Long-Term Efficacy and Safety of RNAi-Mediated Virus Resistance in 'HoneySweet' Plum.

Interfering RNA technology has been established as an effective strategy to protect plants against viral infection. Despite this success, interfering RNA (RNAi) has rarely been applied due to the regulatory barriers that confront genetically engineered plants and concerns over possible environmental and health risks posed by non-endogenous small RNAs. 'HoneySweet' was developed as a virus-resistant plum variety that is protected by an RNAi-mediated process against Sharka disease caused by the plum pox virus. 'HoneySweet' has been approved for cultivation in the United States but not in countries where the plum pox virus is endemic. In this study, we evaluated the long-term efficacy of virus resistance in 'HoneySweet,' the nature and stability of its sRNA profile, and the potential health risks of consuming 'HoneySweet' plums. Graft-challenged 'HoneySweet' trees carrying large non-transgenic infected limbs remained virus-free after more than 10 years in the field, and the viral sequences from the non-transgenic infected limbs showed no evidence of adaptation to the RNAi-based resistance. Small RNA profiling revealed that transgene-derived sRNA levels were stable across different environments and, on average, were more than 10 times lower than those present in symptom-less fruits from virus-infected trees. Comprehensive 90-day mouse feeding studies showed no adverse health impacts in mice, and there was no evidence for potential siRNA off-target pathologies predicted by comparisons of the most abundant transgene-derived sRNAs to the mouse genome. Collectively, the data confirmed that RNAi provides a highly effective, stable, and safe strategy to combat virus diseases in crop plants.

[Idiopathic pulmonary fibrosis prognostic factors - analysis of the Czech registry]. / Prognostické faktory idiopatické plicní fibrózy (IPF) - analýza Ceského registru IPF.

UNLABELLED: Idiopathic pulmonary fibrosis (IPF) is a rare, progressive and usually fatal form of idiopathic interstitial pneumonia. IPF is characterized by failure of alveolar re-epithelization, persistence of fibroblasts, deposition of extracellular matrix, and distortion of lung architecture, which ultimately results in respiratory failure.We analysed 202 consecutive patients with IPF diagnosed at the Departments of Pulmonary Diseases and Tuberculosis in the Czech Republic, who they were included in the nationwide Czech IPF registry. Our aim was to determine prognostic factors of IPF and outcome of the disease.There were 129 males and 73 females who were the median age 67 years. IPF was biopsy-proven in 66 (33 %) of patients. Median time from the first symptom to diagnosis was 12 months. Diagnosis was made in 57 patients (28.3 %) within 6 months from the onset of respiratory symptoms. 8 (4 %) patients had an acute exacerbation during the course of the disease.In uniparametric (univariate) analysis as prognostic factors associated with poorer survival were found: higher age, higher degree dyspnea scores, clubbing fingers, comorbidities (arterial hypertension, osteoporosis), patients without histology biopsy, and bronchoalveolar increased neutrophil count. We found these positive prognostic factors: higher levels of VC (vital capacity), TLC (total lung capacity) and DLCO (diffusing capacity for carbon monooxide).In multiparametric (multivariate) analysis as prognostic factors associated with mortality were found: higher age, higher degree of dyspnoe score. Increased lymphocytes in bronchoalveolar fluid, higher level of VC a DLCO were associated with better survival. There was no difference in survival of patients by sex, by smoking status. No significant difference in survival rates was found between IPF with and without emphysema, between the extent of fibrosis on HRCT (high resolution computed tomography) of thorax and mortality. Median survival was 51.6 months. 58 (28.7 %) patients died. The most frequent reason of dead was IPF progression with respiratory failure. KEY WORDS: Idiopathic pulmonary fibrosis; prognosis; treatment.

High level of full-length cereblon mRNA in lower risk myelodysplastic syndrome with isolated 5q deletion is implicated in the efficacy of lenalidomide.

Downregulation of cereblon (CRBN) gene expression is associated with resistance to the immunomodulatory drug lenalidomide and poor survival outcomes in multiple myeloma (MM) patients. However, the importance of CRBN gene expression in patients with myelodysplastic syndrome (MDS) and its impact on lenalidomide therapy are not clear. In this study, we evaluate cereblon expression in mononuclear cells isolated from bone marrow [23 lower risk MDS patients with isolated 5q deletion (5q-), 37 lower risk MDS patients with chromosome 5 without the deletion of long arms (non-5q-), and 24 healthy controls] and from peripheral blood (38 patients with 5q-, 52 non-5q- patients and 25 healthy controls) to gain insight into, firstly, the role of cereblon in lower risk MDS patients with or without 5q deletion and, secondly, into the mechanisms of lenalidomide action. Patients with 5q- lower risk MDS have the highest levels of CRBN mRNA in comparison with both lower risk MDS without the deletion of long arms of chromosome 5 and healthy controls. CRBN gene expression was measured using the quantitative TaqMan real-time PCR. High levels of CRBN mRNA were detected in all lenalidomide responders during the course of therapy. A significant decrease of the CRBN mRNA level during lenalidomide treatment is associated with loss of response to treatment and disease progression. These results suggest that, similar to the treatment of MM, high levels of full-length CRBN mRNA in lower risk 5q- patients are necessary for the efficacy of lenalidomide.

Minimal residual disease detectable by quantitative assessment of WT1 gene before allogeneic stem cell transplantation in patients in first remission of acute myeloid leukemia has an impact on their future prognosis.

Overall 42 patients (pts) transplanted in hematological CR1 were retrospectively analyzed. Median follow-up was 15 months (range 2-77). The expression of WT1 gene was measured according to the European Leukaemia Net recommendations. At the time of allogeneic stem cell transplantation (allo-SCT) 29 pts were WT1-negative and 13 pts were WT1-positive. In the univariate analysis, significantly better results were observed in the group of WT1 neg in terms of progression-free survival (in three yr 77% vs. 27%, p = 0.001). In multivariate analysis, the only significant feature in terms of better OS was WT1 negativity (p = 0.029). Our results show that minimal residual disease status measured by quantitative assessment of WT1 gene in acute myeloid leukemia pts in CR1 significantly affects their future prognosis after allo-SCT.

Estimation of molecular upper remission limit for monitoring minimal residual disease in peripheral blood of acute myeloid leukemia patients by WT1 expression.

To date, approximately one half of acute myeloid leukaemia (AML) patients do not have a suitable specific molecular marker for monitoring minimal residual disease (MRD). The Wilm's tumour gene (WT1) has been suggested as a possible molecular marker of MRD in AML. The expression of WT1 in peripheral blood (PB) was measured using quantitative real-time reverse transcription-polymerase chain reaction in peripheral leukocytes from 151 patients with AML at diagnosis. WT1 expression was significantly elevated, i.e. up to 3 orders of magnitude in the majority (80%) of AML patients at diagnosis compared to the PB of healthy donors. Sequence samples of the long-term followed-up AML patients treated with chemotherapy and/or allogeneic bone marrow transplantation were analysed for WT1 expression. The results revealed that the hematological relapses were preceded (median, 1.8 months) by an increase in WT1 gene expression. For the practical utility of this gene as a molecular marker of relapse, it was necessary to determine an upper remission limit, crossing which would signal hematological relapse. The upper remission limit was determined in our set of patients to be 0.02 WT1/ABL. The AML patients who consequently relapsed crossed this upper remission limit; however, those in permanent remission did not. Therefore, this upper remission limit could be taken as the border of molecular relapse of AML patients. Moreover, insufficient decline of WT1 expression under the upper remission limit following induction and/or consolidation therapy was associated with markedly high risk of relapse. The results show that our upper remission limit can be taken as the border of molecular relapse of AML patients and WT1 levels following initial therapy as a beneficial prognostic marker.

Expression of four major WT1 splicing variants in acute and chronic myeloid leukemia patients analyzed by newly developed four real-time RT PCRs.

Although the mechanism of action of leukemic oncogene Wilms' tumor gene 1 (WT1) remains unclear, WT1 has already been used in monitoring of patients with acute myeloid leukemia (AML) and it is being tested for immunotherapy. More detailed understanding of the role of WT1 in leukemia may improve its utilization. At least 36 isoforms may be produced. Four major variants denoted as -5/-KTS, -5/+KTS, +5/-KTS and +5/+KTS are produced by combining splicing of exon 5 and KTS sequence. In this study, we report applicability of newly developed real-time RT PCRs enabling for the first time full quantification of the four major WT1 splicing variants. Following careful optimization and testing of quantification reliability of four assays, we analyzed 34 samples of patients with AML and 12 samples of patients with chronic myeloid leukemia (CML) at the time of diagnosis. Analyses of five more CML patients provided insight into WT1 variants expression kinetics. We found predominance of +5/+KTS in both diagnoses. Comparison of WT1 variant expression in AML and CML patients' groups differing in response to therapy suggested possible importance of particular WT1 variant levels as markers of further disease course.

From cryptic chromosomal lesions to pathologically relevant genes: integration of SNP-array with gene expression profiling in myelodysplastic syndrome with normal karyotype.

Myelodysplastic syndrome (MDS), a clonal disorder originating from hematopoietic stem cell, is characterized by a progressive character often leading to transformation to acute myeloid leukemia. We used single nucleotide polymorphism arrays (SNP-A) to identify previously cryptic chromosomal abnormalities such as copy number alterations and uniparental disomies (UPD) in cytogenetically normal MDS. In the aberrant regions, we attempted to localize candidate genes with potential relevance to the disease. Using SNP-A, we analyzed peripheral blood granulocytes from 37 MDS patients. The analysis identified 13 cryptic chromosomal defects in 10 patients (27%). Four UPD (affecting chromosomes 3q, 7q, 17q, and 20p), 5 deletions and 4 duplications were detected. Gene expression data measured on CD34+ cells were available for 4 patients with and 6 patients without SNP-A lesions. We performed an integrative analysis of genotyping and gene expression microarrays and found several genes with an altered expression located in the aberrant regions. The expression microarrays suggested BMP2 and TRIB3 located in 20p UPD as potential candidate genes contributing to MDS. We showed that the genome-wide integrative approach is beneficial to the comprehension of molecular backgrounds of diseases with incompletely understood etiopathology.

Simultaneous detection and identification of four pome fruit viruses by one-tube pentaplex RT-PCR.

A pentaplex reverse-transcription polymerase chain reaction (Pentaplex RT-PCR) in a single tube was developed for the simultaneous detection of the pome fruit viruses: Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple mosaic virus (ApMV). This is the first report of the simultaneous detection of all four viruses and host mRNA as an internal specific control. Pentaplex RT-PCR was applied successfully throughout the year, using different plant organs (leaves or dormant buds). The sensitivity of detection by monoplex- and pentaplex RT-PCR assays was comparable. Different combinations of mixed infections of viruses were identified in samples of infected apple and pear trees from different geographical regions. The pentaplex RT-PCR assay developed was sensitive, simple, rapid, and reliable for simultaneous detection of the four viruses in extracts of leaves or dormant buds.

Type J CBFbeta/MYH11 transcript in the M4Eo subtype of acute myeloid leukemia.

Acute myeloid leukemia (AML) carrying inversion or translocation of chromosome 16 is usually associated with the FAB M4Eo morphological subtype and belongs to AMLs with a relatively favorable prognosis. At the molecular level, it is associated with a disease-specific fusion gene, CBFbeta/MYH11. Previously, 10 different types of CBFbeta/MYH11 fusion transcripts have been described in the literature, 7 of them are still known as unique cases. In the current study, peripheral blood and/or bone marrow samples from 265 AML patients were tested for the presence of the CBFbeta/MYH11 fusion using RT-PCR and 12 (4.5%) positive cases were identified. The most common type A CBFbeta/MYH11 transcript was confirmed in 11 patients. The transcript in the remaining one (a 71-year-old female) was different and sequence analysis allowed us to classify it as CBFbeta/MYH11 type J. In contrast to the first type J case previously reported from Australia, this patient exhibited a typical FAB M4Eo morphology. The evidence of the second case indicates that the type J breakage might be a non-random event within the MYH11 gene.