The process of fracture healing is complex, and poor or incomplete healing remains a significant health problem. Proper fracture healing relies upon resident mesenchymal stem cell (MSC) differentiation into chondrocytes and osteoblasts, which are necessary for callus formation and ossification. Alcohol abuse is a leading contributor to poor fracture healing. Although the mechanism behind this action is unknown, excessive alcohol consumption is known to promote systemic oxidative stress. The family of FoxO transcription factors is activated by oxidative stress, and FoxO activation antagonizes Wnt signaling, which regulates mesenchymal stem cell differentiation. We hypothesize that alcohol exposure increases oxidative stress leading to deficient fracture repair by activating FoxO transcription factors within the fracture callus which disrupts chondrogenesis of mesenchymal stem cells. Our laboratory has developed an experimental model of delayed fracture union in mice using ethanol administration. We have found that ethanol administration significantly decreases external, cartilaginous callus formation, and hallmarks of endochondral ossification, and these changes are concomitant with increases in FoxO expression and markers of activation in fracture callus tissue of these mice. We were able to prevent these alcohol-induced effects with the administration of the antioxidant n-acetyl cysteine (NAC), suggesting that alcohol-induced oxidative stress produces the perturbed endochondral ossification and FoxO expression.

Sialic acid‐binding immunoglobulin‐type lectins (Siglecs) are a family of immunoglobulin‐type lectins that mediate protein‐carbohydrate interactions via sialic acids attached to glycoproteins or glycolipids. Most of the CD33‐related Siglecs (CD33rSiglecs), a major subfamily of rapidly evolving Siglecs, contain a cytoplasmic signaling domain consisting of the immunoreceptor tyrosine‐based inhibitory motif (ITIM) and immunoreceptor tyrosine‐based switch motif (ITSM) and mediate suppressive signals for lymphoid and myeloid cells. While most CD33rSiglecs are expressed by innate immune cells, such as monocytes and neutrophils, to date, the expression of Siglecs in human T cells has not been well appreciated. In this study, we found that Siglec‐5, a member of the CD33rSiglecs, is expressed by most activated T cells upon antigen receptor stimulation. Functionally, Siglec‐5 suppresses T cell activation. In support of these findings, we found that Siglec‐5 overexpression abrogates antigen receptor induced activation of NFAT and AP‐1. Furthermore, we show that GBS β‐protein, a known bacterial ligand of Siglec‐5, reduces the production of cytokines and cytolytic molecules by activated primary T cells in a Siglec‐5 dependent manner. Our data also show that some cancer cell lines express a putative Siglec‐5 ligand(s), and that the presence of soluble Siglec‐5 enhances tumor‐cell specific T cell activation, suggesting that some tumor cells inhibit T cell activation via Siglec‐5. Together, our data demonstrate that Siglec‐5 is a previously unrecognized inhibitory T cell immune checkpoint molecule and suggest that blockade of Siglec‐5 could serve as a new strategy to enhance anti‐tumor T cell functions.

BackgroundAlcohol consumption is a risk factor for impaired fracture healing, though the mechanism(s) by which this occurs are not well understood. Our laboratory has previously shown that episodic alcohol exposure of rodents negatively affects fracture callus development, callus biomechanics, and cellular signaling which regulates stem cell differentiation. Here, we examine whether alcohol alters chemokine expression and/or signaling activity in the mouse fracture callus during early fracture healing.MethodsA mouse model for alcohol-impaired tibia fracture healing was utilized. Early fracture callus was examined for alcohol-effects on tissue composition, expression of chemokines involved in MSC migration to the fracture site, and biomechanics. The effects of alcohol on MSC migration and cell adhesion receptors were examined in an in vitro system.ResultsMice exposed to alcohol showed decreased evidence of external callus formation, decreased callus-related osteopontin (OPN) expression levels, and decreased biomechanical stiffness. Alcohol exposure decreased rOPN-mediated MSC migration and integrin β1 receptor expression in vitro.ConclusionsThe effects of alcohol exposure demonstrated here on fracture callus-associated OPN expression, rOPN-mediated MSC migration in vitro, and MSC integrin β1 receptor expression in vitro have not been previously reported. Understanding the effects of alcohol exposure on the early stages of fracture repair may allow timely initiation of treatment to mitigate the long-term complications of delayed healing and/or fracture non-union.

RNA recognition motif-type RNA-binding domain containing proteins (RBDPs) participate in RNA metabolism including regulating mRNA stability, nuclear-cytoplasmic shuttling, and splicing. Rbm45 is an RBDP first cloned from rat brain and expressed spatiotemporally during rat neural development. More recently, RBM45 has been associated with pathological aggregates in the human neurological disorders amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer’s. Rbm45 and the neural developmental protein musashi-1 are in the same family of RDBPs and have similar expression patterns. In contrast to Musashi-1, which is upregulated during colorectal carcinogenesis, we found no association of RBM45 overexpression in human colon cancer tissue. In order to begin characterizing RNA-binding partners of Rbm45, we have successfully cloned and expressed human RBM45 in an Intein fusion-protein expression system. Furthermore, to gain a better understanding of the molecular genetics and evolution of Rbm45, we used an in silico approach to analyze the gene structure of the human and mouse Rbm45 homologues and explored the evolutionary conservation of Rbm45 in metazoans. Human RBM45 and mouse Rbm45 span ~17 kb and 13 kb, respectively, and contain 10 exons, one of which is non-coding. Both genes have TATA-less promoters with an initiator and a GC-rich element. Downstream of exon 10, both homologues have canonical polyadenylation signals and an embryonic cytoplasmic polyadenylation element. Moreover, our data indicate Rbm45 is conserved across all metazoan taxa from sponges (phylum Porifera) to humans (phylum Chordata), portending a fundamental role in metazoan development.

The fetal and neonatal immune systems are uniquely poised to generate tolerance to self, maternal and environmental antigens encountered in the womb and shortly after birth. However, the tolerogenic nature of fetal and neonatal immunity can be detrimental in the context of pathogens, leading to overwhelming bacterial infections or chronic viral infections. A variety of mechanisms contribute to fetal and neonatal tolerance, including a propensity to generate Foxp3 + regulatory T cells (Treg cells). However, the mechanism(s) of fetal Foxp3 + T-cell differentiation, the specific antigen-presenting cells required and factors that inhibit Treg generation after the neonatal period are poorly understood. Here, we demonstrate that a subset of CD14 + monocytes expressing the scavenger molecule, CD36, can generate CD4 + and CD8 + T cells that coexpress Foxp3 and Tbet from both umbilical cord blood. These Foxp3 + T-bet + T cells potently suppress T-cell proliferation and ameliorate xenogeneic graft-versus-host disease. CD14 + CD36 + monocytes provide known Treg-inducing signals: membrane-bound transforming growth factor-beta and retinoic acid. Unexpectedly, adult peripheral blood monocytes are also capable of inducing Foxp3 + T cells from both cord blood and adult peripheral na€ ıve T cells. The induction of Foxp3 + T cells in umbilical cord blood by monocytes was inhibited by the lymphoid fraction of adult peripheral blood cells. These studies highlight a novel immunoregulatory role of monocytes and suggest that antigen presentation by CD36 hi monocytes may contribute to the peripheral development of Foxp3 + T-bet + T cells with regulatory functions in both neonates and adults.