Azithromycin and high-dose vitamin D for treatment and prevention of asthma-like episodes in hospitalised preschool children: study protocol for a combined double-blind randomised controlled trial.

INTRODUCTION: Previous randomised controlled trials (RCTs) suggest antibiotics for treating episodes of asthma-like symptoms in preschool children. Further, high-dose vitamin D supplementation has been shown to reduce the rate of asthma exacerbations among adults with asthma, while RCTs in preschool children are lacking. The aims of this combined RCT are to evaluate treatment effect of azithromycin on episode duration and the preventive effect of high-dose vitamin D supplementation on subsequent episodes of asthma-like symptoms among hospitalised preschoolers. METHODS AND ANALYSIS: Eligible participants, 1-5 years old children with a history of recurrent asthma-like symptoms hospitalised due to an acute episode, will be randomly allocated 1:1 to azithromycin (10 mg/kg/day) or placebo for 3 days (n=250). Further, independent of the azithromycin intervention participants will be randomly allocated 1:1 to high-dose vitamin D (2000 IU/day+ standard dose 400 IU/day) or standard dose (400 IU/day) for 1 year (n=320). Participants are monitored with electronic diaries for asthma-like symptoms, asthma medication, adverse events and sick-leave. The primary outcome for the azithromycin intervention is duration of asthma-like symptoms after treatment. Secondary outcomes include duration of hospitalisation and antiasthmatic treatment. The primary outcome for the vitamin D intervention is the number of exacerbations during the treatment period. Secondary outcomes include time to first exacerbation, symptom burden, asthma medication and safety. ETHICS AND DISSEMINATION: The RCTs are approved by the Danish local ethical committee and conducted in accordance with the guiding principles of the Declaration of Helsinki. The Danish Medicines Agency has approved the azithromycin RCT, which is monitored by the local Unit for Good Clinical Practice. The vitamin D RCT has been reviewed and is not considered a medical intervention. Results will be published in peer-reviewed journals and presented at international conferences. TRIAL REGISTRATION NUMBERS: NCT05028153, NCT05043116.

MicroRNA-210, MicroRNA-331, and MicroRNA-7 Are Differentially Regulated in Treated HIV-1-Infected Individuals and Are Associated With Markers of Systemic Inflammation.

OBJECTIVE: Inflammation may contribute to an increased risk of cardiovascular disease (CVD) in HIV-1 infection. MicroRNAs (miRNAs) are involved in the regulation of inflammation. In treated HIV-1-infected individuals, we aimed to identify differentially expressed miRNAs with known roles in inflammation and CVD risk and to investigate associations between these and systemic inflammation. METHODS: In a screening cohort including 14 HIV-1-infected individuals and 9 uninfected controls, microarray profiling was performed using peripheral blood mononuclear cells (PBMCs). Differentially regulated miRNAs previously related to inflammation and CVD were validated using real-time quantitative reverse-transcription polymerase chain reaction in 26 HIV-1-infected individuals and 20 uninfected controls. Validated miRNAs were measured in PBMCs, CD4 and CD8 T cells. Interleukin-6, tumor necrosis factor-alpha, high-sensitivity C-reactive protein, lipopolysaccharide (LPS), cytomegalovirus immunoglobulin G, lipids, and fasting glucose were measured, and associations with validated miRNAs were assessed with multiple linear regression analysis. RESULTS: Upregulation of miR-210, miR-7, and miR-331 was found in PBMCs from HIV-1-infected individuals when compared with those from uninfected controls (P < 0.005). In contrast, miR-210 and miR-331 were downregulated in CD8 T cells. In multivariate analysis, miR-210 in CD8 T cells was negatively associated with LPS (P = 0.023) and triglycerides (P = 0.003) but positively associated with tumor necrosis factor-alpha (P = 0.004). MiR-7 in PBMC was positively associated with interleukin-6 (P = 0.025) and fasting glucose (P = 0.005), whereas miR-331 was negatively associated with LPS (P = 0.006). In PBMCs from HIV-1-infected individuals with low cytomegalovirus immunoglobulin G, miR-7, miR-29a, miR-221, and miR-222 were downregulated. CONCLUSION: In 2 independent cohorts, miR-210, miR-7, and miR-331 were differentially regulated in treated HIV-1-infected individuals and associated with markers of systemic inflammation.

Malignant T cells express lymphotoxin α and drive endothelial activation in cutaneous T cell lymphoma.

Lymphotoxin α (LTα) plays a key role in the formation of lymphatic vasculature and secondary lymphoid structures. Cutaneous T cell lymphoma (CTCL) is the most common primary lymphoma of the skin and in advanced stages, malignant T cells spreads through the lymphatic to regional lymph nodes to internal organs and blood. Yet, little is known about the mechanism of the CTCL dissemination. Here, we show that CTCL cells express LTα in situ and that LTα expression is driven by aberrantly activated JAK3/STAT5 pathway. Importantly, via TNF receptor 2, LTα functions as an autocrine factor by stimulating expression of IL-6 in the malignant cells. LTα and IL-6, together with VEGF promote angiogenesis by inducing endothelial cell sprouting and tube formation. Thus, we propose that LTα plays a role in malignant angiogenesis and disease progression in CTCL and may serve as a therapeutic target in this disease.

miR-18b overexpression identifies mantle cell lymphoma patients with poor outcome and improves the MIPI-B prognosticator.

Recent studies show that mantle cell lymphoma (MCL) express aberrant microRNA (miRNA) profiles; however, the clinical effect of miRNA expression has not previously been examined and validated in large prospective homogenously treated cohorts. We performed genome-wide miRNA microarray profiling of 74 diagnostic MCL samples from the Nordic MCL2 trial (screening cohort). Prognostic miRNAs were validated in diagnostic MCL samples from 94 patients of the independent Nordic MCL3 trial (validation cohort). Three miRNAs (miR-18b, miR-92a, and miR-378d) were significantly differentially expressed in patients who died of MCL in both cohorts. MiR-18b was superior to miR-92a and miR-378d in predicting high risk. Thus, we generated a new biological MCL International Prognostic Index (MIPI-B)-miR prognosticator, combining expression levels of miR-18b with MIPI-B data. Compared to the MIPI-B, this prognosticator improved identification of high-risk patients with regard to cause-specific, overall, and progression-free survival. Transfection of 2 MCL cell lines with miR-18b decreased their proliferation rate without inducing apoptosis, suggesting that miR-18b may render MCL cells resistant to chemotherapy by decelerating cell proliferation. We conclude that overexpression of miR-18b identifies patients with poor prognosis in 2 large prospective MCL cohorts and adds prognostic information to the MIPI-B. MiR-18b may reduce the proliferation rate of MCL cells as a mechanism of chemoresistance.

Differential expression of miR-155 and miR-21 in tumor and stroma cells in diffuse large B-cell lymphoma.

OncomiRs miR-21 and miR-155 have been linked to lymphomagenesis, but information on their implication in diffuse large B-cell lymphoma (DLBCL) is limited. Here, we used locked nucleic acid-based in situ hybridization (ISH) detection techniques on formalin-fixed paraffin-embedded DLBCL tissue samples to identify miR-155 and miR-21 at the cellular level in 56 patients diagnosed with DLBCL, and compared them to miR array data. miR-155 was observed in tumor cells in 19/56 (33.9%) of the samples evaluated by ISH. miR-21 was localized to the stromal compartment in 41/56 (73.2%). A subset of these, 16/56 (28.6%), also showed labeling in tumor cells. When comparing ISH-scores and miR array data, miR-155 in tumor cells, identified by ISH, was associated with miR-155 expression in miR array data (P=0.030). Equally, miR-21 expression by miR array data were highly associated with miR-21 ISH-scores in the stromal cells (P=0.002), whereas no association between miR array data and ISH of miR-21 in tumor cells was observed (P=0.673). We found no association of miR-155 and miR-21 with overall survival or germinal center B-cell-like (GCB) versus non-GCB-like subtypes of DLBCL. In conclusion, miR-ISH added to the biological interpretation of miR expression in DLBCL compared with miR array data, but miR-155 and miR-21 ISH did not add prognostic information in this series.

MicroRNA expression in early mycosis fungoides is distinctly different from atopic dermatitis and advanced cutaneous T-cell lymphoma.

Mycosis fungoides (MF) is the most common variant of cutaneous T-cell lymphoma (CTCL). MF is characterized by chronic inflammation dominated by cluster of differentiation 4-positive (CD4(+)) T-cells and T helper 2 cytokines, and as the malignant T-cell clone is initially elusive, early diagnosis is often impossible. MF usually takes an indolent course, but for unknown reasons may turn into an aggressive disease with a poor prognosis. Herein, we used a global quantitative real-time polymerase chain reaction platform to study microRNA (miR) expression in patients with early MF (n=13), more advanced CTCL (n=42), and atopic dermatitis (AD, n=20). Thirty-eight miRs were differentially expressed (≥2-fold) in early MF vs. AD and 36 in early MF vs. more advanced disease. miRs that distinguish early MF from AD included both up-regulated (miR-155, miR-146a, 146b-5p, miR-342-3p, let-7i*) and down-regulated (miR-203, miR-205) miRs previously implicated in advanced CTCL. When comparing early MF to more advanced CTCL, additional miRs were significantly up-regulated including miRs which are part of the oncogenic miR-17/92, 106b/25 and 106a/363 clusters. In 16 patients for whom detailed follow-up data were available, 72 miRs were found differentially expressed between patients with progressive vs. those with non-progressive disease, again including miRs with a known relevance for lymphomagenesis, e.g. miR-155, miR-21, let-7i, miR-16, miR-142-3p, miR-146b-5p, miR-92a, miR-93 and miR-106a. In conclusion, we showed that early MF and AD display very different miR profiles despite their clinical, histological, and immunological similarities. During progression, an additional set of miRs becomes deregulated, suggesting their role in disease progression. These data suggest that miR profiling in CTCL may be a key to improving both diagnosis and risk prediction.

A short acidic motif in ARF guards against mitochondrial dysfunction and melanoma susceptibility.

ARF is a small, highly basic protein that can be induced by oncogenic stimuli and exerts growth-inhibitory and tumour-suppressive activities through the activation of p53. Here we show that, in human melanocytes, ARF is cytoplasmic, constitutively expressed, and required for maintaining low steady-state levels of superoxide under conditions of mitochondrial dysfunction. This mitochondrial activity of ARF is independent of its known autophagic and p53-dependent functions, and involves the evolutionarily conserved acidic motif GHDDGQ, which exhibits weak homology to BCL-2 homology 3 (BH3) domains and mediates interaction with BCL-xL--an important regulator of mitochondrial redox homeostasis. Melanoma-predisposing CDKN2A germline mutations, which affect conserved glycine and aspartate residues within the GHDDGQ motif, impair the ability of ARF to control superoxide production and suppress growth of melanoma cells in vivo. These results reveal an important cell-protective function of ARF that links mitochondrial dysfunction and susceptibility to melanoma.

STAT3 activation and infiltration of eosinophil granulocytes in mycosis fungoides.

Eosinophil granulocytes have been implicated in anticancer immunity but recent data indicate that eosinophils can also promote cancer. Herein, we studied eosinophils in skin lesions from 43 patients with mycosis fungoides (MF). The presence of eosinophils correlated with disease stage: 78% of patients with advanced disease displayed eosinophil infiltration, whereas this was only seen in 11% of patients with patches (p<0.01), and in 48% of those with plaque disease. Importantly, 72% of patients with positive staining for phospho-signal-transducer-and-activator-of-transcription (pY-STAT3) in malignant T-cells also stained positively for eosinophils, whereas this was only observed in 28% of pY-STAT3-negative patients (p<0.01). Notably, malignant T-cells expressed eosinophilic activation and trafficking factors: High-mobility group BOX-1 protein (HMGB1) and interleukin 5 (IL5). STAT3 siRNA profoundly inhibited IL5 but not HMGB1 expression. In conclusion, these data suggest that malignant T-cells orchestrate accumulation and activation of eosinophils supporting the notion of STAT3 being a putative target for therapy.

Diffuse large B-cell lymphoma with combined TP53 mutation and MIR34A methylation: Another "double hit" lymphoma with very poor outcome?

MiR34A, B and C have been implicated in lymphomagenesis, but information on their role in normal CD19+ B-cells (PBL-B) and de novo diffuse large B-cell lymphoma (DLBCL) is limited. We show that in normal and activated B-cells miR34A-5p plays a dominant role compared to other miR34 family members. Only miR34A-5p is expressed in PBL-B, and significantly induced in activated B-cells and reactive lymph nodes. In PBL-B, the MIR34A and MIR34B/C promoters are unmethylated, but the latter shows enrichment for the H3K4me3/H3K27me3 silencing mark. Nine de novo DLBCL cases (n=150) carry both TP53 mutation and MIR34A methylation ("double hit") and these patients have an exceedingly poor prognosis with a median survival of 9.4 months (P<0.0001), while neither TP53 mutation, MIR34A or MIR34B/C promoter methylation alone ("single hit") influence on survival. The TP53/MIR34A "double-hit" is an independent negative prognostic factor for survival (P=0.0002). In 2 DLBCL-cell lines with both TP53 mutation and promoter methylation of MIR34A, miR34A-5p is upregulated by 5-aza-2'deoxycytidine. Thus, the TP53/MIR34A "double hit" characterizes a very aggressive subgroup of DLBCL, which may be treatable with epigenetic therapy prior to or in combination with conventional immunochemotherapy.

Expression of miR-155 and miR-126 in situ in cutaneous T-cell lymphoma.

Recently, miR-155 has been implicated in cutaneous T-cell lymphoma (CTCL). Thus, elevated levels of miR-155 were observed in skin lesions from CTCL patients as judged from qPCR and micro-array analysis and aberrant, high miR-155 expression was associated with severe disease. Moreover, miR-155 promoted proliferation of malignant T cells in vitro. Little is, however, known about which cell types express miR-155 in vivo in CTCL skin lesions. Here, we study miR-155 expression using in situ hybridization (ISH) with a miR-155 probe, a negative control (scrambled), and a miR-126 probe as a positive control in nine patients with mycosis fungoides, the most frequent subtype of CTCL. We provide evidence that both malignant and non-malignant T cells stain weakly to moderately positive with the miR-155 probe, but generally negative with the miR-126 and negative control probes. Reversely, endothelial cells stain positive for miR-126 and negative for miR-155 and the control probe. Solitary T cells with a malignant morphology display brighter staining with the miR-155 probe. Taken together, our findings suggest that both malignant and non-malignant T cells express miR-155 in situ in CTCL. Moreover, they indicate heterogeneity in miR-155 expression among malignant T cells.

MicroRNA profiling in ocular adnexal lymphoma: a role for MYC and NFKB1 mediated dysregulation of microRNA expression in aggressive disease.

PURPOSE: Ocular adnexal lymphoma (i.e., lymphoma with involvement of the orbit, eyelids, conjunctiva, lacrimal gland, and lacrimal sac), although rare, is common among malignant tumors involving the ocular adnexal region. The main subtypes are low-grade extranodal marginal zone lymphoma (EMZL) and aggressive diffuse large B-cell lymphoma (DLBCL). In rare cases, low-grade EMZL are reported to transform to DLBCL. It is unclear, however, which genetic events distinguish low-grade disease from aggressive, potentially fatal disease. METHODS: Using LNA-based arrays from Exiqon, we performed global microRNA (miRNA) expression profiling of 18 EMZLs and 25 DLBCLs involving ocular adnexal sites to investigate changes in the miRNA expression in low- versus high-grade disease. Findings were confirmed by real-time quantitative PCR (RTq-PCR). RESULTS: Our analysis revealed 43 miRNAs with altered expression profiles in DLBCL compared to EMZL. Seven of the miRNAs down-regulated in DLBCL relative to EMZL showed enrichment for a direct transcriptional repression by the oncoprotein MYC. We also report a possible loss-of-regulation of NFKB1 and its downstream miRNAs. In addition, our analysis identified a group of DLBCLs whose expression profiles resembled that of EMZL. Although transformation of EMZL to DLBCL in the ocular adnexal region is rare, we hypothesize that the intermediate group potentially may derive from transformation of EMZL that was not recognized by histology. CONCLUSIONS: We conclude that fundamental differences in miRNA expression exist between ocular adnexal EMZL and DLBCL, mainly due to differences in MYC and NF-ĸB regulatory pathways.

Genome-wide profiling identifies a DNA methylation signature that associates with TET2 mutations in diffuse large B-cell lymphoma.

The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers and recent investigations have shown TET2 mutations in diffuse large B-cell lymphoma. However, the detection rates and the types of TET2 mutations vary, and the relation to global methylation patterns has not been investigated. Here, we show TET2 mutations in 12 of 100 diffuse large B-cell lymphomas with 7% carrying loss-of-function and 5% carrying missense mutations. Genome-wide methylation profiling using 450K Illumina arrays identified 315 differentially methylated genes between TET2 mutated and TET2 wild-type cases. TET2 mutations are primarily associated with hypermethylation within CpG islands (70%; P<0.0001), and at CpG-rich promoters (60%; P<0.0001) of genes involved in hematopoietic differentiation and cellular development. Hypermethylated loci in TET2 mutated samples overlap with the bivalent (H3K27me3/H3K4me3) silencing mark in human embryonic stem cells (P=1.5×10(-30)). Surprisingly, gene expression profiling showed that only 11% of the hypermethylated genes were down-regulated, among which there were several genes previously suggested to be tumor suppressors. A meta-analysis suggested that the 35 hypermethylated and down-regulated genes are associated with the activated B-cell-like type of diffuse large B-cell lymphoma in other studies. In conclusion, our data suggest that TET2 mutations may cause aberrant methylation mainly of genes involved in hematopoietic development, which are silenced but poised for activation in human embryonic stem cells.

STAT5-mediated expression of oncogenic miR-155 in cutaneous T-cell lymphoma.

The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains elusive. Recent discoveries indicate that the oncogenic microRNA miR-155 is overexpressed in affected skin from CTCL patients. Here, we address what drives the expression of miR-155 and investigate its role in the pathogenesis of CTCL. We show that malignant T cells constitutively express high levels of miR-155 and its host gene BIC (B cell integration cluster). Using ChIP-seq, we identify BIC as a target of transcription factor STAT5, which is aberrantly activated in malignant T cells and induced by IL-2/IL-15 in non-malignant T cells. Incubation with JAK inhibitor or siRNA-mediated knockdown of STAT5 decreases BIC/miR-155 expression, whereas IL-2 and IL-15 increase their expression in cell lines and primary cells. In contrast, knockdown of STAT3 has no effect, and BIC is not a transcriptional target of STAT3, indicating that regulation of BIC/miR-155 expression by STAT5 is highly specific. Malignant proliferation is significantly inhibited by an antisense-miR-155 as well as by knockdown of STAT5 and BIC.   In conclusion, we provide the first evidence that STAT5 drives expression of oncogenic BIC/miR-155 in cancer. Moreover, our data indicate that the STAT5/BIC/miR-155 pathway promotes proliferation of malignant T cells, and therefore is a putative target for therapy in CTCL.

Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL).

Cutaneous T-cell lymphomas (CTCLs) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult because of a clinical and histologic resemblance to benign inflammatory skin diseases. To address whether microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays, we show that the most induced (miR-326, miR-663b, and miR-711) and repressed (miR-203 and miR-205) miRNAs distinguish CTCL from benign skin diseases with > 90% accuracy in a training set of 90 samples and a test set of 58 blinded samples. These miRNAs also distinguish malignant and benign lesions in an independent set of 50 patients with PTL and skin inflammation and in experimental human xenograft mouse models of psoriasis and CTCL. Quantitative (q)RT-PCR analysis of 103 patients with CTCL and benign skin disorders validates differential expression of 4 of the 5 miRNAs and confirms previous reports on miR-155 in CTCL. A qRT-PCR-based classifier consisting of miR-155, miR-203, and miR-205 distinguishes CTCL from benign disorders with high specificity and sensitivity, and with a classification accuracy of 95%, indicating that miRNAs have a high diagnostic potential in CTCL.

Malignant cutaneous T-cell lymphoma cells express IL-17 utilizing the Jak3/Stat3 signaling pathway.

IL-17 is a proinflammatory cytokine that is crucial for the host's protection against a range of extracellular pathogens. However, inappropriately regulated expression of IL-17 is associated with the development of inflammatory diseases and cancer. In cutaneous T-cell lymphoma (CTCL), malignant T cells gradually accumulate in skin lesions characterized by massive chronic inflammation, suggesting that IL-17 could be involved in the pathogenesis. In this study we show that IL-17 protein is present in 10 of 13 examined skin lesions but not in sera from 28 CTCL patients. Importantly, IL-17 expression is primarily observed in atypical lymphocytes with characteristic neoplastic cell morphology. In accordance, malignant T-cell lines from CTCL patients produce IL-17 and the synthesis is selectively increased by IL-2 receptor ß chain cytokines. Small-molecule inhibitors or small interfering RNA against Jak3 and signal transducer and activator of transcription 3 (Stat3) reduce the production of IL-17, showing that the Jak3/Stat3 pathway promotes the expression of the cytokine. In summary, our findings indicate that the malignant T cells in CTCL lesions express IL-17 and that this expression is promoted by the Jak3/Stat3 pathway.

Integrated genetic and epigenetic analysis of bladder cancer reveals an additive diagnostic value of FGFR3 mutations and hypermethylation events.

The bladder cancer genome harbors numerous oncogenic mutations and aberrantly methylated gene promoters. The aim of our study was to generate a profile of these alterations and investigate their use as biomarkers in urine sediments for noninvasive detection of bladder cancer. We systematically screened FGFR3, PIK3CA, TP53, HRAS, NRAS and KRAS for mutations and quantitatively assessed the methylation status of APC, ARF, DBC1, INK4A, RARB, RASSF1A, SFRP1, SFRP2, SFRP4, SFRP5 and WIF1 in a prospective series of tumor biopsies (N = 105) and urine samples (N = 113) from 118 bladder tumor patients. We also analyzed urine samples from 33 patients with noncancerous urinary lesions. A total of 95 oncogenic mutations and 189 hypermethylation events were detected in the 105 tumor biopsies. The total panel of markers provided a sensitivity of 93%, whereas mutation and methylation markers alone provided sensitivities of 72% and 70%, respectively. In urine samples, the sensitivity was 70% for all markers, 50% for mutation markers and 52% for methylation markers. FGFR3 mutations occurred more frequently in tumors with no methylation events than in tumors with one or more methylation events (78% vs. 33%; p < 0.0001). FGFR3 mutation in combination with three methylation markers (APC, RASSF1A and SFRP2) provided a sensitivity of 90% in tumors and 62% in urine with 100% specificity. These results suggest an inverse correlation between FGFR3 mutations and hypermethylation events, which may be used to improve noninvasive, DNA-based detection of bladder cancer.

Equitoxic doses of 5-azacytidine and 5-aza-2'deoxycytidine induce diverse immediate and overlapping heritable changes in the transcriptome.

BACKGROUND: The hypomethylating agent 5-Azacytidine (5-Aza-CR) is the first drug to prolong overall survival in patients with myelodysplastic syndrome (MDS). Surprisingly, the deoxyribonucleoside analog 5-Aza-2'deoxycytidine (5-Aza-CdR) did not have a similar effect on survival in a large clinical trial. Both drugs are thought to exert their effects after incorporation into DNA by covalent binding of DNA methyltransferase (DNMT). While 5-Aza-CdR is incorporated into only DNA, 5-Aza-CR is also incorporated into RNA. Here, we have analyzed whether this difference in nucleic acid incorporation may influence the capacities of these drugs to regulate the expression of mRNA and microRNAs (miRNA), which may potentially affect the activities of the drugs in patients. METHODOLOGY/PRINCIPAL FINDINGS: A hematopoietic (HL-60; acute myeloid leukemia) and a solid (T24; transitional cell carcinoma) cancer cell line were treated with equitoxic doses of 5-Aza-CR and 5-Aza-CdR for 24 hrs, and the immediate (day 2) and lasting (day 8) effects on RNA expression examined. There was considerable overlap between the RNAs heritably upregulated by both drugs on day 8 but more RNAs were stably induced by the deoxy analog. Both drugs strongly induced expression of cancer testis antigens. On day 2 more RNAs were downregulated by 5-Aza-CR, particularly at higher doses. A remarkable downregulation of miRNAs and a significant upregulation of tRNA synthetases and other genes involved in amino acid metabolism was observed in T24 cells. CONCLUSIONS/SIGNIFICANCE: Overall, this suggests that significant differences exist in the immediate action of the two drugs, however the dominant pattern of the lasting, and possible heritable changes, is overlapping.

Custom-designed MLPA using multiple short synthetic probes: application to methylation analysis of five promoter CpG islands in tumor and urine specimens from patients with bladder cancer.

Ligation of two oligonucleotide probes hybridized adjacently to a DNA template has been widely used for detection of genome alterations. The multiplex ligation-dependent probe amplification (MLPA) technique allows simultaneous screening of multiple target sequences in a single reaction by using pairs of probes that carry tails for binding of common amplification primers. Resolution of the various targets is achieved by electrophoresis on the basis of predefined differences in amplicon length. In the conventional MLPA approach, one of the two target probes is generated by cloning in a single-stranded bacteriophage vector to introduce a sequence of defined length between the primer binding site and the specific target sequence. Here we demonstrate that differences in amplicon length can be achieved by using multiple short synthetic probes for each target sequence. When joined by a DNA ligase, these probes will form a single amplifiable template whose length is defined by the number and lengths of the individual probes. We have used this principle to establish a methylation-specific MLPA (MS-MLPA) assay that simultaneously determines the methylation status of five promoter CpG islands, and we have used this assay to analyze DNA from tumor tissue and corresponding urine samples from patients with bladder cancer. Our data show that the use of multiple short synthetic probes provides a simple means for custom-designed MS-MLPA analysis.