Quantitative Competitive RT-PCR Research Articles

Photoreceptor phagocytosis selectively activates PPARgamma expression in retinal pigment epithelial cells.

Phagocytosis of tips of rod outer segments (ROS) by retinal pigment epithelial (RPE) cells is vitally important for maintaining structural and functional integrity of the retina. We previously reported that receptor-mediated specific phagocytosis of ROS induces expression of early response genes coding for transcription factors. Here we study the expression of peroxisome proliferator-activated receptors (PPAR) -alpha, -delta (beta) and -gamma during ROS phagocytosis of rat RPE cells in primary cell culture, using competitive quantitative RT-PCR. During phagocytosis of ROS (but not of latex particles) by RPE cells, RT-PCR revealed a transient increase in PPARgamma mRNA expression, that peaked at 4-6 hr. We sequenced and described two alternatively spliced variants of rat PPARgamma: rPPARgamma1a and rPPARgamma1b. Both of these, along with the recently described rPPARgamma2 were induced by ROS phagocytosis. PPARalpha and PPARdelta mRNA expression was also detected in RPE cells, but the level of expression did not change during ROS phagocytosis. All-trans-retinoic acid and prostaglandin E(2) (PGE(2)) selectively potentiated both basal and ROS-phagocytosis-induced PPARgamma expression. All-trans-retinoic acid had the opposite inhibitory effect on PPARalpha and PPARdelta expression. Cycloheximide had a dual action on PPARgamma expression in RPE cells: it enhanced expression under basal conditions but repressed expression induced by ROS phagocytosis. It also stimulated expression of PPARalpha but had no effect on PPARdelta. Selective activation of PPARgamma may play an important role in regulating the expression of target genes that are involved in lipid and fatty acid metabolism in the photoreceptor renewal process.

Isolation and analysis of the 3'-untranslated regions of the human relaxin H1 and H2 genes.

The human has two relaxins, termed H1 and H2, both of which are biologically active and co-expressed in the decidua, placenta and prostate; in the corpus luteum, the main source of circulating relaxin, only the H2 form is expressed. The reasons for this differential expression of the relaxin genes are unknown. The possibility that their 3'-untranslated regions (UTRs) contribute to this differential expression by affecting their mRNA stabilities was investigated. Thus the 3'-UTRs of both relaxin genes were isolated through a combined 3'-rapid amplification of cDNA ends-PCR (RACE-PCR) using poly (A)(+)RNA from human decidua, placenta, prostate and corpus luteum. The sequences obtained for each 3'-UTR were identical in the tissues examined, were AT-rich (72%) and showed 91% homology between relaxin H1 and H2 when maximally aligned to include several gaps, the significance of which is unknown. Relaxin H1 has two, and relaxin H2 has one, poly (A)(+) signal, in addition to one cytoplasmic polyadenylation element 30 nucleotides upstream of this. The mRNA levels of relaxin H1 and H2 in the prostate adenocarcinoma LNCaP.FGC cell line were determined by quantitative competitive RT-PCR. Relaxin H1 had a 10-fold greater number of molecules (approximately 2.5x10(7)) per microgram of total RNA than relaxin H2 (approximately 2.5x10(6)). The stability of relaxin H1 and H2 mRNAs were compared in LNCaP cells treated with the transcription inhibitor actinomycin D (10 mM) for 0, 1, 2, 4, 8, 10, 14, or 24 h. Half-lives of 3.17 days for relaxin H1 mRNA and 11. 4 h for relaxin H2 mRNA were obtained from semi-logarithmic plots. Thus both mRNAs are relatively stable; however, relaxin H1 mRNA is considerably more stable than relaxin H2, at least in LNCaP cells. This difference in their mRNA stability may partly explain the greater level of expression of relaxin H1 in these cells.

Osteogenic protein-1 promotes the synthesis and retention of extracellular matrix within bovine articular cartilage and chondrocyte cultures

Objective We have used recombinant osteogenic protein-1 to investigate our hypothesis that proper repair and maintenance of cartilage requires not only enhanced biosynthesis and replenishment of the extracellular matrix but also the enhancement of components necessary for matrix retention.Design The effects of osteogenic protein-1 were examined on bovine articular cartilage slices as well as isolated chondrocytes grown in alginate beads. Cartilage slices were examined for accumulation of proteoglycan by incorporation of35S-sulfate and staining using Safranin O or, a biotinylated probe specific for hyaluronan. Bovine chondrocytes were characterized by use of a particle exclusion assay, in-situ hybridization, quantitative-competitive RT-PCR and a hyaluronan-binding assay.Results Osteogenic protein-1 treatment substantially enhanced the accumulation of hyaluronan and proteoglycan within cartilage tissue slices. As with the tissue, osteogenic protein-1 enhanced the size of cell-associated matrices assembled and retained by chondrocytes in vitro. This enhanced matrix assembly was paralleled by an increased expression of mRNA for aggrecan, hyaluronan synthase-2 and CD44. Of the two hyaluronan synthase genes expressed by chondrocytes, only hyaluronan synthase-2 was upregulated by osteogenic protein-1. Coupled with the increase in the CD44 mRNA was an increase in functional hyaluronan binding activity present at the chondrocyte cell surface.Conclusions These results demonstrate that osteogenic protein-1 stimulates not only the synthesis of the major cartilage extracellular matrix component aggrecan, but also two associated molecules necessary for the retention of aggrecan, namely hyaluronan and CD44.

Differential Regulation in Human Amnion Epithelial and Fibroblast Cells of Prostaglandin E2Production and Prostaglandin H Synthase-2 mRNA Expression by Dexamethasone but not Tumour Necrosis Factor-α

Previous studies have identified both pro-inflammatory cytokines and glucocorticoids as positive regulators of amnion prostaglandin (PG) biosynthesis. The stimulatory effects of dexamethasone (Dex), a glucocorticoid agonist, on prostaglandin endoperoxide H synthase (PGHS)-2 mRNA expression and PG biosynthesis in amnion have been attributed to an atypical response by the mesenchymal cells of the amnion. The objective of this study was to confirm previous findings concerning cell type-dependant Dex-induced upregulation of PGHS-2 mRNA expression and PG production using separated amnion cell populations, in comparison with the effects of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-α). Amnion cells from placentae delivered at term by caesarian section were isolated by tryptic digestion and epithelial cells were then separated from mesenchymal cells by differential absorption onto plastic. After 24–72h, the two cell populations were passaged and sub-cultured. Cells were treated with Dex (10−9–10−6m) or TNF-α (0.1–50ng/ml) or media alone. Thereafter, PGE2production was determined and PGHS-2 mRNA content analysed by a competitive quantitative RT-PCR method established and validated for this study. PGE2production in fibroblast-enriched cultures was increased to 310±41 per cent (mean±sem, n=4 wells per treatment point) of control in the presence of 10−8m Dex. Conversely, PGE2production in Dex-treated amnion epithelial cells was decreased to 67±24 per cent of control. Altered PGE2biosynthesis was accompanied by the upregulation of PGHS-2 mRNA in amnion fibroblasts but not in epithelial cells. TNF-α increased PG output and PGHS-2 expression independent of cell type. Glucocorticoids therefore appear to have opposing effects on PG biosynthesis in the two major cellular components of the human amnion.

Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of rare target RNA.

Reverse transcription polymerase chain reaction (RT-PCR) analysis is increasingly becoming part of the diagnostic and prognostic evaluation for hematologic and oncologic disorders. Currently, different RNA isolation methods are used in the diagnostic laboratories. No data are available on their suitability for sensitive detection of breakpoint cluster region-abelson (BCR-ABL) gene transcripts. We have extracted RNA from mononuclear cell (MNC) fractions and from lysed blood samples of 4 patients (1 with leukocytosis, 1 with chronic myelogeneous leukemia (CML) under interferon treatment, and 2 CML patients after bone marrow transplantation) with 3 RNA isolation reagents (TRIzol, RNAzol, FastTube reagent). RNA yield was slightly higher with RNAzol than with TRIzol as indicated by agarose gel electrophoresis and spectrophotometric measurement at 260 nm. The FastTube reagent was unsuitable for RNA isolation from MNC, and was not evaluated for lysed blood. Quantitative competitive RT-PCR amplification of the ABL gene showed comparable results for RNA isolated with RNAzol and TRIzol. In RNA samples extracted from lysed whole blood, the presence of amplifiable RNA/cDNA was confirmed by amplification of 4 selected reference genes (porphobilinogen deaminase (PBGD), ABL, the gene spanning the BCR on chromosome 22 and retinoic acid receptor alpha (RARA)) in a multiplex PCR. High quality, DNA-free RNA was obtained with RNAzol, and 1 BCR-ABL-positive (specific for translocation t [9; 221) cell among 2x10(4) normal cells was successfully detectable by single step RT-PCR. In RNA isolated with TRIzol, major contaminations with genomic DNA were observed which significantly impaired the interpretation of the results of RT-PCR analysis.

Age-Related Decline in Osteoprotegerin Expression by Human Bone Marrow Cells Cultured in Three-Dimensional Collagen Sponges

With advancing age, an increase in bone resorption relative to bone formation results in bone loss. Bone marrow stromal cells and their products support osteoclastogenesis from hematopoietic progenitors. Another of their products, osteoprotegerin (OPG), blocks the osteoclast–stimulatory effects of OPG ligand. We tested the hypothesis that with advancing age there is a decrease in OPG expression by human bone marrow cells. Bone marrow cells were obtained from 18 subjects (age range 38–84 years). Expression of mRNA transcripts of OPG was assessed by quantitative competitive RT-PCR. Median number of OPG transcripts in the younger group was 0.3 zetptomoles (range 0.01 to 1.30) and was higher than in the older group's median of 0.06 (range 0 to 0.5; p < 0.05). The decline in the expression of OPG with age may increase the capacity of stromal/osteoblast cells to support osteoclastogenesis.

Preoperative treatment with doxycycline reduces aortic wall expression and activation of matrix metalloproteinases in patients with abdominal aortic aneurysms

Purpose: Matrix metalloproteinases (MMPs) are considered to play a central role in the pathogenesis of abdominal aortic aneurysms (AAAs). Doxycycline (Dox) has direct MMP-inhibiting properties in vitro, and it effectively suppresses the development of elastase-induced AAAs in rodents. The purpose of this study was to determine if treatment with Dox suppresses AMPS within human aneurysm tissue and to elucidate the molecular mechanisms underlying this effect.Methods: Aneurysm tissues were obtained from 15 patients with an AAA, eight of whom had been treated with Dox before surgery (100 mg orally twice a day for 7 days). Protein extracts were examined by means of gelatin zymography and immunoblot analysis, and RNA was examined by means of reverse transcription-polymerase chain reaction (R-TPCR). The effects of Dox on MMP production were further examined in human THP1 mononuclear phagocytes in vitro.Results: No detectable difference was found between groups by using substrate zymography as a means of assessing total AMP activity, but Dox treatment was associated with a slight (24.4%) reduction in the activated fraction of 72-kDa gelatinase (MMP-2; P < .05). In.contrast, a 2.5-fold reduction in the amount of extractable 92-kDa gelatinase (MMP-9) protein in Dox-treated patients was revealed by means of immunoblot analysis (P < .05). Also, a 5.5-fold (81.9%) reduction in MMP-9 messenger RNA (mRNA) in Dox-treated patients was demonstrated by means of quantitative competitive RT-PCR (mean ± SE, mol MMP-9/mol β-actin: 1.3 ± 0.5 vs 7.2 ± 3.1; P < .04). There was no significant difference between groups in the relative expression of MMP-2 protein or mRNA. In cultured THP-1 monocytes stimulated with phorbol ester, the expression of MMP-9 protein and mRNA were both decreased after exposure to relevant concentrations of Dox in vitro.Conclusion: In addition to its recognized effects as a direct MMP antagonist, Dox may influence connective tissue degradation within human aneurysm tissue by reducing monocyte/macrophage expression of MMP-9 mRNA and by suppressing the post-translational processing (activation) of proMMP-2. Through this complementary combination of mechanisms, treatment with Dox may be a particularly effective strategy for achieving MAP inhibition in patients with an AAA.

Gene expression of neuronal nitric oxide synthase and adrenomedullin in human neuroblastoma using real-time PCR.

The objective of our study was to assess the gene expression of the antiproliferative systems neuronal nitric oxide synthase (nNOS) and adrenomedullin (AM) in human neuroblastoma. A novel real-time PCR method was evaluated using neuropeptide Y (NPY) for validation. Glyceraldehyd-3-phospate dehydrogenase (GAPDH) and NPY gene expression in neuroblastomas of 50 patients were measured in parallel by competitive quantitative and TaqMan real-time RT-PCR. AM and nNOS mRNA were determined by real-time PCR. Our results showed a linear relationship between competitive quantitative and real-time RT-PCR measurements of NPY and GAPDH (r = 0.87 and r = 0.92, respectively). AM and nNOS mRNA was found in all tumor samples. AM/GAPDH mRNA increased with higher differentiation according to Shimada (p = 0.013). There was no relation between MYCN amplification nor with the tumor stage (p = 0.78 and p = 0.30, respectively). AM/GAPDH did not relate to recurrence or death in a 5-year follow-up period. Neuronal NOS/GAPDH expression did not relate to any biological or clinical parameter of prognosis or differentiation. Similar results were obtained when the neuronal marker protein gene product 9.5 (PGP9.5) was used to normalize mRNA concentration. In conclusion, TaqMan real-time PCR appears to be a reliable method to quantify gene expression in neuroblastomas. Adrenomedullin mRNA in neuroblastoma is linked to tumor differentiation but not to prognostic markers.

Characterisation of Acute Murine Dextran Sodium Sulphate Colitis: Cytokine Profile and Dose Dependency

Background/Aims: In our experience with the acute murine dextran sodium sulphate (DSS) model of experimental colitis, we noted both interstrain and interanimal variations in daily water consumption. One might critically question whether observed differences in injuries are just a dose dependency phenomenon reflecting variations in DSS intake. To clarify this important topic, we performed a dose and concentration dependency study of DSS in Balb/c mice. We also determined Th1 and Th2 cytokine levels to compare the cytokine profile to that from inflammatory bowel disease (IBD). Methods: In four groups (14 animals each group) different concentrations of DSS (0, 2.5, 5 and 7.5%) were given for 7 days ad libitum. Mucosal injury of the entire colon was histologically assessed and graded. Cytokine levels were determined by competitive quantitative RT-PCR. Results: A linear increase in the crypt damage score was noted with increasing concentrations (0, 4.9 ± 0.7, 11.9 ± 0.5 and 18.9 ± 1.3, respectively), but the total dose of DSS intake did not correlate with mucosal damage. Progressive upregulation in the transcripts for Th1 cytokines (IL-12, IFN-γ, IL-1, TNF-α) was observed with increasing dosage of DSS. Interestingly, an increase in IL-10, but not IL-4 mRNA transcripts was also noted. Discussion: Acute DSS-induced mucosal injury is dependent on the administered DSS water concentration but not on the consumed DSS dose. The cytokine profile is a classic Th1 response and is similar to that of various inflammatory conditions in the colon. Conclusions: Minor variations in fluid consumption do not affect the severity of DSS-induced injury in mice. The acute murine DSS colitis model is useful for studying the pathophysiological aspects of colonic inflammatory diseases as IBD and for evaluating new potential therapeutic agents

Quantification of GABA A receptor subunit mRNAs by non-radioisotopic competitive RT-PCR utilizing plate-based EIA methodology

We developed a non-radioisotopic quantitative competitive RT-PCR method for the measurement of γ-aminobutyric acid (GABA) type A receptor subunit mRNA levels. The specificity of the method was optimized by the use of four subunit-specific oligonucleotides in the sequential steps: reverse transcription, polymerase chain reaction (PCR), and detection. The biotinylated PCR products were bound on streptavidin-coated microtiter plates allowing detection of the products using dinitrophenyl (DNP)-labeled probes and anti-DNP alkaline phosphatase conjugate. The method was set up for the six major cerebellar GABA A receptor subunits: α1; α6; β2; β3; γ2 and δ. The method is quantitative and rapid. With a large dynamic range from 10 fg to 1 ng of subunit mRNA, the accuracy was 12 and 19% (intra- and interassay coefficients of variation, respectively), which might be improved by using a smaller range of standards. The use of a double logarithmic standard curve [log (standard to competitor signal) vs. log (standard mRNA originally present)] requires only one reaction from each sample, allowing the analysis of a large number of samples in one experiment.

Lack of IRF-1 expression in acute promyelocytic leukemia and in a subset of acute myeloid leukemias with del(5)(q31).

One allele of interferon regulatory factor-1 (IRF-1), a transcriptional activator of genes critical for growth suppression, differentiation, and apoptosis, is usually deleted in acute myeloid leukemias (AML) and myelodysplasias (MDS) with deletion of chromosome 5q31. Accelerated exon skipping of IRF-1, resulting in transcripts lacking a translation initiation site, has been hypothesized as a means of functional inactivation of IRF-1 in AML/MDS. To test this hypothesis, we developed quantitative competitive RT-PCR assays to measure levels of full length and exon-skipped IRF-1 transcripts and measured IRF-1 proteins by Western blotting in a series of 45 samples of AML (13: -5/del5(q); 11: t(15;17); 7: t(8;21); and 7: inv(16)), normal blood and marrow, and myeloid cell lines. In contrast to AMLs with inv(16) or t(8;21), two AML samples with del(5q) had accelerated exon skipping and relatively low levels of full-length transcripts, while a third sample had very low transcript levels; IRF-1 proteins were not expressed and could not be induced by interferon gamma (IFNgamma). An additional six AML cases with -5/del(5q) had moderate exon-skipping and lacked constitutive IRF-1 proteins; however IRF-1 proteins were IFNgamma-inducible. Unexpectedly, all primary acute promyelocytic leukemia (APL) samples lacked IRF-1 protein and most exhibited accelerated exon skipping; furthermore, IRF-1 could not be induced by IFNgamma or all-trans retinoic acid (ATRA) which both induce IRF-1 in the NB4 APL cell line. Thus, accelerated exon skipping results in a loss of IRF-1 expression and function that cannot be overcome by exposure to inducing agents in a subset of AML patients with -5/del(5q) and in APL.

Regulation of transforming growth factor-beta1 expression by granulocyte macrophage-colony-stimulating factor in leiomyoma and myometrial smooth muscle cells.

Human myometrium and leiomyomas express granulocyte macrophage-colony-stimulating factor (GM-CSF), transforming growth factor-beta (TGFbeta), and their receptors. Overexpression of TGFbeta and, to a limited extent, GM-CSF has been associated with tissue fibrosis throughout the body, including leiomyomas. The objective of the present study was to determine the action of GM-CSF on leiomyoma and myometrial smooth muscle cells (LSMC and MSMC) and examine whether the action of GM-CSF is mediated through the induction of TGFbeta1 expression. Using competitive quantitative RT-PCR and enzyme-linked immunosorbent assay, we found that LSMC express significantly higher GM-CSF messenger ribonucleic acid (mRNA; 0.6 +/- 0.1 x 10(3) copies of mRNA/microg total RNA) and protein (0.75 +/- 0.2 ng/mL) than MSMC (0.5 +/- 0.1 x 10(2) copies of mRNA and 0.45 +/- 0.07 ng/mL protein; P < 0.05). In addition, LSMC expressed significantly higher TGFbeta1 mRNA (1.6 +/- 0.3 x 10(4) copies of mRNA/microg total RNA) than MSMC (2.4 +/- 0.4 x 10(3) copies) and synthesized and secreted more TGFbeta1 protein (1.7 +/- 0.2 vs. 0.5 +/- 0.02 ng/mL); whereas MSMC contained more cell-associated TGFbeta1 (56.2 +/- 1.2 ng/mL) than LSMC (35.2 +/- 1.2 ng/mL; P < 0.05). We found that GM-CSF (0.01-100 ng/mL) has limited mitogenic activity for LSMC but not for MSMC determined by the rate of [3H]thymidine incorporation and cell proliferation assay. However, GM-CSF at 1 ng/mL increased its own production, the expression of TGFbeta1 mRNA, the cell-associated TGFbeta1 protein content in both cell types, and TGFbeta1 released into the culture-conditioned medium of LSMC (P < 0.05). TGFbeta1 also increased its own mRNA and protein expression, but had no effect on cell-associated TGFbeta1 in both cell types (P < 0.05). Cotreatment of LSMC and MSMC with GM-CSF and TGFbeta1 induced changes similar to those produced by GM-CSF in both cells. In conclusion, our data suggest that GM-CSF is not a mitogen for MSMC and LSMC, but it regulates its own expression and the expression of TGFbeta1 by these cells, a regulatory interaction that may account for the GM-CSF-induced tissue fibrosis that occurs in leiomyomas.

Investigation of recombinant human insulin-like growth factor type I in thymus regeneration in the acute stage of experimental FIV infection in juvenile cats.

Thymus involvement and the development of thymic lesions in HIV-1 infection is hypothesized to suppress thymus function and limit T cell maturation and replenishment of the peripheral lymphoid pool. Therapeutic modulation to protect or enhance thymus function may therefore ameliorate peripheral lymphocytopenia and retard disease progression. Thymotrophic agents, such as insulin-like growth factor type I (IGF-I), may therefore represent adjunctive but important methods of treatment to protect or promote thymus function. The assessment of rhIGF-I in lentiviral infection and its impact on the thymus was performed using the feline immunodeficiency virus (FIV) model. Regeneration of the thymus in juvenile cats and amelioration of the thymic lesion after FIV infection was assessed by multiple measurements including thymic weight, stereologic analysis of the thymus cortex and medulla, histologic and immunohistologic analysis, quantitation of thymocyte and peripheral lymphocyte subsets, and quantitative competitive RT-PCR. Evidence of thymic cortical regeneration was observed in FIV-inoculated cats after 12 and 20 weeks of rhIGF-I treatment. Inflammation in the thymus was reduced during this period of treatment in this group of rhIGF-I/FIV-inoculated cats as evidenced by the reduced numbers of B cells detected. Viral replication rates in peripheral lymph nodes were not altered by rhIGF-I treatment and were decreased by 1 log in the thymus after 20 weeks of treatment. Peripheral blood CD4+ T cell counts also increased after 14 weeks of treatment. This suggests that rhIGF-I treatment can enhance thymus function and replenishment of the peripheral T cell pool.

Differential down-regulation of CD95 or CD95L in chronically HIV-infected cells of monocytic or lymphocytic origin: cellular studies and molecular analysis by quantitative competitive RT-PCR

We analysed the expression of CD95/CD95L in two widely used models for studying the cellular effects of chronic infection with human immunodeficiency virus type 1 (HIV-1), i.e. ACH-2 cells, derived from the lymphocytic cell line A301, and U1, derived from monocytic U937 cells. A301 and ACH-2 mounted the same amount of plasma membrane CD95, while U1 had a consistent decrease in CD95 expression. Using different antibodies, we failed to detect the plasma membrane form of its ligand, CD95L, but we could see the intracellular presence of that molecule in A301 cells and, to a lesser extent, in ACH-2 cells, but not in U937 or U1 cells. To confirm the cytofluorimetric data and quantify the expression of CD95L at the RNA level, we developed a quantitative competitive RT-PCR assay. The HUT78 cell line had about 50 000 copies mRNA/1000 cells, three times more after induction with a phorbol ester and ionomycin. ACH-2 expressed about 400- (basal) or 10- (induced) fold less CD95L mRNA than the parental cell line A301; U937 and U1 were below the limit of detection. In cells of lymphoid origin (ACH-2) chronic HIV infection inhibits the expression of CD95L, the phenomenon occurring at the transcriptional level. In cells of monocytic origin (U1) the infection decreases the plasma membrane expression of CD95. This suggests that HIV could trigger different anti-apoptotic strategies which likely depend upon the cell line which is infected. In monocytic cells which act as a viral reservoir, the expression of the molecule whose binding triggers apoptosis decreases, while in lymphoid cells, capable of exerting cytotoxicity, the expression of a molecule which induces apoptosis is reduced.

Regulation of Glucose Transporters by Estradiol in the Immature Rat Uterus

A series of metabolic changes within the immature rat uterus begins minutes after the administration of microgram quantities of estradiol (E2). One of the earliest effects that has been measured is an increase in the rate at which the uterus takes up glucose. To characterize the effect of E2 on glucose transport stimulation, whole protein preparations were examined for the presence of mammalian glucose transport proteins Glut1 through Glut5. E2-stimulated changes in the steady state levels of messenger RNA (mRNA) and protein were measured for Glut1 and Glut4 by quantitative competitive RT-PCR and Western blots. Both Glut1 mRNA and protein increased approximately 3- to 4-fold within 4–8 h. This increase in Glut1 mRNA and protein agrees with the maximal stimulation of the glucose transport rate that was observed. No translocation of either Glut1 or Glut4 was observed 2 h after E2 injection, indicating that translocation is not the mechanism responsible for the initial E2-stimulated increase in glucose transport observed in immature rat uterus. These data support the conclusion that the prolonged increase in glucose transport rate is due to either the transcriptional activation of Glut1 and/or the increased Glut1 mRNA half-life.

Regulation of glucose transporters by estradiol in the immature rat uterus.

A series of metabolic changes within the immature rat uterus begins minutes after the administration of microgram quantities of estradiol (E2). One of the earliest effects that has been measured is an increase in the rate at which the uterus takes up glucose. To characterize the effect of E2 on glucose transport stimulation, whole protein preparations were examined for the presence of mammalian glucose transport proteins Glut1 through Glut5. E2-stimulated changes in the steady state levels of messenger RNA (mRNA) and protein were measured for Glut1 and Glut4 by quantitative competitive RT-PCR and Western blots. Both Glut1 mRNA and protein increased approximately 3- to 4-fold within 4-8 h. This increase in Glut1 mRNA and protein agrees with the maximal stimulation of the glucose transport rate that was observed. No translocation of either Glut1 or Glut4 was observed 2 h after E2 injection, indicating that translocation is not the mechanism responsible for the initial E2-stimulated increase in glucose transport observed in immature rat uterus. These data support the conclusion that the prolonged increase in glucose transport rate is due to either the transcriptional activation of Glut1 and/or the increased Glut1 mRNA half-life.

Quantitative competitive reverse transcriptase-polymerase chain reaction for BCR-ABL on Philadelphia-negative leukaphereses allows the selection of low-contaminated peripheral blood progenitor cells for autografting in chronic myelogenous leukemia.

The Philadelphia (Ph) translocation t(9;22) results in the creation of the BCR-ABL gene, which is now regarded as central to the mechanism that underlies the chronic phase of chronic myelogenous leukemia (CML). From a clinical point of view, BCR-ABL mRNA detection has become the basis for the study of minimal residual disease in CML, particularly when a complete cytogenetic remission is achieved after interferon-alpha (IFN-alpha) therapy or allogeneic stem cell transplantation. We have recently demonstrated that it is possible to mobilize normal peripheral blood progenitor cells (PBPC) in higher rates if this procedure is performed during the early chronic phase. In an attempt to monitor the leukemic cell content of PBPC collections, we used quantitative-competitive RT-PCR (QC-RT-PCR). Thirty consecutive Philadelphia (Ph) chromosome positive patients were enrolled in this study. After chemotherapy and G-CSF, 14 patients achieved 100% Ph-negative metaphases, nine patients had < or =34% and seven patients >34% leukemic metaphases. A total of 116 collection samples were studied. For each sample, BCR-ABL transcript numbers and BCR-ABL/ABL ratio were evaluated. A highly significant correlation between Ph-positive metaphases and BCR-ABL transcript numbers (r = 0.84, P < 0.0001) or BCR-ABL/ABL ratio (r = 0.86, P < 0.0001) was found. For patients that underwent the procedure in early chronic phase, Ph-negative collections showed different levels of BCR-ABL expression. BCR-ABL transcript numbers varied from a median of 100/microg RNA in the first and second leukaphereses, to 500/microg RNA in the third and fourth leukaphereses, and 1500/microg RNA in the fifth leukapheresis (P = 0.002). BCR-ABL/ABL ratio values showed similar kinetics. We have also demonstrated that there is a correlation between low values in BCR-ABL/ABL ratio (< or =0.01) in the reinfused PBPC and the achievement of cytogenetic remission after autografting (chi2 test, P = 0.01). In conclusion, this study demonstrates that QC-RT-PCR for BCR-ABL is a reliable and helpful method for monitoring residual leukemic load in mobilized PBPC, particularly in Ph-negative collections. Moreover, QC-RT-PCR allows selection of the best available collections for reinfusion into patients after myeloablative therapy.

Comparative effects of interleukin-12 and interleukin-4 on cytokine responses by antigen-stimulated memory CD4+ T cells of cattle: IL-12 enhances IFN-gamma production, whereas IL-4 has marginal effects on cytokine expression.

Interleukin-12 (IL-12) and IL-4 are important immunoregulatory cytokines that determine the fate of naive T cells during antigen priming in mice and also influence cytokine synthesis by differentiated murine and human T cells. The roles of these cytokines in regulating the differentiation and effector function of bovine T cells are less well studied. We investigated the ability of human IL-12 and bovine IL-4 to modify cytokine expression by antigen-stimulated T cells from cattle immune to the protozoal parasites Babesia bovis and Babesia bigemina or reactive with Mycobacterium bovis purified protein derivative. Peripheral blood mononuclear cells (PBMC) were cultured with specific antigen and IL-4 or IL-12 for 1 week. Then viable lymphoblasts consisting of predominantly CD4+ T cells were restimulated with antigen and antigen-presenting cells (APC) with or without cytokine. Cell lines were cultured for several weeks, and following restimulation with antigen and APC in the absence of exogenous cytokine, the cell lines were analyzed for proliferation, interferon-gamma (IFN-gamma) production, and expression of IL-2, IL4-, IL-10, or IFN-gamma transcript levels using a quantitative competitive RT-PCR. IL-12 and IL-4 had no effect on the composition of CD4, CD8, or gammadelta T cells in the cell lines or on the level of antigen-induced proliferation. IL-12 stimulated enhanced levels of IFN-gamma protein and transcript expression in all cell lines, with no consistent effects on IL-2 or IL-4 expression. In two B. bovis-specific cell lines, IL-12 suppressed IL-10 expression. IL-4 had no consistent effect on expression of any cytokine. These results indicate the use of IL-12 as an adjuvant to enhance type 1 cytokine responses in cattle during antigen priming.

Alterations in RANTES Gene Expression and T-Cell Prevalence in Intestinal Mucosa during Pathogenic or Nonpathogenic Simian Immunodeficiency Virus Infection

RANTES, a β-chemokine, can suppress human immunodeficiency virus (HIV) as well as simian immunodeficiency virus (SIV) infections in T-lymphocyte cultures in vitro. However, the association of RANTES levels in peripheral blood with viral loads and disease outcome in HIV infection has been inconclusive. SIV-infected rhesus macaques were evaluated to determine whether RANTES gene expression correlated with suppression of viral infection in intestinal lymphoid tissues. Intestinal tissues were obtained from rhesus macaques infected with either pathogenic or nonpathogenic SIVmac variants at various stages of infection (primary acute, asymptomatic, and terminal). We examined the level of SIV infection (in situ hybridization), RANTES expression (quantitative competitive RT-PCR), and T-cell counts (immunohistochemistry). The most pronounced increase in RANTES gene expression in intestinal tissues was observed in primary SIV infection, which correlated with the pathogenicity of the infecting virus and not the tissue viral loads. Our results demonstrated that in contrast to the occurrence of viral suppression by RANTES in vitro, there was no direct correlation between high RANTES gene expression and suppression of viral loads in intestinal lymphoid tissues. Thus RANTES expression in the gut lymphoid tissue may not be a correlate for viral suppression. However, RANTES gene expression in primary SIV infection may be part of early host immune response to viral infection.

Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using a 'real time' quantitative RT-PCR assay.

Quantitative competitive RT-PCR techniques have been developed to detect BCR-ABL fusion transcripts in CML but they are hardly reproducible. In this work, we have developed BCR-ABL quantification by real time RT-PCR using the ABI PRISM 7700 (Perkin Elmer), a new technique which allows simple and rapid quantification of a target sequence during the extension phase of PCR amplifications. A fluorogenic probe labeled with both a reporter dye at the 5' end and a quencher-dye at the 3' end hybridizes to the target sequence on the third exon of the ABL gene. The exonuclease activity of the Taq DNA polymerase cleaves the probe and releases the reporter dye, resulting in an increase in the fluorescence signal. The absolute copy number of the target sequence (BCR-ABL) or a control gene (ABL) in an unknown sample can then be calculated using a calibration curve prepared from a set of BCR-ABL RNA standards, and results are expressed as a BCR-ABL/ABL ratio. In our hands, the sensitivity of a serial dilution of total RNA from a positive cell line (K562) in a negative cell line (HL60) was 10(-4). Fifteen CML patients in cytogenetic CR, including 11 allografted patients, two autografted patients and two patients treated by IFN were studied sequentially by this new real time quantitative RT-PCR technique in parallel with conventional qualitative two round nested RT-PCR. The two autografted patients showed high BCR-ABL/ABL ratio in all samples. The two patients treated by IFN showed a progressive decrease in the ratio. In the 11 allografted patients, four were sequentially studied 2 years or more after allo-BMT, and all ratios were below 10(-4). The four patients remained in clinical and cytogenetic CR. The seven other allografted patients were studied immediately after the procedure. Three of them showed a progressive decrease in the BCR-ABL/ABL ratio which reached 10(-4) 7 months after allo-BMT. The three patients remained in hematologic and cytogenetic CR. The remaining four allografted patients had progressive increase of BCR-ABL ratio; three developed cytogenetic relapse 9, 11, 28 months after allo-BMT, and the last patient remained in cytogenetic CR in the bone marrow but developed granulocytic sarcoma. Results of real-time quantitative RT-PCR were in agreement with those of qualitative two round nested PCR. However, evolution changes in the results of real-time quantitative RT-PCR often preceded those of the conventional technique: a decrease of the BCR-ABL/ABL ratio preceded progression from first round to second round positivity and then negativity with the classical technique; conversely, an increase in the ratio preceded evolution with the classical technique. Thus, real-time quantitative RT-PCR may show better correlation with clinical and cytogenetic evolution than conventional qualitative techniques and may help in making early therapeutic decisions in CML, especially after molecular relapse.