Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transfer of therapeutic genes to human hematopoietic stem cells (HSCs) using complex vectors at clinically relevant efficiencies remains a major challenge. Recently we described a stable retroviral vector that sustains long-term expression of green fluorescent protein (GFP) and a human beta-globin gene in the erythroid progeny of transduced murine HSCs. We now report the efficient transduction of primitive human CD34(+) fetal liver or cord blood cells with this vector and expression of the beta-globin transgene in the erythroid progeny of these human cells for at least 2 months. After growth factor prestimulation and then a 2- to 3-day exposure to the virus, 35% to 55% GFP(+) progeny were seen in assays of transduced colony-forming cells, primitive erythroid precursors that generate large numbers of glycophorin A(+) cells in 3-week suspension cultures, and 6-week long-term culture-initiating cells. In immunodeficient mice injected with unselected infected cells, 5% to 15% of the human cells regenerated in the marrow (including the erythroid cells) were GFP(+) 3 and 6 weeks after transplantation. Importantly, the numbers of GFP(+) human lymphoid and either granulopoietic or erythroid cells in individual mice 6 weeks after transplantation were significantly correlated, indicative of the initial transduction of human multipotent cells with in vivo repopulating activity. Expression of the transduced beta-globin gene in human cells obtained directly from the mice or after their differentiation into erythroid cells in vitro was demonstrated by reverse transcriptase-polymerase chain reaction using specific primers. These experiments represent a significant step toward the realization of a gene therapy approach for human beta-globin gene disorders.
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PMID:Expression of a human beta-globin transgene in erythroid cells derived from retrovirally transduced transplantable human fetal liver and cord blood cells. 1214 6

Little is known about the clinical significance of interleukin (IL)-18, a novel immunoregulatory cytokine, in acute myeloid leukemia (AML). Using reverse transcriptase polymerase chain reaction (RT-PCR) analysis, levels of IL-18 mRNA were assessed in bone marrow mononuclear cells (BMMC) from 47 adult patients with de novo or CR AML in order to explore the clinical significance of IL-18. The relationship between expression levels and the established prognostic factors such as age, cytogenetic aberrations, CD34 expression and FAB subtypes was investigated. Either disease status, age or CD34 expression were found to significantly correlate with the expression of IL-18. With respect to FAB cytotypes, expression of IL-18 gene in M4/M5 (n=15) was statistically higher than that in other subtypes (n=32, P<0.001). Moreover, a significant difference in IL-18 gene expression was obtained between the high risk group and the intermediate risk group (0.5627 versus 0.3111, P=0.038). In addition, a relationship between IL-18 expression of BMMC and initial white blood cell (WBC) was clearly demonstrated by a statistical analysis (r=0.806, P<0.001). These observations suggest that IL-18 gene over-expression might reflect the convergence of several important unfavorable prognostic factors in AML.
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PMID:Clinical significance of IL-18 gene over-expression in AML. 1236 62

SHP-2 (Src homology phosphatase type-2) is essential for haematopoietic skeletal and vascular development. Thus the identification of its binding partners is critically important. In the present study, we describe a unique monoclonal antibody, WM78, which interacts with PZR, a SHP-2 binding partner. Furthermore, we identify two novel isoforms of PZR, PZRa and PZRb, derived by differential splicing from a single gene transcription unit on human chromosome 1q24. All are type 1 transmembrane glycoproteins with identical extracellular and transmembrane domains, but differ in their cytoplasmic tails. The PZR intracellular domain contains two SHP-2 binding immunoreceptor tyrosine-based inhibitory motifs (VIY(246)AQL and VVY(263)ADI) which are not present in PZRa and PZRb. Using the WM78 monoclonal antibody, which recognizes the common extracellular domain of the PZR isoforms, we demonstrate that the PZR molecules are expressed on mesenchymal and haematopoietic cells, being present on the majority of CD34(+)CD38(+) and early clonogenic progenitors, and at lower levels on CD34(+)CD38(-) cells and the hierarchically more primitive pre-colony forming units. Interestingly, we show by reverse transcriptase-PCR that the PZR isoforms are differentially expressed in haematopoietic, endothelial and mesenchymal cells. Both PZR and PZRb are present in CD133(+) precursors and endothelial cells, PZRb predominates in mesenchymal and committed myelomonocytic progenitor cells, and all three isoforms occur in erythroid precursor cell lines. Importantly, using SHP-2 mutant (Delta 46-110) and SHP-2 rescue of embryonic fibroblasts stably expressing the PZR isoforms, we demonstrate for the first time that PZR, but not PZRa or PZRb, facilitates fibronectin- dependent migration of cells expressing a competent SHP-2 molecule. These observations will be instrumental in determining the mechanisms whereby PZR isoforms regulate cell motility.
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PMID:Novel mesenchymal and haematopoietic cell isoforms of the SHP-2 docking receptor, PZR: identification, molecular cloning and effects on cell migration. 1241 Jun 37

High-dose chemotherapy combined with autologous stem cell support has improved response rates in high-risk and metastatic breast cancer, but has failed to improve long-term survival. Breast cancer has a tendency to metastasize to the bone marrow, and live tumor cells are known to circulate in the peripheral blood of breast cancer patients. Sensitive immunohistochemical, culture-based, and reverse transcriptase polymerase chain reaction (RT-PCR)-based methods have shown that about 50% of histologically normal stem cell grafts from breast cancer patients are contaminated with occult tumor cells, which may cause or contribute to tumor recurrences. Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) inactivates a wide range of leukemia and lymphoma cells and is well tolerated by normal hematopoietic stem and progenitor cells. Unfortunately, most solid tumor cells (including breast cancer cells) are only moderately sensitive or refractory to MC540-PDT. We report here that if MC540-PDT is followed by a 1-h incubation with the alkyl-lysophospholipid, Edelfosine (ET-18-OCH(3)), the depletion of murine and human breast cancer cells is greatly enhanced whereas the recovery of normal hematopoietic stem and progenitor cells is only minimally degraded. When used under conditions that reduce CD34-positive human bone marrow cells only 5.1-fold, and murine and human granulocyte/macrophage progenitors 6.8- and 3-fold, respectively, combination purging with MC540-PDT and Edelfosine depletes murine (Mm5MT) and human (MDA-MB-435S) breast cancer cells >17,000- and >125,000-fold, respectively. These data suggest that combination purging with MC540-PDT and Edelfosine may offer a simple, safe and effective method for the ex vivo purging of autologous stem cell grafts from breast cancer patients.
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PMID:Anti-tumor effect of Merocyanine 540-mediated photochemotherapy combined with Edelfosine: potential implications for the ex vivo purging of hematopoietic stem cell grafts from breast cancer patients. 1246 4

Adult marrow-derived cells have been shown to contribute to various nonhematologic tissues and, conversely, primitive cells isolated from nonhematopoietic tissues have been shown to reconstitute hematopoiesis. Circulating endothelial progenitor cells (EPCs) have been reported to be at least partially donor derived after allogeneic bone marrow transplantation, and shown to contribute to neovascularization in murine ischemia models. However, it is unknown whether these EPCs are actually clonally derived from the same population of stem and progenitor cells that reconstitute hematopoiesis, or from another cell population found in the marrow or mobilized blood that is transferred during transplantation. To approach this question, we characterized circulating EPCs and also endothelial cells from large vessels harvested at autopsy from rhesus macaques previously transplanted with retrovirally transduced autologous CD34-enriched peripheral blood stem cells (PBSCs). Endothelial cells were grown in culture for 21-28 days and were characterized as CD31(+) CD14(-) via flow cytometry, as acLDL(+) UEA-1(+) via immunohistochemistry, and as Flk-1(+) by reverse transcriptase-polymerase chain reaction (RT-PCR). Animals had stable vector marking in hematopoietic lineages of 2-15%. Neither cultured circulating EPCs collected in steady state (n = 3), nor endothelial cells grown from large vessels (n = 2), had detectable retroviral marking. EPCs were CD34(+) and could be mobilized into the circulation with granulocyte colony-stimulating factor. Under ex vivo culture conditions, in which CD34(+) cells were optimized to transduce hematopoietic progenitor and stem cells, there was a marked depletion of EPCs. Transduction of EPCs was much more efficient under conditions supporting endothelial cell growth. Further elucidation of the origin and in vivo behavior of EPCs may be possible, using optimized transduction conditions and a vascular injury model.
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PMID:Analysis of origin and optimization of expansion and transduction of circulating peripheral blood endothelial progenitor cells in the rhesus macaque model. 1248 99

Vascular endothelial growth factor (VEGF) is a glycoprotein that plays an important role in neovascularization and increases vascular permeability. We reported that VEGF is involved in motion pain of patients with rotator cuff disease by causing synovial proliferation in the subacromial bursa (SAB). The present study investigates whether VEGF is also involved in the development of shoulder contracture in diabetics with rotator cuff disease. We examined 67 patients with rotator cuff disease, including 36 with complete cuff tears, 20 with incomplete tears, and 11 without apparent tears (subacromial bursitis). The patients were into groups according to the presence or absence of diabetes (14 type II diabetics and 53 non-diabetics). Specimens of the synovium of the SAB were obtained from all patients during surgery. Expression of the VEGF gene in the synovium of the subacromial bursa was evaluated by using the reverse transcriptase polymerase chain reaction. The VEGF protein was localized by immunohistochemistry, and the number of vessels was evaluated based on CD34 immunoreactivity. The results showed that VEGF mRNA was expressed in significantly more diabetics (100%, 14/14) than in non-diabetics (70%, 37/53) (P=0.0159, Fisher's test). Investigation of VEGF isoform expression revealed VEGF121 in all 14 diabetics and in 37 of the 53 non-diabetics, VEGF165 in 12 of the 14 diabetics and in 21 of the 53 non-diabetics, and VEGF189 in 1 of the 14 diabetics and in 2 of the 53 non-diabetics. No VEGF206 was expressed in either group. VEGF protein was localized in both vascular endothelial cells and synovial lining cells. The mean number of VEGF-positive vessels and the vessel area were also significantly greater in the diabetics (p<0.015, Mann-Whitney U test). Synovial proliferation and shoulder joint contracture were more common in the diabetics (P=0.0329 and P=0.073, respectively; Fisher's test). The mean preoperative range of shoulder motion significantly differed in terms of elevation between two groups: 103.8 degrees in diabetics and 124.9 degrees in no diabetics (p=0.0039 Mann-Whitney U test). In contrast, external rotation did not significantly differ: 44 degrees in diabetics and 49 degrees in non-diabetics (p=0.4957, Mann-Whitney U test). These results suggest that VEGF121 and VEGF165 expression in the SAB is responsible for the development of shoulder joint contracture, especially in elevation, among type II diabetic patients with rotator cuff disease.
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PMID:Vascular endothelial growth factor 121 and 165 in the subacromial bursa are involved in shoulder joint contracture in type II diabetics with rotator cuff disease. 1455 30

The intracoelomic route for in utero hematopoietic stem cell transplantation has been evaluated in pre-immune fetal sheep and the engraftment characteristics defined. Twelve ovine fetuses (gestational ages: 40-45 days) received intracoelomic transplants of human CD3-depleted (50 x 10(6) per lamb) or CD34-selected (1-2 x 10(5) per lamb) cord blood hematopoietic stem cells. Engraftment was evaluated from cell suspension of the liver, spleen, bone marrow and thymus by flow cytometry, cloning assays and polymerase chain reaction (PCR) analysis for human beta(2)-microglobulin gene. The engraftment of liver samples was also evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescent in situ hybridization (FISH) and immunohistochemistry. Four fetuses (33%) aborted shortly after intracoelomic transplantation and were not evaluable for engraftment. Engraftment was detected in 4 fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells. The degree of engraftment in these 4 fetuses ranged from 6 to 22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry). Three fetuses obtained after cesarean section at 102 (No. 435184) and 105 (Nos 915293, 037568) days and 1 fetus delivered at term, which received CD34-selected cord blood cells, had human engraftment with 10, 32, 20 and 10% CD45+ cells in bone marrow, respectively. A further check of human chimerism was done at 1 year after birth of the fetus delivered at term and 7.6% of bone marrow chimerism was detected. In 6 out of 8 fetuses evaluable for human engraftment, chimerism was confirmed by PCR analysis for human beta(2)-microglobulin which also identified human cells in brain, spinal cord, heart, lung and skeletal muscle. On liver samples, FISH and RT-PCR confirmed the xenograft of human cells and the immunohistochemical analysis detected human markers of hematopoietic and hepatic lineage of differentiation. This preliminary study indicates that intracoelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment.
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PMID:The intracoelomic route: a new approach for in utero human cord blood stem cell transplantation. 1464 12

We describe a case of a poorly differentiated monophasic synovial sarcoma arising in the lung of a 50-year-old man. The tumor, which was located in the right upper lobe, was lobulated, relatively well-circumscribed, and whitish to yellowish in color. Microscopically, it was composed exclusively of ovoid to polygonal or short spindle cells, with a high nuclear to cytoplasmic ratio and relatively scant cytoplasm, arranged in solid sheets or in a hemangiopericytomatous pattern with intervening wiry collagen fibers. At the periphery of the tumor, entrapped benign alveolar epithelium produced a pseudo-biphasic appearance. In some areas, an abundance of keloidal collagen imparted a close resemblance to a solitary fibrous tumor, making it difficult to establish the diagnosis on the initial needle biopsy, although the malignant nature of the tumor was suggested because of nuclear anaplasia. Immunohistochemically, the tumor was positive for cytokeratin AE1/AE3, CAM5.2, EMA, vimentin, bcl-2 protein, calretinin, and CD34. The reverse transcriptase-polymerase chain reaction (RT-PCR), using RNA extracted from fresh-frozen tissue, demonstrated SYT/SSX-1 fusion transcripts, confirming the diagnosis of synovial sarcoma. Microscopic examination demonstrated metastatic deposits in hilar lymph nodes. This case indicates that a primary pulmonary synovial sarcoma, particularly in its poorly differentiated form, is a diagnostically challenging and highly aggressive neoplasm typically found at an advanced stage.
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PMID:Primary poorly differentiated monophasic synovial sarcoma of the lung. A case report with immunohistochemical and genetic studies. 1864 80

Human herpesvirus 8 (HHV-8) is recognized as a major causal agent of Kaposi sarcoma (KS), and it has been detected in all epidemiologic variants of KS. Until now, detection of HHV-8 in paraffin-embedded sections was done mostly by using reverse transcriptase-polymerase chain reaction. To assess the sensitivity and specificity of an anti-HHV-8 antibody and its potential usefulness for separating KS from its mimickers, we immunostained 72 KS samples and 108 samples of potential mimickers of KS with the monoclonal antibody latent nuclear antigen-1 (LNA-1; Advanced Biotechnologies, Columbia, MD). Cases of KS included all epidemiologic variants of the disease. Non-KS lesions included 34 angiosarcomas, 4 kaposiform hemangioendotheliomas, and 70 various benign vascular lesions. Immunostaining for CD31, CD34, and/or von Willebrand factor (factor VIII) also were performed in all cases. All but 1 case of KS (sensitivity, 99%) and none of the non-KS lesions (specificity, 100%) stained with the LNA-1 anti-HHV-8 antibody. The LNA-1 anti-HHV-8 antibody is a reliable marker of all variants of KS. Because KS mimickers are consistently negative for this marker, its use for diagnostic purposes is recommended.
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PMID:Human herpesvirus 8 immunostaining: a sensitive and specific method for diagnosing Kaposi sarcoma in paraffin-embedded sections. 1502 36

We studied the immunophenotype of 100 cases of acute promyelocytic leukemia (APL) with cytogenetic evidence of t(15;17)(q22;q21), 72 hypergranular (M3) and 28 microgranular (M3v), and correlated the results with molecular and clinical features. Most neoplasms (75/100 [75%]) had a typical immunophenotype: CD13+CD33+CD34-HLA-DR-. CD64, CD2, CD34, and HLA-DR were expressed in 27% (24/88), 23% (22/94), 21% (21/100), and 9% (9/98), respectively. CD34 expression was restricted to M3v; HLA-DR and CD2 were expressed more often in M3v than in M3 (P < .001). PML-RARalpha fusion transcripts were detected by reverse transcriptase-polymerase chain reaction in all 70 patients assessed. The short form of PML-RARalpha transcripts was found more frequently in M3v (P < .002) and CD2+ APL (P < .0001) than in M3 and CD2- APL, respectively. The median follow-up was 128 weeks. CD2+ APL was associated significantly with leukocytosis (P = .004), shorter complete remission duration (P = .03), and a trend toward shorter overall survival (P = .07) than CD2- APL. Overall survival for M3v vs M3 (P = .68) and short vs long transcripts (P = .21) was not significantly different. Immunophenotyping is useful for predicting the biologic and clinical behavior of APL.
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PMID:Expression of CD2 in acute promyelocytic leukemia correlates with short form of PML-RARalpha transcripts and poorer prognosis. 1502 45


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