Epicardial deletion of Sox9 leads to myxomatous valve degeneration and identifies Cd109 as a novel gene associated with valve development.

Epicardial-derived cells (EPDCs) are involved in the regulation of myocardial growth and coronary vascularization and are critically important for proper development of the atrioventricular (AV) valves. SOX9 is a transcription factor expressed in a variety of epithelial and mesenchymal cells in the developing heart, including EPDCs. To determine the role of SOX9 in epicardial development, an epicardial-specific Sox9 knockout mouse model was generated. Deleting Sox9 from the epicardial cell lineage impairs the ability of EPDCs to invade both the ventricular myocardium and the developing AV valves. After birth, the mitral valves of these mice become myxomatous with associated abnormalities in extracellular matrix organization. This phenotype is reminiscent of that seen in humans with myxomatous mitral valve disease (MVD). An RNA-seq analysis was conducted in an effort to identify genes associated with this myxomatous degeneration. From this experiment, Cd109 was identified as a gene associated with myxomatous valve pathogenesis in this model. Cd109 has never been described in the context of heart development or valve disease. This study highlights the importance of SOX9 in the regulation of epicardial cell invasion-emphasizing the importance of EPDCs in regulating AV valve development and homeostasis-and reports a novel expression profile of Cd109, a gene with previously unknown relevance in heart development.

Peripheral Auditory Nerve Impairment in a Mouse Model of Syndromic Autism.

Dysfunction of the peripheral auditory nerve (AN) contributes to dynamic changes throughout the central auditory system, resulting in abnormal auditory processing, including hypersensitivity. Altered sound sensitivity is frequently observed in autism spectrum disorder (ASD), suggesting that AN deficits and changes in auditory information processing may contribute to ASD-associated symptoms, including social communication deficits and hyperacusis. The MEF2C transcription factor is associated with risk for several neurodevelopmental disorders, and mutations or deletions of MEF2C produce a haploinsufficiency syndrome characterized by ASD, language, and cognitive deficits. A mouse model of this syndromic ASD (Mef2c-Het) recapitulates many of the MEF2C haploinsufficiency syndrome-linked behaviors, including communication deficits. We show here that Mef2c-Het mice of both sexes exhibit functional impairment of the peripheral AN and a modest reduction in hearing sensitivity. We find that MEF2C is expressed during development in multiple AN and cochlear cell types; and in Mef2c-Het mice, we observe multiple cellular and molecular alterations associated with the AN, including abnormal myelination, neuronal degeneration, neuronal mitochondria dysfunction, and increased macrophage activation and cochlear inflammation. These results reveal the importance of MEF2C function in inner ear development and function and the engagement of immune cells and other non-neuronal cells, which suggests that microglia/macrophages and other non-neuronal cells might contribute, directly or indirectly, to AN dysfunction and ASD-related phenotypes. Finally, our study establishes a comprehensive approach for characterizing AN function at the physiological, cellular, and molecular levels in mice, which can be applied to animal models with a wide range of human auditory processing impairments.SIGNIFICANCE STATEMENT This is the first report of peripheral auditory nerve (AN) impairment in a mouse model of human MEF2C haploinsufficiency syndrome that has well-characterized ASD-related behaviors, including communication deficits, hyperactivity, repetitive behavior, and social deficits. We identify multiple underlying cellular, subcellular, and molecular abnormalities that may contribute to peripheral AN impairment. Our findings also highlight the important roles of immune cells (e.g., cochlear macrophages) and other non-neuronal elements (e.g., glial cells and cells in the stria vascularis) in auditory impairment in ASD. The methodological significance of the study is the establishment of a comprehensive approach for evaluating peripheral AN function and impact of peripheral AN deficits with minimal hearing loss.

A molecular perspective on TH2-promoting cytokine receptors in patients with allergic disease.

The cytokines IL-4, IL-13, and thymic stromal lymphopoietin play a key role in allergic disease by virtue of their ability to initiate, maintain, and augment TH2 responses. These molecules mediate their effects through type 1 cytokine receptors, which bind cytokines with a characteristic structure. Receptors are expressed on a broad array of immune cell types and are integral to complex cytokine networks operating in health and disease. TH2-promoting cytokines bind different configurations of receptors. Receptor subunits can exist in surface-bound or soluble forms, as well as in isolation or in partnership with other subunits. Sharing of receptor subunits among different cytokine receptor complexes adds to the intricate landscape. This article describes the characteristics of receptors for IL-4, IL-13, and thymic stromal lymphopoietin and their respective ligands from a structure-function perspective. We detail the mechanisms of receptor complex assembly, the interrelated nature of these receptors, and the effect on allergic inflammation. The ability for novel and atypical types of receptors to modulate inflammatory processes is also discussed. We highlight current and emerging treatments that target TH2-promoting receptor complexes. Understanding the molecular features of these receptors provides insight into different disease phenotypes and the variable clinical outcomes arising from targeted therapies. These considerations can be used to inform future directions for research and creative strategies for treating individual patients.

High affinity chimeric antigen receptor signaling induces an inflammatory program in human regulatory T cells.

Regulatory T cells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) T-cell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here, we compared CAR with endogenous T cell receptor (TCR)/CD28 activation in human Tregs. Strikingly, CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28 activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed, CAR Tregs secreted high levels of inflammatory cytokines, with a subset of FOXP3+ CAR Tregs uniquely acquiring CD40L surface expression and producing IFNγ. Interestingly, decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy.

MYC is sufficient to generate mid-life high-grade serous ovarian and uterine serous carcinomas in a p53-R270H mouse model.

Genetically engineered mouse models (GEMM) have fundamentally changed how ovarian cancer etiology, early detection, and treatment is understood. However, previous GEMMs of high-grade serous ovarian cancer (HGSOC) have had to utilize genetics rarely or never found in human HGSOC to yield ovarian cancer within the lifespan of a mouse. MYC, an oncogene, is amongst the most amplified genes in HGSOC, but it has not previously been utilized to drive HGSOC GEMMs. We coupled Myc and dominant negative mutant p53-R270H with a fallopian tube epithelium-specific promoter Ovgp1 to generate a new GEMM of HGSOC. Female mice developed lethal cancer at an average of 15.1 months. Histopathological examination of mice revealed HGSOC characteristics including nuclear p53 and nuclear MYC in clusters of cells within the fallopian tube epithelium and ovarian surface epithelium. Unexpectedly, nuclear p53 and MYC clustered cell expression was also identified in the uterine luminal epithelium, possibly from intraepithelial metastasis from the fallopian tube epithelium (FTE). Extracted tumor cells exhibited strong loss of heterozygosity at the p53 locus, leaving the mutant allele. Copy number alterations in these cancer cells were prevalent, disrupting a large fraction of genes. Transcriptome profiles most closely matched human HGSOC and serous endometrial cancer. Taken together, these results demonstrate the Myc and Trp53-R270H transgene was able to recapitulate many phenotypic hallmarks of HGSOC through the utilization of strictly human-mimetic genetic hallmarks of HGSOC. This new mouse model enables further exploration of ovarian cancer pathogenesis, particularly in the 50% of HGSOC which lack homology directed repair mutations. Histological and transcriptomic findings are consistent with the hypothesis that uterine serous cancer may originate from the fallopian tube epithelium.

Targeting TACC3 represents a novel vulnerability in highly aggressive breast cancers with centrosome amplification.

Centrosome amplification (CA) is a hallmark of cancer that is strongly associated with highly aggressive disease and worse clinical outcome. Clustering extra centrosomes is a major coping mechanism required for faithful mitosis of cancer cells with CA that would otherwise undergo mitotic catastrophe and cell death. However, its underlying molecular mechanisms have not been fully described. Furthermore, little is known about the processes and players triggering aggressiveness of cells with CA beyond mitosis. Here, we identified Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) to be overexpressed in tumors with CA, and its high expression is associated with dramatically worse clinical outcome. We demonstrated, for the first time, that TACC3 forms distinct functional interactomes regulating different processes in mitosis and interphase to ensure proliferation and survival of cancer cells with CA. Mitotic TACC3 interacts with the Kinesin Family Member C1 (KIFC1) to cluster extra centrosomes for mitotic progression, and inhibition of this interaction leads to mitotic cell death via multipolar spindle formation. Interphase TACC3 interacts with the nucleosome remodeling and deacetylase (NuRD) complex (HDAC2 and MBD2) in nucleus to inhibit the expression of key tumor suppressors (e.g., p21, p16 and APAF1) driving G1/S progression, and its inhibition blocks these interactions and causes p53-independent G1 arrest and apoptosis. Notably, inducing CA by p53 loss/mutation increases the expression of TACC3 and KIFC1 via FOXM1 and renders cancer cells highly sensitive to TACC3 inhibition. Targeting TACC3 by guide RNAs or small molecule inhibitors strongly inhibits growth of organoids and breast cancer cell line- and patient-derived xenografts with CA by induction of multipolar spindles, mitotic and G1 arrest. Altogether, our results show that TACC3 is a multifunctional driver of highly aggressive breast tumors with CA and that targeting TACC3 is a promising approach to tackle this disease.

Heparan sulfate modification of the transmembrane receptor CD47 is necessary for inhibition of T cell receptor signaling by thrombospondin-1.

Cell surface proteoglycans on T cells contribute to retroviral infection, binding of chemokines and other proteins, and are necessary for some T cell responses to the matricellular glycoprotein thrombospondin-1. The major cell surface proteoglycans expressed by primary T cells and Jurkat T cells have an apparent M(r) > 200,000 and are modified with chondroitin sulfate and heparan sulfate chains. Thrombospondin-1 bound in a heparin-inhibitable manner to this proteoglycan and to a soluble form released into the medium. Based on mass spectrometry, knockdown, and immunochemical analyses, the proteoglycan contains two major core proteins as follows: amyloid precursor-like protein-2 (APLP2, apparent M(r) 230,000) and CD47 (apparent M(r) > 250,000). CD47 is a known thrombospondin-1 receptor but was not previously reported to be a proteoglycan. This proteoglycan isoform of CD47 is widely expressed on vascular cells. Mutagenesis identified glycosaminoglycan modification of CD47 at Ser(64) and Ser(79). Inhibition of T cell receptor signaling by thrombospondin-1 was lost in CD47-deficient T cells that express the proteoglycan isoform of APLP2, indicating that binding to APLP2 is not sufficient. Inhibition of CD69 induction was restored in CD47-deficient cells by re-expressing CD47 or an S79A mutant but not by the S64A mutant. Therefore, inhibition of T cell receptor signaling by thrombospondin-1 is mediated by CD47 and requires its modification at Ser(64).

STAT3 in tumor fibroblasts promotes an immunosuppressive microenvironment in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is associated with an incredibly dense stroma, which contributes to its recalcitrance to therapy. Cancer-associated fibroblasts (CAFs) are one of the most abundant cell types within the PDAC stroma and have context-dependent regulation of tumor progression in the tumor microenvironment (TME). Therefore, understanding tumor-promoting pathways in CAFs is essential for developing better stromal targeting therapies. Here, we show that disruption of the STAT3 signaling axis via genetic ablation of Stat3 in stromal fibroblasts in a Kras G12D PDAC mouse model not only slows tumor progression and increases survival, but re-shapes the characteristic immune-suppressive TME by decreasing M2 macrophages (F480+CD206+) and increasing CD8+ T cells. Mechanistically, we show that loss of the tumor suppressor PTEN in pancreatic CAFs leads to an increase in STAT3 phosphorylation. In addition, increased STAT3 phosphorylation in pancreatic CAFs promotes secretion of CXCL1. Inhibition of CXCL1 signaling inhibits M2 polarization in vitro. The results provide a potential mechanism by which CAFs promote an immune-suppressive TME and promote tumor progression in a spontaneous model of PDAC.

Modeling ex vivo tumor-infiltrating lymphocyte expansion from established solid malignancies.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) elicits the regression of metastatic malignancies, yet a low proportion of patients achieve complete durable responses. The high incidence of relapse in these patients highlights the need to better understand mechanisms of tumor escape from T cell control. While melanoma has provided the foundation for developing TIL therapy, much less is known about TIL efficacy and relapse in other malignancies. We sought to investigate TIL characteristics in mouse tumors which have not been studied in this setting. Here, we expanded murine TIL ex vivo in IL-2 from fragments of multiple tumor models, including oral cavity cancer models of varying immunogenicity. Additionally, TIL was expanded from pmel-1 mice bearing B16F10 melanoma, yielding an enriched population of tumor-infiltrating TCR transgenic T cells. Murine TIL are similar to human TIL in that they express high levels of inhibitory receptors (PD-1, Tim-3, etc.) and can be expanded ex vivo in IL-2 extensively. Of clinical relevance, we draw parallels between murine and human oral cavity cancer TIL, evaluating relationships between inhibitory receptor expression and function. This platform can be used by labs even in the absence of clinical specimens or clean cell facilities and will be important to more broadly understand TIL phenotypes across many different malignancies.

Response and recurrence correlates in individuals treated with neoadjuvant anti-PD-1 therapy for resectable oral cavity squamous cell carcinoma.

Neoadjuvant PD-1 blockade may be efficacious in some individuals with high-risk, resectable oral cavity head and neck cancer. To explore correlates of response patterns to neoadjuvant nivolumab treatment and post-surgical recurrences, we analyzed longitudinal tumor and blood samples in a cohort of 12 individuals displaying 33% responsiveness. Pretreatment tumor-based detection of FLT4 mutations and PTEN signature enrichment favors response, and high tumor mutational burden improves recurrence-free survival. In contrast, preexisting and/or acquired mutations (in CDKN2A, YAP1, or JAK2) correlate with innate resistance and/or tumor recurrence. Immunologically, tumor response after therapy entails T cell receptor repertoire diversification in peripheral blood and intratumoral expansion of preexisting T cell clones. A high ratio of regulatory T to T helper 17 cells in pretreatment blood predicts low T cell receptor repertoire diversity in pretreatment blood, a low cytolytic T cell signature in pretreatment tumors, and innate resistance. Our study provides a molecular framework to advance neoadjuvant anti-PD-1 therapy for individuals with resectable head and neck cancer.

Neoadjuvant presurgical PD-1 inhibition in oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OCSCC) is a prevalent surgically treated subset of head and neck cancer with frequent recurrence and poor survival. Immunotherapy has demonstrated efficacy in recurrent/metastatic head and neck cancer. However, whether antitumor responses could be fostered by neoadjuvant presurgical immunotherapy remains unclear. Using a Simon's two-stage design, we present results of a single-arm phase-II trial where 12 patients with stage II-IVA OCSCC received 3 to 4 biweekly doses of 3 mg/kg nivolumab followed by definitive surgical resection with curative intent. Presurgical nivolumab therapy in this cohort shows an overall response rate of 33% (n = 4 patients; 95% CI: 12%-53%). With a median follow up of 2.23 years, 10 out of 12 treated patients remain alive. Neoadjuvant nivolumab is safe, well-tolerated, and is not associated with delays in definitive surgical treatment in this study. This work demonstrates feasibility and safety for incorporation of nivolumab in the neoadjuvant setting for OCSCC (ClinicalTrials.gov: NCT03021993).

Increasing survival of ischemic tissue by targeting CD47.

Thrombospondin-1 (TSP1) limits the angiogenic and vasodilator activities of NO. This activity of TSP1 can be beneficial in some disease states, but endogenous TSP1 limits recovery of tissue perfusion following fixed ischemic injury in dorsal skin flaps in mice. Using mice lacking the TSP1 receptors CD36 or CD47, we now show that CD47 is the necessary receptor for limiting NO-mediated vascular smooth muscle relaxation and tissue survival following ischemic injury in skin flaps and hindlimbs. We further show that blocking CD47 or TSP1 using monoclonal antibodies and decreasing CD47 expression using an antisense morpholino oligonucleotide are effective therapeutic approaches to dramatically increase survival of soft tissue subjected to fixed ischemia. These treatments facilitate rapid vascular remodeling to restore tissue perfusion and increase skin and muscle viability. Thus, limiting CD47-dependent antagonism of NO-mediated vasodilation and vascular remodeling is a promising therapeutic modality to preserve tissues subject to ischemic stress.

Gene silencing of CD47 and antibody ligation of thrombospondin-1 enhance ischemic tissue survival in a porcine model: implications for human disease.

BACKGROUND: Insufficient tissue perfusion underlies many acute and chronic diseases. Tissue perfusion in turn requires adequate blood flow, determined in large part by the relative state of relaxation or constriction of arterial vessels. Nitric oxide (NO) produced by vascular cells modulates blood flow and tissue perfusion by relaxing and dilating arteries. Recently, we reported that the secreted protein thrombospondin-1 (TSP1), through its cell surface receptor CD47, limits the ability of NO to relax and dilate blood vessels and thus decreases tissue perfusion. In the present study, we tested the hypothesis that blocking TSP1-CD47 signaling increases ischemic tissue survival in random cutaneous porcine flaps. METHODS: Random cutaneous flaps 2 x 10 cm2 were raised in white hairless Yucatan miniature pigs and were treated with a monoclonal antibody to TSP1, an antisense morpholino oligonucleotide to CD47 or control agents and tissue survival assessed. Primary vascular smooth muscle cell cultured from Yucatan pigs were also treated with the same agents +/- and an NO donor (DEA/NO) and cGMP quantified. RESULTS: Antibody blockade of TSP1 or morpholino suppression of CD47 dramatically enhanced survival of random tissue flaps. These responses correlated with increased blood vessel patency and tissue blood flow on vessel injection studies. NO-stimulated cGMP flux in Yucatan vascular smooth muscle cell was abrogated after antibody or morpholino treatment. CONCLUSION: Antibody ligation of TSP1 or antisense morpholino knock down of CD47 greatly increased tissue survival to ischemia. Given the similarity between porcine and human soft tissues these results suggest significant therapeutic potential for people.

A novel Ras inhibitor, Eri1, engages yeast Ras at the endoplasmic reticulum.

Ras oncoproteins are monomeric GTPases that link signals from the cell surface to pathways that regulate cell proliferation and differentiation. Constitutively active mutant forms of Ras are found in ca. 30% of human tumors. Here we report the isolation of a novel gene from Saccharomyces cerevisiae, designated ERI1 (for endoplasmic reticulum-associated Ras inhibitor 1), which behaves genetically as an inhibitor of Ras signaling. ERI1 encodes a 68-amino-acid protein that associates in vivo with GTP-bound Ras in a manner that requires an intact Ras-effector loop, suggesting that Eri1 competes for the same binding site as Ras target proteins. We show that Eri1 localizes primarily to the membrane of the endoplasmic reticulum (ER), where it engages Ras. The recent demonstration that signaling from mammalian Ras is not restricted to the cell surface but can also proceed from the cytoplasmic face of the ER suggests a regulatory function for Eri1 at that membrane.

HTL1 encodes a novel factor that interacts with the RSC chromatin remodeling complex in Saccharomyces cerevisiae.

RSC is an essential chromatin remodeling complex in Saccharomyces cerevisiae that performs central roles in transcriptional regulation and cell cycle progression. Here we identify Htl1 as a novel factor that associates with the RSC complex both physically and functionally. We isolated HTL1 through a genetic screen for mutants that displayed additive growth defects with a conditional mutation in the protein kinase C gene (PKC1), which has been suggested through genetic connections to interact functionally with RSC. Several lines of evidence connect HTL1 to RSC function. First, an htl1Delta mutant displayed temperature-sensitive growth and a G(2)/M cell cycle arrest at restrictive temperatures, a phenotype similar to that of strains with conditional mutations in essential RSC components. Second, we isolated RSC3, which encodes a component of the RSC complex, as a dosage suppressor of the htl1Delta growth arrest. Third, an htl1Delta mutant displayed additive growth defects with conditional rsc3 alleles. Fourth, overexpression of HTL1 suppressed the growth defect of a strain with a conditional mutation in another RSC component, RSC8. Finally, we demonstrate that Htl1 is a nuclear protein that can associate in vivo with a fraction of the RSC complex. We propose that an RSC-Htl1 complex acts coordinately with protein kinase C to regulate the G(2)/M transition.

Measuring tissue-based biomarkers by immunochromatography coupled with reverse-phase lysate microarray.

PURPOSE: There is a need for new technologies to study tissue-based biomarkers. The current gold standard, immunohistochemistry, is compromised by variability in tissue processing and observer bias. Reverse transcription-PCR (RT-PCR), immunocytochemistry, and reverse-phase lysate microarrays (RPM) are promising alternative technologies but have not yet been validated, or correlated, on the same patient-derived tissues. Furthermore, RPM is currently limited by time-consuming microdissection and low amounts of evaluable protein lysates. EXPERIMENTAL DESIGN: Metastatic melanoma was surgically excised from 30 patients and macroscopically dissected from surrounding stroma. Each specimen was processed by formalin-fixation (immunohistochemistry), cytospin (immunocytochemistry), or disaggreagation and enrichment (RT-PCR and RPM). The latter protocol uses immunochromatography to remove hematopoetic-derived cells, thus enriching for melanoma cells. Each sample was measured for the expression of gp100 or MART-1 normalized to actin. RESULTS: Immunochromatography coupled with RPM (I-RPM) is reproducible (r >/= 0.70) and, for gp100, correlates strongly with immunohistochemistry and immunocytochemistry (r = 0.78 and 0.76, respectively) and moderately with transcript levels, measured by RT-PCR (r = 0.61). In contrast, for MART-1, I-RPM correlates strongly with transcript level (r = 0.78) but only moderately strong correlations are noted with immunohistochemistry and immunocytochemistry (r = 0.64 and 0.59, respectively). In general, transcript levels show only moderately strong correlations with immunohistochemistry and immunocytochemistry (r = 0.41-0.64). CONCLUSION: I-RPM is a promising technology for quantitative grading of tissue biomarkers; however, antigen-dependent correlations are noted.

CSF proteome: a protein repository for potential biomarker identification.

Proteomic analysis is not limited to the analysis of serum or tissues. Synovial, peritoneal, pericardial and cerebrospinal fluid represent unique proteomes for disease diagnosis and prognosis. In particular, cerebrospinal fluid serves as a rich source of putative biomarkers that are not solely limited to neurologic disorders. Peptides, proteolytic fragments and antibodies are capable of crossing the blood-brain barrier, thus providing a repository of pathologic information. Proteomic technologies such as immunoblotting, isoelectric focusing, 2D gel electrophoresis and mass spectrometry have proven useful for deciphering this unique proteome. Cerebrospinal fluid proteins are generally less abundant than their corresponding serum counterparts, necessitating the development and use of sensitive analytical techniques. This review highlights some of the promising areas of cerebrospinal fluid proteomic research and their clinical applications.

Validation of ELISA methods for quantification of total tau and phosporylated-tau181 in human cerebrospinal fluid with measurement in specimens from two Alzheimer's disease studies.

Tau measurements in cerebrospinal fluid (CSF) are gaining acceptance as aids to diagnosis of Alzheimer's disease (AD) and differentiation from other dementias. Two ELISA assays, the INNOTEST® hTAU Ag and the INNOTEST® PHOSPHO-TAU(181P) for quantification of t-tau and p-tau181 respectively, have been validated to regulatory standards. Validation parameters were determined by repeated testing of human CSF pools. Specimens from Phase 2 studies of the γ-secretase inhibitor semagacestat and the therapeutic antibody solanezumab at baseline and following 12-14 weeks of treatment were also tested. Estimated intra-assay CV for repeated testing of 3 CSF pools were ≤11.5% and RE varied between -14.1% and +6.4%. Inter-assay CV for t-tau was <5% and RE was within ±8%. For p-tau181, inter-assay CV was <9% and RE was within ±2.5%. Total CV (intra-assay plus inter-assay) were below 10% for both analytes. Up to 20-fold dilutional linearity was demonstrated for both analytes. Stability of t-tau and p-tau181 was demonstrated in CSF during five freeze-thaw cycles at ≤-20 °C and ≤-70 °C and at 18-22 °C for up to 24 h. Neither semagacestat nor solanezumab interfered with either assay. Inter-individual t-tau and p-tau181 concentrations were highly variable but intra-individual variations were small. These assays are suitable for analysis of CSF t-tau and p-tau181 in a single laboratory supporting multi-center AD clinical trials. No effect of treatment with semagacestat or solanezumab was observed in response to three months of drug administration.

Blockade of thrombospondin-1-CD47 interactions prevents necrosis of full thickness skin grafts.

BACKGROUND: Skin graft survival and healing requires rapid restoration of blood flow to the avascular graft. Failure or delay in the process of graft vascularization is a significant source of morbidity and mortality. One of the primary regulators of blood flow and vessel growth is nitric oxide (NO). The secreted protein thrombospondin-1 (TSP1) limits NO-stimulated blood flow and growth and composite tissue survival to ischemia. We herein demonstrate a role for TSP1 in regulating full thickness skin graft (FTSG) survival. METHODS AND RESULTS: FTSG consistently fail in wild type C57BL/6 mice but survive in mice lacking TSP1 or its receptor CD47. Ablation of the TSP1 receptor CD36, however, did not improve FTSG survival. Remarkably, wild type FTSG survived on TSP1 null or CD47 null mice, indicating that TSP1 expression in the wound bed is the primary determinant of graft survival. FTSG survival in wild type mice could be moderately improved by increasing NO flux, but graft survival was increased significantly through antibody blocking of TSP1 binding to CD47 or antisense morpholino oligonucleotide suppression of CD47. CONCLUSIONS: TSP1 through CD47 limits skin graft survival. Blocking TSP1 binding or suppressing CD47 expression drastically increases graft survival. The therapeutic applications of this approach could include burn patients and the broader group of people requiring grafts or tissue flaps for closure and reconstruction of complex wounds of diverse etiologies.