 cases). Therefore, this technique may provide an important element in understanding this congenital disease.

Animals↗
PubMedDate not supplied

Evaluation of new linkers and synthetic methods for internal modified oligonucleotides.

Several fluorescence resonance energy transfer (FRET) oligonucleotide probes were made with different internal linkages between the DNA and the quencher dye. In one example, a 5'-fluorescein beta-actin-based 26-mer DNA sequence was synthesized bearing an internal Tamra quencher. Two different versions were prepared using either conventional C5 [N-(6-aminohexyl)-3-acrylamido]pyrimidine-modified uridine and solution-phase Tamra active ester coupling or solid-phase addition of a Tamra amidite to a C5 [N-(6-hydroxyhexyl)-3-acrylamido]pyrimidine-modified uridine. The products were compared in functional assays. They performed very similarly both in a fluorescence-based melting point assay as well as in quantitative PCR. Another set of beta-actin probes were synthesized utilizing N4 [N-2-(ethylene glycol ethyl)-5-methyl]cytidine and solid-phase Tamra amidite addition at positions flanking those of the uridine. These versions gave lower T(m)s than either uridine-labeled probe and did not work as well in quantitative PCR. A control experiment using oligonucleotides with the same modified residues but without fluorophores attached revealed the same trend as the T(m) study of internal Tamra-labeled probes. Experimental details for the synthesis, purification, and testing are presented.

Base Sequence↗
PubMed2006

Hepatic ENPP1 expression is induced in diabetic rabbits.

The ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an inhibitor of the insulin receptor. Variants of ENPP1 are associated with infantile arterial calcification, obesity, and insulin resistance. To study the functional relevance of this protein in vivo, we cloned rabbit ENPP1 and studied its regulation in experimentally induced diabetes mellitus. We amplified and sequenced the complete coding sequence of rabbit ENPP1 gene out of a liver cDNA library using redundant primers deduced from other species. Next, we performed quantitative PCR of ENPP1 to study the tissue distribution of ENPP1 expression and its regulation in an alloxan-dependent diabetes model. The putative rabbit ENPP1 protein contains 873 amino acids and is highly conserved when compared with human ENPP1 (90% amino acid identity). Particularly high levels of ENPP1 mRNA expression were found in adipose tissue. Quantitative PCR analysis revealed a significant upregulation of ENPP1 transcription in liver (p = 0.025) and brain (p = 0.034) of diabetic rabbits compared with controls. Hepatic ENPP1 expression is induced in diabetic rabbits when compared with controls. This approximately twofold upregulation of ENPP1 mRNA in rabbit liver parallels previous findings in patients with type 2 diabetes mellitus. We provide further molecular information about ENPP1 as a potential pharmacologic target and characterize its regulation in an insulin-dependent diabetes mellitus animal model.

Amino Acid Sequence↗
PubMed2005

TGF-beta1, IL-1beta, and Th2 cytokines stimulate vascular endothelial growth factor production from conjunctival fibroblasts.

Giant papillary formation containing newly formed vessels is a major characteristic of severe allergic conjunctivitis, such as atopic keratoconjunctivitis (AKC) or vernal keratoconjunctivitis (VKC). We examined production of vascular endothelial growth factor (VEGF) from cultured conjunctival fibroblasts from normal volunteers under stimulation with type 1-, type 2-helper T cell derived and proinflammatory cytokines to investigate the mechanism of giant papillae formation in AKC/VKC. Primary cultured conjunctival fibroblasts were incubated with interleukin (IL)-4, IL-13, IL-1beta, IL-2, tumor necrotizing factor (TNF)-alpha, interferon (IFN)-gamma, or transforming growth factor (TGF)-beta1. Effects of cytokines on VEGF protein secretion in supernatant were assessed by ELISA, and VEGF mRNA expression in cultured cells were assessed by quantitative PCR. TGF-beta1 most effectively increased VEGF concentration with dose- and time-dependent manner IL-1beta, IL-4, and IL-13 significantly increased VEGF concentration. Though IL-2 also showed slight increase of VEGF concentration, it was not statistically significant. TNF-alpha and INF-gamma did not increase VEGF concentration. Quantitative PCR showed significant increase of VEGF mRNA in TGF-beta1, IL-1beta, and IL-4 stimulated fibroblasts. TGF-beta1, IL-1beta, and Th2 cytokines from allergic inflammatory cells induced VEGF production in conjunctival fibroblasts, and may play a crucial role in neovascularization and formation of giant papillae in AKC/VKC.

Cell Division↗
PubMed2003

Identification of epithelial cell adhesion molecule autoantibody in patients with ovarian cancer.

The epithelial cell adhesion molecule (Ep-CAM) exhibited an ovarian cancer:normal human ovarian surface epithelium ratio of 444. For validation studies, real-time quantitative PCR analysis and immunohistochemistry were performed in normal and malignant ovarian epithelial cell lines and tissues. To evaluate the potential of the Ep-CAM autoantibody as a tumor marker, we examined the amount of Ep-CAM autoantibody in serum samples obtained from ovarian cancer patients and normal controls by an ELISA. Real-time quantitative PCR analysis revealed significant overexpression of Ep-CAM mRNA in cancer cell lines (P < 0.001) and microdissected cancer tissues (P < 0.05), compared with that in cultured normal human ovarian surface epithelium and microdissected germinal epithelium, respectively. Immunolocalization of the Ep-CAM autoantibody showed that the sera of ovarian cancer patients expressed higher levels of Ep-CAM autoantibody than benign tumor patients and normal controls (P < 0.05). The levels of Ep-CAM autoantibody found were as follows: 0.132 in 52 patients with ovarian cancer, 0.098 in 26 cases with benign gynecologic disease, and 0.090 in 26 normal women. This investigation has shown that the Ep-CAM autoantibody was found to be associated with ovarian cancer and suggested that future research assessing its clinical usefulness would be worthwhile.

Antigens, Neoplasm↗
PubMed2006

Techniques in gerontology: cell lines as standards for telomere length and telomerase activity assessment.

The length of telomeres is believed to critically influence cellular aging processes and disease development. In order to reliably monitor telomere length and the corresponding cellular telomerase activity by optimized procedures, either based on flow cytometry or quantitative PCR technique, we here propose three commonly used cell lines, HEK293, K562 and TCL1301 as standards. In this contribution, efficient methods to determine mean telomere length of eukaryotic chromosomal DNA and determination of the corresponding telomeras activity are outlined. In particular, wide-range standard curves for a precise assessment of telomere length of genomic DNA by quantitative PCR technique are presented, measures, which greatly simplify the evaluation of respective functional roles of telomeres when studying biological processes such as disease progression and aging.

Cell Line↗
PubMed2004

Expression of therapeutic levels of factor VIII in hemophilia A mice using a novel adeno/adeno-associated hybrid virus.

We have generated an E1a/E1b/E3-deleted adeno/adeno-associated (Ad/AAV) hybrid virus driven by a small nuclear RNA (pHU1-1) promoter for expression of a B domain-deleted (Thr761-Asn1639) factor VIII transgene (FVIIIDelta761-1639). Productive replication of Ad/AAV/FVIIIDelta761-1639 in AAV rep-expressing cells resulted in generation of monomeric and dimeric mini-adenoviral (mAd) replicative forms that retained the AAV integration elements (mAd/FVIIIDelta761-1639). In vitro studies using Ad/AAV/FVIIIDelta761-1639 generated approximately 2-logs greater FVIII activity than mAd/FVIIIDelta761-1639. To determine its capacity for in vivo excision and/or genomic integration, Ad/AAV/FVIIIDelta761-1639 was injected by tail vein into three groups of hemophilia A mice (2 x 10(11) vp [n = 3]; 4 x 10(11) vp [n = 3]; 8 x 10(11) vp [n = 3]), with clear concentration-dependent increase in FVIII activity (range 160-510 mU/ml; plasma activity 16%-51% of normal). Peak activity was seen by Day (D) 5, with slow return to baseline by D28 (0.1-0.9% activity); in only 3/9 mice was loss of FVIII activity associated with development of anti-FVIII antibodies. Quantitative-PCR using genomic DNA isolated from D28 liver, spleen, heart, lungs, and kidney demonstrated the highest concentration in liver (approximately 10 genomes/cell), with little to no organ toxicity at early (D5 or 6) or late (D28) post-infusion time points. There was no evidence for spontaneous transgene excision or genomic integration in vivo as evaluated by quantitative PCR and genomic blotting. These data establish (i) the feasibility and applicability of developing high-titer Ad/AAV hybrid viruses for FVIII delivery using a small cellular promoter, (ii) the potential utility of this virus for generation of "gutted" monomeric and dimeric mAD/FVIII retaining AAV integration elements, and (iii) that the development of strategies for regulated Rep68/78 co-expression may provide a novel approach for excision, integration, and long-term FVIII transgene expression.

Adenoviridae↗
PubMed2000

Conditions that enable human hematopoietic stem cell engraftment in all NOD-SCID mice.

BACKGROUND: Transplantation of human hematopoietic stem cells is the only true test of their long-term repopulation potential. Models are readily available to evaluate murine hematopoietic stem cells, but few exist that allow reliable quantification of human stem cells. The non-obese diabetic-severe combined immunodeficient (NOD-SCID) mouse model enables quantification of human hematopoietic stem cells, but the conditions that permit human engraftment in all animals have yet to be defined. The aims of the project were, therefore, to describe the variables that allow human engraftment in the NOD-SCID mouse model and the techniques that accurately quantify this engraftment. METHODS: NOD-SCID mice that had or had not received 250, 325, or 400 cGy irradiation received cord blood (CB) mononuclear or CD34+ cells i.v. or i.p. Mice were killed 6 weeks after transplantation, and the bone marrow, spleen, and thymus were harvested. Four-color flow cytometric analysis, semi-quantitative PCR, myeloid and erythroid progenitor, and stem cell assays were used to monitor human engraftment. RESULTS: A 250 or 325 cGy and i.v. injection of CB mononuclear or CD34+ cells is required to detect multilineage human engraftment in the bone marrow, spleen, or thymus of NOD-SCID mice. Four-color flow cytometric analysis and semi-quantitative PCR enable accurate detection of 0.1% human cells. Progenitor and stem cell assays provide functional information about the engrafted cells. CONCLUSIONS: Successful development of the NOD-SCID mouse model and techniques to assess human engraftment now allow it to be used reliably to analyze the effects of short-term cytokine exposure on the long-term repopulating capacity of CB stem cells.

Animals↗
PubMed2003

Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes.

BACKGROUND: Toxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid. METHODS: Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity. RESULTS: Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii. CONCLUSION: We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis.

Animals↗
PubMed2005

Selective down-regulation of sub-endocardial ryanodine receptor expression in a rabbit model of left ventricular dysfunction.

Defective sarcoplasmic reticulum (SR) Ca2+ handling is evident in cardiomyopathy and may be mediated by selective dysregulation of SR Ca2+ handling proteins. To assess whether regulation of SR Ca2+ release may vary regionally within the normal and diseased heart, left ventricular transmural expression and activity of the ryanodine receptor (RyR2) was studied in a rabbit coronary artery ligation model of left ventricular dysfunction (LVD). Tissue/cells were isolated from both the sub-endocardial and subepicardial layers of the left ventricular free wall from sham-operated and coronary artery ligated rabbit hearts. Three independent methods were used to study alterations in RyR2 mRNA (real-time quantitative PCR (RT-PCR)) and protein expression (quantitative immunoblotting and [3H] ryanodine binding). These biochemical data were compared with functional measurements of fractional SR Ca2+ release from both of these regions. Data from RT-PCR revealed lower RyR2 mRNA levels in the sub-endocardium compared with subepicardium in both experimental groups with the reduction being significantly lower in the sub-endocardium from the LVD group. Quantitative analysis of RyR2 protein levels revealed the same expression patterns. Calsequestrin mRNA and protein levels showed no significant changes. This study demonstrates a lower expression level of RyR2 in the sub-endocardium of the left ventricle of rabbit hearts and is the first to show a further specific reduction in LVD. There is a corresponding decrease in fractional SR Ca2+ release in cells isolated from the sub-endocardium of hearts from the LVD group, relating these selective biochemical alterations to changes in function.

Animals↗
PubMed2006

Detection of Borrelia burgdorferi DNA in tissues from dogs with presumptive Lyme borreliosis.

OBJECTIVE: To develop a quantitative PCR assay for detection of Borrelia burgdorferi DNA in formalin-fixed, paraffin-embedded tissues; compare results of this assay with results of immunohistochemical staining of tissues from seropositive dogs; and determine whether B burgdorferi DNA could be detected in renal tissues from dogs with presumptive Lyme nephritis. DESIGN: Cohort study. SAMPLE POPULATION: Archived tissue samples from 58 dogs. PROCEDURES: A quantitative PCR assay was performed on formalin-fixed, paraffin-embedded tissue sections from the dogs. Results were compared with results of immunohistochemical staining, B burgdorferi serostatus, clinical signs, and necropsy findings. RESULTS: 38 dogs were classified as having positive or equivocal results for Lyme borreliosis, and 20 were classified as having negative results on the basis of clinical signs, serologic findings, and pathologic abnormalities. Borrelia burgdorferi DNA was amplified from tissue samples from only 4 (7%) dogs, all of which had been classified as having positive or equivocal results for Lyme borreliosis and had signs of presumptive Lyme nephritis. Results of PCR assays of renal tissue were positive for only 1 dog, and there was no agreement between results of immunohistochemical staining (ie, detection of B burgdorferi antigen) and results of the PCR assay (ie, detection of B burgdorferi DNA) for renal tissues. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that detection of B burgdorferi DNA in formalin-fixed, paraffin-embedded tissues is feasible, but that intact B burgdorferi DNA is rarely found in tissues from naturally infected dogs, even tissues from dogs with presumptive Lyme borreliosis. Further, findings support the contention that Lyme nephritis may be a sterile, immune complex disease.

Animals↗
PubMedDate not supplied

Upregulation of the oncogenic helix-loop-helix protein Id2 in Ewing sarcoma.

AIMS AND BACKGROUND: Id helix-loop-helix proteins function as regulators of cell growth and differentiation. However, they can induce malignant transformation when overexpressed. The EWS/ETS chimeric proteins in Ewing sarcoma act as aberrant transcription factors leading to tumorigenic processes. An enhanced expression of the Id2 gene in Ewing sarcoma cells was previously shown by gene array techniques. We investigated the expression of Id2 at the protein and gene level in Ewing sarcoma. METHODS: We evaluated the expression of Id2 protein using immunohistochemistry in formalin-fixed, paraffin-embedded specimens from a total of 71 cases of Ewing sarcoma. Additionally, a Ewing sarcoma cell line was examined by real-time quantitative PCR. RESULTS: Id2 expression was observed in 65 cases (91.5%) of the 71 total cases examined and a high level of Id2 expression was observed in 45 of these cases (63.8%). In tumor cells, Id2 proteins displayed cytoplasmic as well as nuclear localization. The amplification of the Id2 gene was not noted in a Ewing sarcoma cell line using real-time quantitative PCR. The crossing points of Id2 in the Ewing sarcoma cell line, control fibroblast, and osteosarcoma cell line were 18.54 +/- 0.16, 18.25, and 18.34, respectively. CONCLUSIONS: Our data support a role for increased Id2 protein expression in Ewing sarcoma. However, this overexpression of the Id2 protein could not be confirmed by a corresponding change at the gene level in a Ewing sarcoma cell line.

Biomarkers, Tumor↗
PubMed2005

Intrapulmonary and intramyocardial gene transfer in rhesus monkeys (Macaca mulatta): safety and efficiency of HIV-1-derived lentiviral vectors for fetal gene delivery.

Fetal gene transfer was studied using intrapulmonary and intramyocardial transfer of SIN HIV-1-derived lentiviral vectors expressing EGFP in rhesus monkeys. Fetuses were monitored sonographically during gestation and tissue analyses performed at term or 3 months postnatal age. Animals remained healthy during the study period as evidenced by normal growth, development, hematology, clinical chemistry, echocardiography, and pulmonary function tests. Strong pulmonary fluorescence was observed postnatally after fetal intrapulmonary delivery of lenti-CMV, but not lenti-SP-C, and compared to nontransferred controls. High EGFP copy numbers were found by quantitative PCR with both vector constructs in lung lobes (<or=15%) and EGFP copies were also detected in the diaphragm, pericardium, and thorax. No differences were found in lung:body weight ratios, percentage lung parenchyma, or overall morphology when compared to controls. For intramyocardial gene delivery, strong transgene expression was found within the myocardium and pericardium, and high EGFP copy numbers were found by quantitative PCR (3-36%). EGFP was also detected in the aorta, thorax, and diaphragm. These studies indicate that postnatal heart and lung development and function were not altered after fetal intraorgan gene transfer and subsequent transgene expression prenatally and postnatally, and gene transfer was restricted to the thoracic cavity with intrapulmonary and intramyocardial lentiviral vector-mediated gene delivery.

Animals↗
PubMed2003

Gene expression analysis of atopic dermatitis-like skin lesions induced in NC/Nga mice by mite antigen stimulation under specific pathogen-free conditions.

BACKGROUND: Atopic dermatitis (AD) is a chronic relapsing inflammation characterized by pruritic and eczematous skin lesions usually observed in patients with a familial history of atopic diseases, but its exact etiology is unclear. An animal model is indispensable for the analysis of the pathogenesis and the development of new drugs to treat this disease. Here, we compare changes in gene expression profiles in the AD-like skin lesions of NC/Nga or BALB/c mice stimulated intradermally by mite antigen under specific pathogen-free (SPF) conditions. METHODS: Mite Extract-Dp was injected intradermally into the right and left pinnae and into the skin of the back of NC/Nga or BALB/c mice in 2 places once per 3 days, and the clinical symptoms and the ear thickness were measured. On day 14 or day 28 after starting mite extract injection, we collected plasma and pinnae from NC/Nga or BALB/c mice. The amount of total immunoglobulin E (IgE) in plasma was assayed. We analyzed mRNA transcripts in pinnae using real-time quantitative PCR for the murine counterparts of several known allergy-related genes. Moreover, genome-wide gene expression in pinnae from NC/Nga mice was analyzed using high-density oligonucleotide arrays (GeneChip, Affymetrix). RESULTS: From 2 weeks after stimulation, marked skin inflammation was induced in the pinnae of NC/Nga but not BALB/c mice. However, IgE levels in sera rose equally in both strains. Quantitative PCR analysis and comprehensive GeneChip analysis of the AD-like pinna skin lesions revealed that their development was accompanied by changes in expression of more than 1,000 genes. These included cytokines, cytokine receptors, proteases, and adhesion molecules. Furthermore, genes thus far not reported in association with AD were also affected. CONCLUSION: From these results, the NC/Nga mouse model using mite sensitization under SPF conditions could be useful for elucidating the mechanisms of AD pathogenesis and developing more effective therapy for AD.

Animals↗