Rat-specific decreases in platelet count caused by a humanized monoclonal antibody against sclerostin.

LY2541546 is a humanized monoclonal antibody (IgG(4)) that has been optimized for neutralizing activity against sclerostin. In 5-week and 6-month nonclinical safety studies in rats, LY2541546 caused dose-dependent reversible decreases in platelet counts accompanied by accelerated platelet production, increased megakaryocytes, and altered megakaryocyte morphology. These treatment-related effects resulted in altered primary hemostasis as manifested by prolonged bleeding after phlebotomy or incidental toenail break. In some cases, the defects in hemostasis were sufficient to result in death of the affected rats. There was no evidence in rats of general bone marrow suppression or processes (e.g., disseminated intravascular coagulopathy) that may result in thrombocytopenia. Cynomolgus monkeys given LY2541546 for 5 weeks or 9 months had no changes in platelet count or megakaryocytes. In vitro cross-reactivity studies in rats, cynomolgus monkeys, and humans revealed LY2541546-bound rat but not cynomolgus monkey or human platelets and megakaryocytes. These data taken together demonstrated that the platelet and megakaryocyte effects in rats had a species-specific pathogenesis which likely involved LY2541546 binding of a rat-specific antigen on the surface of platelets and megakaryocytes resulting in the increased clearance of platelets and megakaryocyte hyperplasia. The species-specific nature of these reversible toxicological findings combined with the ease of clinical monitoring using standard hematology enabled the safe initiation of clinical studies in human volunteers.

IL-22 induces an acute-phase response.

IL-22 is made by a unique set of innate and adaptive immune cells, including the recently identified noncytolytic NK, lymphoid tissue-inducer, Th17, and Th22 cells. The direct effects of IL-22 are restricted to nonhematopoietic cells, its receptor expressed on the surface of only epithelial cells and some fibroblasts in various organs, including parenchymal tissue of the gut, lung, skin, and liver. Despite this cellular restriction on IL-22 activity, we demonstrate that IL-22 induces effects on systemic biochemical, cellular, and physiological parameters. By utilizing adenoviral-mediated delivery of IL-22 and systemic administration of IL-22 protein, we observed that IL-22 modulates factors involved in coagulation, including fibrinogen levels and platelet numbers, and cellular constituents of blood, such as neutrophil and RBC counts. Furthermore, we observed that IL-22 induces thymic atrophy, body weight loss, and renal proximal tubule metabolic activity. These cellular and physiological parameters are indicative of a systemic inflammatory state. We observed that IL-22 induces biochemical changes in the liver including induction of fibrinogen, CXCL1, and serum amyloid A that likely contribute to the reported cellular and physiological effects of IL-22. Based on these findings, we propose that downstream of its expression and impact in local tissue inflammation, circulating IL-22 can further induce changes in systemic physiology that is indicative of an acute-phase response.

Computing expectation values for RNA motifs using discrete convolutions.

BACKGROUND: Computational biologists use Expectation values (E-values) to estimate the number of solutions that can be expected by chance during a database scan. Here we focus on computing Expectation values for RNA motifs defined by single-strand and helix lod-score profiles with variable helix spans. Such E-values cannot be computed assuming a normal score distribution and their estimation previously required lengthy simulations. RESULTS: We introduce discrete convolutions as an accurate and fast mean to estimate score distributions of lod-score profiles. This method provides excellent score estimations for all single-strand or helical elements tested and also applies to the combination of elements into larger, complex, motifs. Further, the estimated distributions remain accurate even when pseudocounts are introduced into the lod-score profiles. Estimated score distributions are then easily converted into E-values. CONCLUSION: A good agreement was observed between computed E-values and simulations for a number of complete RNA motifs. This method is now implemented into the ERPIN software, but it can be applied as well to any search procedure based on ungapped profiles with statistically independent columns.

Profile-based detection of microRNA precursors in animal genomes.

MOTIVATION: MicroRNAs (miRNA) are essential 21-22 nt regulatory RNAs produced from larger hairpin-like precursors. Local sequence alignment tools such as BLAST are able to identify new members of known miRNA families, but not all of them. We set out to estimate how many new miRNAs could be recovered using a profile-based strategy such as that implemented in the ERPIN program. RESULTS: We constructed alignments for 18 miRNA families and performed ERPIN searches on animal genomes. Results were compared to those of a WU-BLAST search at the same E-value cutoff. The two combined approaches produced 265 new miRNA candidates that were not found in miRNA databases. About 17% of hits were ERPIN specific. They showed better structural characteristics than BLAST-specific hits and included interesting candidates such as members of the miR-17 cluster in Tetraodon. Profile-based RNA detection will be an important complement of similarity search programs in the completion of miRNA collections.

The ERPIN server: an interface to profile-based RNA motif identification.

ERPIN is an RNA motif identification program that takes an RNA sequence alignment as an input and identifies related sequences using a profile-based dynamic programming algorithm. ERPIN differs from other RNA motif search programs in its ability to capture subtle biases in the training set and produce highly specific and sensitive searches, while keeping CPU requirements at a practical level. In its latest version, ERPIN also computes E-values, which tell biologists how likely they are to encounter a specific sequence match by chance-a useful indication of biological significance. We present here the ERPIN online search interface (http://tagc.univ-mrs.fr/erpin/). This web server automatically performs ERPIN searches for different RNA genes or motifs, using predefined training sets and search parameters. With a couple of clicks, users can analyze an entire bacterial genome or a genomic segment of up to 5Mb for the presence of tRNAs, 5S rRNAs, SRP RNA, C/D box snoRNAs, hammerhead motifs, miRNAs and other motifs. Search results are displayed with sequence, score, position, E-value and secondary structure graphics. An example of a complete genome scan is provided, as well as an evaluation of run times and specificity/sensitivity information for all available motifs.

The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice.

Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults.

A gene expression profile for endochondral bone formation: oligonucleotide microarrays establish novel connections between known genes and BMP-2-induced bone formation in mouse quadriceps.

Endochondral bone formation has been fairly well characterized from a morphological perspective and yet this process remains largely undefined at molecular and biochemical levels. In vitro and in vivo studies have shown that human bone morphogenetic protein-2 (hBMP-2) is an important developmental growth and differentiation factor, capable of inducing ectopic bone formation in vivo. This study evaluated several aspects of the osteogenic effect of hBMP-2 protein injected into quadriceps of female C57B1/6J SCID mice. Mice were euthanized 1, 2, 3, 4, 7, and 14 days postinjection and muscles were collected for several methods of analysis. Hematoxylin and eosin-stained sections of muscles injected with formulation buffer showed no evidence of osteogenesis. In contrast, sections of muscles injected with hBMP-2 showed evidence of endochondral bone formation that progressed to mineralized bone by day 14. In addition, radiographs of mice injected with hBMP-2 showed that much of the quadriceps muscle had undergone mineralization by day 14. Labeled mRNA solutions were prepared and hybridized to oligonucleotide arrays designed to monitor approximately 1300 murine, full-length genes. Changes in gene expression associated with hBMP-2 were determined from time-matched comparisons between buffer and hBMP-2 samples. A gene expression profile was created for 215 genes that showed greater than 4-fold changes at one or more of the indicated time points. One hundred twenty-two of these genes have previously been associated with bone or cartilage metabolism and showed significant increases in expression, e.g., aggrecan (Agc1), runt related transcription factor 2 (Runx2), bone Gla protein 1 (Bglap1), and procollagens type II (Col2a1) and X (Col10a1). In addition, there were 93 genes that have not been explicitly associated with bone or cartilage metabolism. Two of these genes, cytokine receptor-like factor-1 (Crlf1) and matrix metalloproteinase 23 (Mmp23), showed peak changes in gene expression of 15- and 40-fold on days 4 and 7, respectively. In situ hybridizations of muscle sections showed that Mmp23 and Crlf1 mRNAs were expressed in chondrocytes and osteoblasts, suggesting a role for both proteins in some aspect of cartilage or bone formation. In conclusion, oligonucleotide arrays enabled a broader view of endochondral bone formation than has been reported to date. An increased understanding of the roles played by these gene products will improve our understanding of skeletogenesis, fracture repair, and pathological conditions such as osteoporosis.

A survey of metazoan selenocysteine insertion sequences.

The computational detection of novel selenoproteins in genomic sequences is usually achieved through identification of SECIS, a conserved secondary structure element found in the 3' UTR of animal selenoprotein mRNAs. Previous studies have used "descriptors" specifying the number of base pairs and the conserved nucleotides in SECIS to identify this element. A major drawback of the "descriptor" approach is that the number of detections in current genomic or transcript databases largely exceeds the number of true selenoproteins. In this study, we use instead the ERPIN program to detect SECIS elements. ERPIN is based on a lod-score profile algorithm that uses a training-set of aligned RNA sequences as input. From an initial alignment of 44 animal SECIS sequences, we performed a series of iterative searches in which the training set was progressively enriched up to 117 confirmed SECIS elements, from a large collection of metazoan species. About 200 high-scoring candidates were also detected. We show that ERPIN scores for these candidates can be converted into expect values, thus enabling their statistical evaluation. The most interesting SECIS candidates are presented.