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A new variant of the HLA-A*010101 allele designated as HLA-A*0111N, previously known as HLA-A*010101var, was identified in a patient requiring a stem-cell transplantation. The patient was typed by serologic methods as HLA-A2 homozygous and by sequence-based typing (SBT) as A*010101,020601. Flow-cytometric (FCM) analysis with 11 human monoclonal antibodies (mAbs) for the A1 molecule confirmed lack of any cell membrane expression of the A*0111N allele. One-dimensional isoelectric focusing (1D-IEF) of total cell lysate from the patient’s cells revealed no cell surface and cytoplasmic A1 protein expression, whereas the HLA-A2 molecule was identified by both FCM analysis and 1D-IEF. DNA sequence analysis showed the presence of a synonymous substitution from G to T at position 597 in codon 175. RNA SBT revealed a deletion of 24 bp in exon 3, position 596 through 619, encoding codons 175 through 182 of the HLA-A*0111N allele. The synonymous substitution introduced a new splice site, resulting in an efficient splicing, because no classical A1 protein could be detected in the patient. This alternative splicing prevented the translation into a correct and stable class I molecule expression on the cell surface.

Objective Angiogenesis and inflammation are important features in atherosclerotic plaque destabilization. The transcription factor hypoxia-inducible factor-1 alpha (HIF-1α) is a key regulator of angiogenesis and is also involved in inflammatory reactions. We studied HIF-1α expression in different atherosclerotic plaque phenotypes. Methods and results HIF-1α expression was observed in 18/37 (49%) carotid and in 9/15 (60%) femoral endarterectomy specimens. Expression of HIF-1α was associated with the presence of a large extracellular lipid core (P = 0.03) and macrophages (P = 0.02). HIF-1α co-localized with vascular endothelial growth factor (VEGF), an important downstream target of HIF-1α. In addition, a strong association was observed between expression levels of HIF-1α and VEGF (P = 0.001). The average number of plaque microvessels was higher in plaques with no or minor HIF-1α staining than in plaques with moderate or heavy HIF-1α staining (P = 0.03). In human macrophages, lipopolysaccharide activation induced HIF-1α expression. In embryonic fibroblasts derived from wild-type mice, lipopolysaccharide activation induced an increase in HIF-1α mRNA, whereas in Toll-like receptor 4 defective embryonic fibroblasts no effect was observed after lipopolysaccharide stimulation. Conclusions In atherosclerotic plaque, the transcription factor HIF-1α is associated with an atheromatous inflammatory plaque phenotype and with VEGF expression. HIF-1α expression is upregulated in activated macrophages under normoxic conditions.

Background: MicroRNAs (miRNAs) are established regulators of development, cell identity and disease. Although nearly two thousand human miRNA genes are known and new ones are continuously discovered, no attempt has been made to gauge the total miRNA content of the human genome. Results: Employing an innovative computational method on massively pooled small RNA sequencing data, we report 2,469 novel human miRNA candidates of which 1,098 are validated by in-house and published experiments. Almost 300 candidates are robustly expressed in a neuronal cell system and are regulated during differentiation or when biogenesis factors Dicer, Drosha, DGCR8 or Ago2 are silenced. To improve expression profiling, we devised a quantitative miRNA capture system. In a kidney cell system, 400 candidates interact with DGCR8 at transcript positions that suggest miRNA hairpin recognition, and 1,000 of the new miRNA candidates interact with Ago1 or Ago2, indicating that they are directly bound by miRNA effector proteins. From kidney cell CLASH experiments, in which miRNA-target pairs are ligated and sequenced, we observe hundreds of interactions between novel miRNAs and mRNA targets. The novel miRNA candidates are specifically but lowly expressed, raising the possibility that not all may be functional. Interestingly, the majority are evolutionarily young and overrepresented in the human brain. Conclusions: In summary, we present evidence that the complement of human miRNA genes is substantially larger than anticipated, and that more are likely to be discovered in the future as more tissues and experimental conditions are sequenced to greater depth. This project was funded by the Spanish Plan Nacional SAF2008-00357 (NOVADIS); the Generalitat de Catalunya AGAUR 2009 SGR-1502; the Instituto de Salud Carlos III (FIS/FEDER PI11/00733); National Institutes of Health (R00HG004515 to KCC) and the European Commission 7th Framework Program, Projects N. 03790 (SIROCCO), N. 282510 (BLUEPRINT), N. 261123 (GEUVADIS) and N. 262055 (ESGI). MRF is supported by EMBO Long-Term fellowship ALTF 225–2011; EL is supported by the ICGC CLL-Genome project funded by the Spanish Ministry of Economy and Competiveness through the Instituto de Salud Carlos III. AJSH is a Marie Curie postdoctoral fellow supported by the European Commission 7th Framework Program under grant agreement N. 330133. MB-C is a Sara Borrell postdoctoral fellow supported by the Spanish Ministry of Economy and Competiveness. GK was supported by the Wellcome Trust Grant 097383 and by the MRC. EM–H is a PhD student from LaCaixa