Surgical restabilization reduces the progression of post-traumatic osteoarthritis initiated by ACL rupture in mice.

OBJECTIVE: People who sustain joint injuries such as anterior cruciate ligament (ACL) rupture often develop post-traumatic osteoarthritis (PTOA). In human patients, ACL injuries are often treated with ACL reconstruction. However, it is still unclear how effective joint restabilization is for reducing the progression of PTOA. The goal of this study was to determine how surgical restabilization of a mouse knee joint following non-invasive ACL injury affects PTOA progression. DESIGN: In this study, 187 mice were subjected to non-invasive ACL injury or no injury. After injury, mice underwent restabilization surgery, sham surgery, or no surgery. Mice were then euthanized on day 14 or day 49 after injury/surgery. Functional analyses were performed at multiple time points to assess voluntary movement, gait, and pain. Knees were analyzed ex vivo with micro-computed tomography, RT-PCR, and whole-joint histology to assess articular cartilage degeneration, synovitis, and osteophyte formation. RESULTS: Both ACL injury and surgery resulted in loss of epiphyseal trabecular bone (-27-32%) and reduced voluntary movement at early time points. Joint restabilization successfully lowered OA score (-78% relative to injured at day 14, p < 0.0001), and synovitis scores (-37% relative to injured at day 14, p = 0.042), and diminished the formation of chondrophytes/osteophytes (-97% relative to injured at day 14, p < 0.001, -78% at day 49, p < 0.001). CONCLUSIONS: This study confirmed that surgical knee restabilization was effective at reducing articular cartilage degeneration and diminishing chondrophyte/osteophyte formation after ACL injury in mice, suggesting that these processes are largely driven by joint instability in this mouse model. However, restabilization was not able to mitigate the early inflammatory response and the loss of epiphyseal trabecular bone, indicating that these processes are independent of joint instability.

CD8ß Depletion Does Not Prevent Control of Viral Replication or Protection from Challenge in Macaques Chronically Infected with a Live Attenuated Simian Immunodeficiency Virus.

We evaluated the contribution of CD8αß+ T cells to control of live-attenuated simian immunodeficiency virus (LASIV) replication during chronic infection and subsequent protection from pathogenic SIV challenge. Unlike previous reports with a CD8α-specific depleting monoclonal antibody (mAb), the CD8ß-specific mAb CD8ß255R1 selectively depleted CD8αß+ T cells without also depleting non-CD8+ T cell populations that express CD8α, such as natural killer (NK) cells and γδ T cells. Following infusion with CD8ß255R1, plasma viremia transiently increased coincident with declining peripheral CD8αß+ T cells. Interestingly, plasma viremia returned to predepletion levels even when peripheral CD8αß+ T cells did not. Although depletion of CD8αß+ T cells in the lymph node (LN) was incomplete, frequencies of these cells were 3-fold lower (P = 0.006) in animals that received CD8ß255R1 than in those that received control IgG. It is possible that these residual SIV-specific CD8αß+ T cells may have contributed to suppression of viremia during chronic infection. We also determined whether infusion of CD8ß255R1 in the LASIV-vaccinated animals increased their susceptibility to infection following intravenous challenge with pathogenic SIVmac239. We found that 7/8 animals infused with CD8ß255R1, and 3/4 animals infused with the control IgG, were resistant to SIVmac239 infection. These results suggest that infusion with CD8ß255R1 did not eliminate the protection afforded to LASIV vaccination. This provides a comprehensive description of the impact of CD8ß255R1 infusion on the immunological composition in cynomolgus macaques, compared to an isotype-matched control IgG, while showing that the control of LASIV viremia and protection from challenge can occur even after CD8ß255R1 administration.IMPORTANCE Studies of SIV-infected macaques that deplete CD8+ T cells in vivo with monoclonal antibodies have provided compelling evidence for their direct antiviral role. These studies utilized CD8α-specific mAbs that target both the major (CD8αß+) and minor (CD8αα+) populations of CD8+ T cells but additionally deplete non-CD8+ T cell populations that express CD8α, such as NK cells and γδ T cells. In the current study, we administered the CD8ß-specific depleting mAb CD8ß255R1 to cynomolgus macaques chronically infected with a LASIV to selectively deplete CD8αß+ T cells without removing CD8αα+ lymphocytes. We evaluated the impact on control of virus replication and protection from pathogenic SIVmac239 challenge. These results underscore the utility of CD8ß255R1 for studying the direct contribution of CD8αß+ T cells in various disease states.

Two-color in situ hybridization of whole-mount mouse embryos.

RNA in situ hybridization is a powerful technique used to identify the spatial localization of a specific RNA in a tissue section or whole tissue. In this protocol, we describe a reliable method for two-color in situ hybridization that can be used to accurately assess the expression of multiple genes with contrasting or overlapping expression patterns in whole mouse embryos.

The Wnt3a/ß-catenin target gene Mesogenin1 controls the segmentation clock by activating a Notch signalling program.

Segmentation is an organizing principle of body plans. The segmentation clock, a molecular oscillator best illustrated by the cyclic expression of Notch signalling genes, controls the periodic cleavage of somites from unsegmented presomitic mesoderm during vertebrate segmentation. Wnt3a controls the spatiotemporal expression of cyclic Notch genes; however, the underlying mechanisms remain obscure. Here we show by transcriptional profiling of Wnt3a (-/-) embryos that the bHLH transcription factor, Mesogenin1 (Msgn1), is a direct target gene of Wnt3a. To identify Msgn1 targets, we conducted genome-wide studies of Msgn1 activity in embryonic stem cells. We show that Msgn1 is a major transcriptional activator of a Notch signalling program and synergizes with Notch to trigger clock gene expression. Msgn1 also indirectly regulates cyclic genes in the Fgf and Wnt pathways. Thus, Msgn1 is a central component of a transcriptional cascade that translates a spatial Wnt3a gradient into a temporal pattern of clock gene expression.

The extracellular domain of Lrp5/6 inhibits noncanonical Wnt signaling in vivo.

Lrp5/6 are crucial coreceptors for Wnt/beta-catenin signaling, a pathway biochemically distinct from noncanonical Wnt signaling pathways. Here, we examined the possible participation of Lrp5/6 in noncanonical Wnt signaling. We found that Lrp6 physically interacts with Wnt5a, but that this does not lead to phosphorylation of Lrp6 or activation of the Wnt/beta-catenin pathway. Overexpression of Lrp6 blocks activation of the Wnt5a downstream target Rac1, and this effect is dependent on intact Lrp6 extracellular domains. These results suggested that the extracellular domain of Lrp6 inhibits noncanonical Wnt signaling in vitro. In vivo, Lrp6-/- mice exhibited exencephaly and a heart phenotype. Surprisingly, these defects were rescued by deletion of Wnt5a, indicating that the phenotypes resulted from noncanonical Wnt gain-of-function. Similarly, Lrp5 and Lrp6 antisense morpholino-treated Xenopus embryos exhibited convergent extension and heart phenotypes that were rescued by knockdown of noncanonical XWnt5a and XWnt11. Thus, we provide evidence that the extracellular domains of Lrp5/6 behave as physiologically relevant inhibitors of noncanonical Wnt signaling during Xenopus and mouse development in vivo.

Wnt3a/beta-catenin signaling controls posterior body development by coordinating mesoderm formation and segmentation.

Somitogenesis is thought to be controlled by a segmentation clock, which consists of molecular oscillators in the Wnt3a, Fgf8 and Notch pathways. Using conditional alleles of Ctnnb1 (beta-catenin), we show that the canonical Wnt3a/beta-catenin pathway is necessary for molecular oscillations in all three signaling pathways but does not function as an integral component of the oscillator. Small, irregular somites persist in abnormally posterior locations in the absence of beta-catenin and cycling clock gene expression. Conversely, Notch pathway genes continue to oscillate in the presence of stabilized beta-catenin but boundary formation is delayed and anteriorized. Together, these results suggest that the Wnt3a/beta-catenin pathway is permissive but not instructive for oscillating clock genes and that it controls the anterior-posterior positioning of boundary formation in the presomitic mesoderm (PSM). The Wnt3a/beta-catenin pathway does so by regulating the activation of the segment boundary determination genes Mesp2 and Ripply2 in the PSM through the activation of the Notch ligand Dll1 and the mesodermal transcription factors T and Tbx6. Spatial restriction of Ripply2 to the anterior PSM is ensured by the Wnt3a/beta-catenin-mediated repression of Ripply2 in posterior PSM. Thus, Wnt3a regulates somitogenesis by activating a network of interacting target genes that promote mesodermal fates, activate the segmentation clock, and position boundary determination genes in the anterior PSM.

Mouse Ripply2 is downstream of Wnt3a and is dynamically expressed during somitogenesis.

Somites are blocks of mesoderm that form when segment boundaries are periodically generated in the anterior presomitic mesoderm (PSM). Periodicity is thought to be driven by an oscillating Notch-centered segmentation clock, whereas boundaries are spatially positioned by the secreted signaling molecules Wnt3a and Fgf8. We identified the putative transcriptional corepressor Ripply2 as a differentially expressed gene in wild-type and Wnt3a(-/-) embryos. Here, we show that Ripply2 is expressed in the anterior PSM and that it indeed lies downstream of Wnt3a. Dynamic Ripply2 expression in prospective somites S0 and S-I overlaps with the rostral expression of cycling genes in the Notch pathway, suggesting that Ripply2 may be controlled by the segmentation clock. Continued expression of Ripply2 in embryos lacking Hes7, a molecular oscillator in the Notch clock, indicates that Hes7 is not a major regulator of Ripply2. Our data are consistent with Ripply2 functioning as a segment boundary determination gene during mammalian embryogenesis. Developmental