Cells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. While a wide range of putative biological functions have been attributed to exosomes, they are assumed to represent a homogenous population of EVs. We hypothesized the existence of subpopulations of exosomes with defined molecular compositions and biological properties. Density gradient centrifugation of isolated exosomes revealed the presence of two distinct subpopulations, differing in biophysical properties and their proteomic and RNA repertoires. Interestingly, the subpopulations mediated differential effects on the gene expression programmes in recipient cells. In conclusion, we demonstrate that cells release distinct exosome subpopulations with unique compositions that elicit differential effects on recipient cells. Further dissection of exosome heterogeneity will advance our understanding of exosomal biology in health and disease and accelerate the development of exosome-based diagnostics and therapeutics.

MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression and are implicated in wide-ranging cellular processes and pathological conditions including Duchenne muscular dystrophy (DMD). We have compared differential miRNA expression in proximal and distal limb muscles, diaphragm, heart and serum in the mdx mouse relative to wild-type controls. Global transcriptome analysis revealed muscle-specific patterns of differential miRNA expression as well as a number of changes common between tissues, including previously identified dystromirs. In the case of miR-31 and miR-34c, upregulation of primary-miRNA transcripts, precursor hairpins and all mature miRNAs derived from the same transcript or miRNA cluster, strongly suggests transcriptional regulation of these miRNAs. The most striking differences in differential miRNA expression were between muscle tissue and serum. Specifically, miR-1, miR-133a, and miR-206 were highly abundant in mdx serum but downregulated or modestly upregulated in muscle, suggesting that these miRNAs are promising disease biomarkers. Indeed, the relative serum levels of these miRNAs were normalized in response to peptide-phosphorodiamidate morpholino oligonucleotide (PMO) mediated dystrophin restoration therapy. This study has revealed further complexity in the miRNA transcriptome of the mdx mouse, an understanding of which will be valuable in the development of novel therapeutics and for monitoring their efficacy.

Bruton's tyrosine kinase (Btk) is encoded by the gene that when mutated causes the primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk is a member of the Tec family of protein tyrosine kinases (PTKs) and plays a vital, but diverse, modulatory role in many cellular processes. Mutations affecting Btk block B-lymphocyte development. Btk is conserved among species, and in this review, we present the sequence of the full-length rat Btk and find it to be analogous to the mouse Btk sequence. We have also analyzed the wealth of information compiled in the mutation database for XLA (BTKbase), representing 554 unique molecular events in 823 families and demonstrate that only selected amino acids are sensitive to replacement (P < 0.001). Although genotype-phenotype correlations have not been established in XLA, based on these findings, we hypothesize that this relationship indeed exists. Using short interfering-RNA technology, we have previously generated active constructs downregulating Btk expression. However, application of recently established guidelines to enhance or decrease the activity was not successful, demonstrating the importance of the primary sequence. We also review the outcome of expression profiling, comparing B lymphocytes from XLA-, Xid-, and Btk-knockout (KO) donors to healthy controls. Finally, in spite of a few genes differing in expression between Xid- and Btk-KO mice, in vivo competition between cells expressing either mutation shows that there is no selective survival advantage of cells carrying one genetic defect over the other. We conclusively demonstrate that for the R28C-missense mutant (Xid), there is no biologically relevant residual activity or any dominant negative effect versus other proteins.

IntroductionBruton tyrosine kinase (Btk) is a nonreceptor tyrosine kinase belonging to the Tec family of protein tyrosine kinases (PTKs). This family consists of 5 mammalian members: Btk, Itk, Tec, Bmx, and Txk. These kinases are also present in other species. 1,2 Btk is expressed in almost all the hematopoietic cells, except T lymphocytes and plasma cells, and in the B-cell lineage, from the very early up to the mature cell stages. [3][4][5] Btk is required for Blymphocyte survival, proliferation, and differentiation in response to BCR activation. Accordingly, mutations in the gene coding for Btk lead to X-linked agammaglobulinemia (XLA) in humans 6-9 and X-linked immunodeficiency (xid) in mice. 10,11 Btk and its direct substrate, phospholipase C␥2 (PLC-␥2), are essential for the activation of the transcription factor nuclear factor-B (NF-B) in response to BCR engagement. [12][13][14] Moreover, Shinners et al recently found that Btk mediates B-cell-activating factor receptor (BAFF-R)-dependent activation of NF-B, thereby promoting B-cell survival. 15 NF-B is known to be essential for both innate and adaptive immunity. Moreover, it is crucial for the initial responses of sentinel cells to pathogens, as well as for the subsequent events that lead to T cell-and B cell-mediated antigen-specific defense. The role of NF-B in the acute, innate immune response is evolutionarily conserved. 16 NF-B has also been shown to be crucial for the development of several mammalian hematopoietic cell lineages and for the formation of secondary lymphoid-organ structures.The NF-B/Rel family of proteins include NF-B1 (p50), NF-B2 (p52), RelA (p65), c-Rel, and RelB, which can form functional homodimer or heterodimer complexes. 16,17 In resting cells, NF-B is sequestered in the cytoplasm by the inhibitory proteins of the I-B family. Following stimulation of cells by inflammatory cytokines, and bacterial (eg, LPS) and viral products, the inhibitor of B (I-B) is phosphorylated by the I-B kinases (IKKs), leading to its degradation through the ubiquitin-proteasome pathway. Thus, in the absence of I-B, NF-B dimers (p50-p65 and p52-RelB) are released, translocated to the nucleus, and subsequently bound to their cognate elements on target genes. 18 Mice deficient in individual NF-B/Rel family members have demonstrated the essential role of these transcription factors in CD40, TLR4, and IgM receptor pathways leading to B-cell proliferation. In particular, B cells from mice deficient in the NF-B members c-Rel or p65 have decreased responses to antigen cross-linking. 19,20 c-Rel Ϫ/Ϫ B cells also failed to respond to CD40 ligation. An examination of B cells in mice expressing a transdominant form of I-B␣ revealed xid-like defects, including lack of proliferation in response to anti-IgM. 21 The transcriptional regulation of the Btk gene has been studied quite extensively. Accordingly, a number of transcription factors including Sp1, Sp3, Spi-B, PU.1, [22][23][24] and OCT1/OBF1 25 have been shown to bind and activate the Btk promoter. However, most...

innate-like lymphocytes ͉ T cell development ͉ transgenics

Duchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here, we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNA–target interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle.

Transgenically introduced laminin (LN) a1 chain prevents muscular dystrophy in LNa2 chain deficient mice. We now report increased integrin a7Bb1D synthesis in dystrophic LNa2 chain deficient muscle. Yet, immunofluorescence demonstrated a reduced expression of integrin a7B subunit at the sarcolemma. Transgenic expression of LNa1 chain reconstituted integrin a7B at the sarcolemma. Expression of a-and b-dystroglycan is enhanced in LNa2 chain deficient muscle and normalized by transgenic expression of LNa1 chain. We suggest that LNa1 chain in part ameliorates the development of LNa2 chain deficient muscular dystrophy by retaining the binding sites for integrin a7Bb1D and a-dystroglycan, respectively.

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