Direct tumor recognition by a human CD4(+) T-cell subset potently mediates tumor growth inhibition and orchestrates anti-tumor immune responses.

Tumor antigen-specific CD4(+) T cells generally orchestrate and regulate immune cells to provide immune surveillance against malignancy. However, activation of antigen-specific CD4(+) T cells is restricted at local tumor sites where antigen-presenting cells (APCs) are frequently dysfunctional, which can cause rapid exhaustion of anti-tumor immune responses. Herein, we characterize anti-tumor effects of a unique human CD4(+) helper T-cell subset that directly recognizes the cytoplasmic tumor antigen, NY-ESO-1, presented by MHC class II on cancer cells. Upon direct recognition of cancer cells, tumor-recognizing CD4(+) T cells (TR-CD4) potently induced IFN-γ-dependent growth arrest in cancer cells. In addition, direct recognition of cancer cells triggers TR-CD4 to provide help to NY-ESO-1-specific CD8(+) T cells by enhancing cytotoxic activity, and improving viability and proliferation in the absence of APCs. Notably, the TR-CD4 either alone or in collaboration with CD8(+) T cells significantly inhibited tumor growth in vivo in a xenograft model. Finally, retroviral gene-engineering with T cell receptor (TCR) derived from TR-CD4 produced large numbers of functional TR-CD4. These observations provide mechanistic insights into the role of TR-CD4 in tumor immunity, and suggest that approaches to utilize TR-CD4 will augment anti-tumor immune responses for durable therapeutic efficacy in cancer patients

MicroRNA profiling of rhesus macaque embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) play important roles in embryonic stem cell (ESC) self-renewal and pluripotency. Numerous studies have revealed human and mouse ESC miRNA profiles. As a model for human-related study, the rhesus macaque is ideal for delineating the regulatory mechanisms of miRNAs in ESCs. However, studies on rhesus macaque (r)ESCs are lacking due to limited rESC availability and a need for systematic analyses of fundamental rESC characteristics.</p> <p>Results</p> <p>We established three rESC lines and profiled microRNA using Solexa sequencing resulting in 304 known and 66 novel miRNAs. MiRNA profiles were highly conserved between rESC lines and predicted target genes were significantly enriched in differentiation pathways. Further analysis of the miRNA-target network indicated that gene expression regulated by miRNAs was negatively correlated to their evolutionary rate in rESCs. Moreover, a cross-species comparison revealed an overall conservation of miRNA expression patterns between human, mouse and rhesus macaque ESCs. However, we identified three miRNA clusters (miR-467, the miRNA cluster in the imprinted Dlk1-Dio3 region and C19MC) that showed clear interspecies differences.</p> <p>Conclusions</p> <p>rESCs share a unique miRNA set that may play critical roles in self-renewal and pluripotency. MiRNA expression patterns are generally conserved between species. However, species and/or lineage specific miRNA regulation changed during evolution.</p

Sequencing and Bioinformatics-Based Analyses of the microRNA Transcriptome in Hepatitis B–Related Hepatocellular Carcinoma

MicroRNAs (miRNAs) participate in crucial biological processes, and it is now evident that miRNA alterations are involved in the progression of human cancers. Recent studies on miRNA profiling performed with cloning suggest that sequencing is useful for the detection of novel miRNAs, modifications, and precise compositions and that miRNA expression levels calculated by clone count are reproducible. Here we focus on sequencing of miRNA to obtain a comprehensive profile and characterization of these transcriptomes as they relate to human liver. Sequencing using 454 sequencing and conventional cloning from 22 pair of HCC and adjacent normal liver (ANL) and 3 HCC cell lines identified reliable reads of more than 314000 miRNAs from HCC and more than 268000 from ANL for registered human miRNAs. Computational bioinformatics identified 7 novel miRNAs with high conservation, 15 novel opposite miRNAs, and 3 novel antisense miRNAs. Moreover sequencing can detect miRNA modifications including adenosine-to-inosine editing in miR-376 families. Expression profiling using clone count analysis was used to identify miRNAs that are expressed aberrantly in liver cancer including miR-122, miR-21, and miR-34a. Furthermore, sequencing-based miRNA clustering, but not individual miRNA, detects high risk patients who have high potentials for early tumor recurrence after liver surgery (P = 0.006), and which is the only significant variable among pathological and clinical and variables (P = 0,022). We believe that the combination of sequencing and bioinformatics will accelerate the discovery of novel miRNAs and biomarkers involved in human liver cancer

Prevalence and Risk Factors of Intestinal Parasitism in Rural and Remote West Malaysia

Intestinal parasitic infections (IPIs) are among the most prevalent human afflictions; these infections still have major impact on the socioeconomic and public health of the bottom billion of the world's poorest people. Although Malaysia has a thriving economy, IPIs are still very much prevalent and causing major health problems among the poor and in deprived communities especially in rural and remote areas. A comprehensive study is paramount to determine the current prevalent and factors closely linked to IPIs so that effective control measures can be instituted. In view of this, we conducted this study to provide detailed data of the existing status of IPIs among 716 participants living in rural and remote areas in Peninsular Malaysia. The establishment of such data is beneficial for the public health service to justify and facilitate the reassessment of control strategies and policies in terms of reducing intestinal parasitism. With effective control measures in place, these communities (especially children) will have a greater opportunity for a better future in terms of health and educational achievement and eventually will be at par socially and economically with urban communities in Malaysia

MiR-34a Represses Numbl in Murine Neural Progenitor Cells and Antagonizes Neuronal Differentiation

MicroRNA (miRNA) function is required for normal animal development, in particular in differentiation pathways from stem cell and precursor populations. In neurogenesis, it is becoming increasingly appreciated that miRNAs act at many stages to ensure proper progression. In this study we examined the role of miR-34a in neural progenitor cells (NPC) derived from murine embryonic cortex. We found that over-expression of miR-34a in NPC significantly reduced the neuron yield upon in vitro induction of differentiation. MiR-34a has several predicted targets in the Notch pathway, which operates to balance progenitor self-renewal and differentiation during cortical neurogenesis. We tested several Notch pathway players for regulation by miR-34a in undifferentiated NPC, and found that mRNA and protein levels of Numbl, a negative regulator of Notch signaling, as well as two downstream pro-neural genes usually blocked by Notch signaling, NeuroD1 and Mash1, were diminished, while Notch1 and Cbf1 transcripts were enhanced by miR-34a over-expression. Using a luciferase reporter assay, we verified the Numbl 3′-UTR as a direct miR-34a target. Correspondingly, knock-down of endogenous miR-34a resulted in increased Numbl, NeuroD1 and Mash1, and reduced Notch1 transcript levels. Together these results implicate Numbl as a physiologically relevant target of miR-34a in NPC, allowing for enhanced Notch signaling and inhibition of neuronal differentiation. This work extends our understanding of miR-34a-mediated control of cell differentiation from cancer to mammalian nervous system development

Postnatal Survival of Mice with Maternal Duplication of Distal Chromosome 7 Induced by a Igf2/H19 Imprinting Control Region Lacking Insulator Function

The misexpressed imprinted genes causing developmental failure of mouse parthenogenones are poorly defined. To obtain further insight, we investigated misexpressions that could cause the pronounced growth deficiency and death of fetuses with maternal duplication of distal chromosome (Chr) 7 (MatDup.dist7). Their small size could involve inactivity of Igf2, encoding a growth factor, with some contribution by over-expression of Cdkn1c, encoding a negative growth regulator. Mice lacking Igf2 expression are usually viable, and MatDup.dist7 death has been attributed to the misexpression of Cdkn1c or other imprinted genes. To examine the role of misexpressions determined by two maternal copies of the Igf2/H19 imprinting control region (ICR)—a chromatin insulator, we introduced a mutant ICR (ICRΔ) into MatDup.dist7 fetuses. This activated Igf2, with correction of H19 expression and other imprinted transcripts expected. Substantial growth enhancement and full postnatal viability was obtained, demonstrating that the aberrant MatDup.dist7 phenotype is highly dependent on the presence of two unmethylated maternal Igf2/H19 ICRs. Activation of Igf2 is likely the predominant correction that rescued growth and viability. Further experiments involved the introduction of a null allele of Cdkn1c to alleviate its over-expression. Results were not consistent with the possibility that this misexpression alone, or in combination with Igf2 inactivity, mediates MatDup.dist7 death. Rather, a network of misexpressions derived from dist7 is probably involved. Our results are consistent with the idea that reduced expression of IGF2 plays a role in the aetiology of the human imprinting-related growth-deficit disorder, Silver-Russell syndrome

MiR-10 Represses HoxB1a and HoxB3a in Zebrafish

BACKGROUND: The Hox genes are involved in patterning the anterior-posterior axis. In addition to the protein coding Hox genes, the miR-10, miR-196 and miR-615 families of microRNA genes are conserved within the vertebrate Hox clusters. The members of the miR-10 family are located at positions associated with Hox-4 paralogues. No function is yet known for this microRNA family but the genomic positions of its members suggest a role in anterior-posterior patterning. METHODOLOGY/PRINCIPAL FINDINGS: Using sensor constructs, overexpression and morpholino knockdown, we show in Zebrafish that miR-10 targets HoxB1a and HoxB3a and synergizes with HoxB4 in the repression of these target genes. Overexpression of miR-10 also induces specific phenotypes related to the loss of function of these targets. HoxB1a and HoxB3a have a dominant hindbrain expression domain anterior to that of miR-10 but overlap in a weaker expression domain in the spinal cord. In this latter domain, miR-10 knockdown results in upregulation of the target genes. In the case of a HoxB3a splice variant that includes miR-10c within its primary transcript, we show that the microRNA acts in an autoregulatory fashion. CONCLUSIONS/SIGNIFICANCE: We find that miR-10 acts to repress HoxB1a and HoxB3a within the spinal cord and show that this repression works cooperatively with HoxB4. As with the previously described interactions between miR-196 and HoxA7 and Hox-8 paralogues, the target genes are located in close proximity to the microRNA. We present a model in which we postulate a link between the clustering of Hox genes and post-transcriptional gene regulation. We speculate that the high density of transcription units and enhancers within the Hox clusters places constraints on the precision of the transcriptional control that can be achieved within these clusters and requires the involvement of post-transcriptional gene silencing to define functional domains of genes appropriately

Mammalian MicroRNA Prediction through a Support Vector Machine Model of Sequence and Structure

BACKGROUND: MicroRNAs (miRNAs) are endogenous small noncoding RNA gene products, on average 22 nt long, found in a wide variety of organisms. They play important regulatory roles by targeting mRNAs for degradation or translational repression. There are 377 known mouse miRNAs and 475 known human miRNAs in the May 2007 release of the miRBase database, the majority of which are conserved between the two species. A number of recent reports imply that it is likely that many mammalian miRNAs remain to be discovered. The possibility that there are more of them expressed at lower levels or in more specialized expression contexts calls for the exploitation of genome sequence information to accelerate their discovery. METHODOLOGY/PRINCIPAL FINDINGS: In this article, we describe a computational method-mirCoS-that uses three support vector machine models sequentially to discover new miRNA candidates in mammalian genomes based on sequence, secondary structure, and conservation. mirCoS can efficiently detect the majority of known miRNAs and predicts an extensive set of hairpin structures based on human-mouse comparisons. In total, 3476 mouse candidates and 3441 human candidates were found. These hairpins are more similar to known miRNAs than to negative controls in several aspects not considered by the prediction algorithm. A significant fraction of predictions is supported by existing expression evidence. CONCLUSIONS/SIGNIFICANCE: Using a novel approach, mirCoS performs comparably to or better than existing miRNA prediction methods, and contributes a significant number of new candidate miRNAs for experimental verification