Haploinsufficiency of phosphodiesterase 10A activates PI3K/AKT signaling independent of PTEN to induce an aggressive glioma phenotype.

Glioblastoma is universally fatal and characterized by frequent chromosomal copy number alterations harboring oncogenes and tumor suppressors. In this study, we analyzed exome-wide human glioblastoma copy number data and found that cytoband 6q27 is an independent poor prognostic marker in multiple data sets. We then combined CRISPR-Cas9 data, human spatial transcriptomic data, and human and mouse RNA sequencing data to nominate PDE10A as a potential haploinsufficient tumor suppressor in the 6q27 region. Mouse glioblastoma modeling using the RCAS/tv-a system confirmed that Pde10a suppression induced an aggressive glioma phenotype in vivo and resistance to temozolomide and radiation therapy in vitro. Cell culture analysis showed that decreased Pde10a expression led to increased PI3K/AKT signaling in a Pten-independent manner, a response blocked by selective PI3K inhibitors. Single-nucleus RNA sequencing from our mouse gliomas in vivo, in combination with cell culture validation, further showed that Pde10a suppression was associated with a proneural-to-mesenchymal transition that exhibited increased cell adhesion and decreased cell migration. Our results indicate that glioblastoma patients harboring PDE10A loss have worse outcomes and potentially increased sensitivity to PI3K inhibition.

The Implant-Induced Foreign Body Response Is Limited by CD13-Dependent Regulation of Ubiquitination of Fusogenic Proteins.

Implanted medical devices, from artificial heart valves and arthroscopic joints to implantable sensors, often induce a foreign body response (FBR), a form of chronic inflammation resulting from the inflammatory reaction to a persistent foreign stimulus. The FBR is characterized by a subset of multinucleated giant cells (MGCs) formed by macrophage fusion, the foreign body giant cells (FBGCs), accompanied by inflammatory cytokines, matrix deposition, and eventually deleterious fibrotic implant encapsulation. Despite efforts to improve biocompatibility, implant-induced FBR persists, compromising the utility of devices and making efforts to control the FBR imperative for long-term function. Controlling macrophage fusion in FBGC formation presents a logical target to prevent implant failure, but the actual contribution of FBGCs to FBR-induced damage is controversial. CD13 is a molecular scaffold, and in vitro induction of CD13KO bone marrow progenitors generates many more MGCs than the wild type, suggesting that CD13 regulates macrophage fusion. In the mesh implant model of FBR, CD13KO mice produced significantly more peri-implant FBGCs with enhanced TGF-ß expression and increased collagen deposition versus the wild type. Prior to fusion, increased protrusion and microprotrusion formation accompanies hyperfusion in the absence of CD13. Expression of fusogenic proteins driving cell-cell fusion was aberrantly sustained at high levels in CD13KO MGCs, which we show is due to a novel CD13 function, to our knowledge, regulating ubiquitin/proteasomal protein degradation. We propose CD13 as a physiologic brake limiting aberrant macrophage fusion and the FBR, and it may be a novel therapeutic target to improve the success of implanted medical devices. Furthermore, our data directly implicate FBGCs in the detrimental fibrosis that characterizes the FBR.

CD13 facilitates immune cell migration and aggravates acute injury but promotes chronic post-stroke recovery.

INTRODUCTION: Acute stroke leads to the activation of myeloid cells. These cells express adhesion molecules and transmigrate to the brain, thereby aggravating injury. Chronically after stroke, repair processes, including angiogenesis, are activated and enhance post-stroke recovery. Activated myeloid cells express CD13, which facilitates their migration into the site of injury. However, angiogenic blood vessels which play a role in recovery also express CD13. Overall, the specific contribution of CD13 to acute and chronic stroke outcomes is unknown. METHODS: CD13 expression was estimated in both mice and humans after the ischemic stroke. Young (8-12 weeks) male wild-type and global CD13 knockout (KO) mice were used for this study. Mice underwent 60 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. For acute studies, the mice were euthanized at either 24- or 72 h post-stroke. For chronic studies, the Y-maze, Barnes maze, and the open field were performed on day 7 and day 28 post-stroke. Mice were euthanized at day 30 post-stroke and the brains were collected for assessment of inflammation, white matter injury, tissue loss, and angiogenesis. Flow cytometry was performed on days 3 and 7 post-stroke to quantify infiltrated monocytes and neutrophils and CXCL12/CXCR4 signaling. RESULTS: Brain CD13 expression and infiltrated CD13+ monocytes and neutrophils increased acutely after the stroke. The brain CD13+lectin+ blood vessels increased on day 15 after the stroke. Similarly, an increase in the percentage area CD13 was observed in human stroke patients at the subacute time after stroke. Deletion of CD13 resulted in reduced infarct volume and improved neurological recovery after acute stroke. However, CD13KO mice had significantly worse memory deficits, amplified gliosis, and white matter damage compared to wild-type animals at chronic time points. CD13-deficient mice had an increased percentage of CXCL12+cells but a reduced percentage of CXCR4+cells and decreased angiogenesis at day 30 post-stroke. CONCLUSIONS: CD13 is involved in the trans-migration of monocytes and neutrophils after stroke, and acutely, led to decreased infarct size and improved behavioral outcomes. However, loss of CD13 led to reductions in post-stroke angiogenesis by reducing CXCL12/CXCR4 signaling.

Coronavirus Receptors as Immune Modulators.

The Coronaviridae family includes the seven known human coronaviruses (CoV) that cause mild to moderate respiratory infections (HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1) as well as severe illness and death (MERS-CoV, SARS-CoV, SARS-CoV-2). Severe infections induce hyperinflammatory responses that are often intensified by host adaptive immune pathways to profoundly advance disease severity. Proinflammatory responses are triggered by CoV entry mediated by host cell surface receptors. Interestingly, five of the seven strains use three cell surface metallopeptidases (CD13, CD26, and ACE2) as receptors, whereas the others employ O-acetylated-sialic acid (a key feature of metallopeptidases) for entry. Why CoV evolved to use peptidases as their receptors is unknown, but the peptidase activities of the receptors are dispensable, suggesting the virus uses/benefits from other functions of these molecules. Indeed, these receptors participate in the immune modulatory pathways that contribute to the pathological hyperinflammatory response. This review will focus on the role of CoV receptors in modulating immune responses.

Segmenting Skin Biopsy Images with Coarse and Sparse Annotations using U-Net.

The number of melanoma diagnoses has increased dramatically over the past three decades, outpacing almost all other cancers. Nearly 1 in 4 skin biopsies is of melanocytic lesions, highlighting the clinical and public health importance of correct diagnosis. Deep learning image analysis methods may improve and complement current diagnostic and prognostic capabilities. The histologic evaluation of melanocytic lesions, including melanoma and its precursors, involves determining whether the melanocytic population involves the epidermis, dermis, or both. Semantic segmentation of clinically important structures in skin biopsies is a crucial step towards an accurate diagnosis. While training a segmentation model requires ground-truth labels, annotation of large images is a labor-intensive task. This issue becomes especially pronounced in a medical image dataset in which expert annotation is the gold standard. In this paper, we propose a two-stage segmentation pipeline using coarse and sparse annotations on a small region of the whole slide image as the training set. Segmentation results on whole slide images show promising performance for the proposed pipeline.

Proteoglycan 4 (PRG4) expression and function in dry eye associated inflammation.

Dry eye disease (DED) affects hundreds of millions of people worldwide. It is characterized by the production of inflammatory cytokines and chemokines as well as damaging matrix metalloproteinases (MMPs) at the ocular surface. While proteoglycan 4 (PRG4), a mucin-like glycoprotein present at the ocular surface, is most well known as a boundary lubricant that contributes to ocular surface integrity, it has been shown to blunt inflammation in various cell types, suggesting a dual mechanism of action. Recently, full-length recombinant human PRG4 (rhPRG4) has been shown to improve signs and symptoms of DED in humans. However, there remains a significant need for basic science research on rhPRG4's biological properties and its potential therapeutic mechanisms of action in treating DED. Therefore, the objectives of this study were to characterize endogenous PRG4 expression by telomerase-immortalized human corneal epithelial (hTCEpi) cells, examine whether exogenous rhPRG4 modulates cytokine and chemokine secretion in response to dry eye associated inflammation (TNFα and IL-1ß), explore interactions between rhPRG4 and MMP-9, and understand how experimental dry eye (EDE) in mice affects PRG4 expression. PRG4 secretion from hTCEpi cells was quantified by Western blot and expression visualized by immunocytochemistry. Cytokine/chemokine production was measured by ELISA and Luminex, while rhPRG4's effect on MMP-9 activity, binding, and expression was quantified using an MMP-9 inhibitor kit, surface plasmon resonance, and reverse transcription polymerase chain reaction (RT-PCR), respectively. Finally, EDE was induced in mice, and PRG4 was visualized by immunohistochemistry in the cornea and by Western blot in lacrimal gland lysate. In vitro results demonstrate that hTCEpi cells synthesize and secrete PRG4, and PRG4 secretion is inhibited by TNFα and IL-1ß. In response to these pro-inflammatory stresses, exogenous rhPRG4 significantly reduced the stimulated production of IP-10, RANTES, ENA-78, GROα, MIP-3α, and MIG, and trended towards a reduction of MIP-1α and MIP-1ß. The hTCEpi cells were also able to internalize fluorescently-labelled rhPRG4, consistent with a mechanism of action that includes downstream biological signaling pathways. rhPRG4 was not digested by MMP-9, and it did not modulate MMP-9 gene expression in hTCEpi cells, but it was able to bind to MMP-9 and inhibited in vitro activity of exogenous MMP-9 in the presence of human tears. Finally, in vivo results demonstrate that EDE significantly decreased immunolocalization of PRG4 on the corneal epithelium and trended towards a reduction of PRG4 in lacrimal gland lysate. Collectively these results demonstrate rhPRG4 has anti-inflammatory properties on corneal epithelial cells, particularly as it relates to mitigating chemokine production, and is an inhibitor of MMP-9 activity, as well as that in vivo expression of PRG4 can be altered in preclinical models of DED. In conclusion, these findings contribute to our understanding of PRG4's immunomodulatory properties in the context of DED inflammation and provide the foundation and motivation for further mechanistic research of PRG4's properties on the ocular surface as well as expanding clinical evaluation of its ability as a multifunctional therapeutic agent to effectively provide relief to those who suffer from DED.

Detection and classification of cancer in whole slide breast histopathology images using deep convolutional networks.

Generalizability of algorithms for binary cancer vs. no cancer classification is unknown for clinically more significant multi-class scenarios where intermediate categories have different risk factors and treatment strategies. We present a system that classifies whole slide images (WSI) of breast biopsies into five diagnostic categories. First, a saliency detector that uses a pipeline of four fully convolutional networks, trained with samples from records of pathologists' screenings, performs multi-scale localization of diagnostically relevant regions of interest in WSI. Then, a convolutional network, trained from consensus-derived reference samples, classifies image patches as non-proliferative or proliferative changes, atypical ductal hyperplasia, ductal carcinoma in situ, and invasive carcinoma. Finally, the saliency and classification maps are fused for pixel-wise labeling and slide-level categorization. Experiments using 240 WSI showed that both saliency detector and classifier networks performed better than competing algorithms, and the five-class slide-level accuracy of 55% was not statistically different from the predictions of 45 pathologists. We also present example visualizations of the learned representations for breast cancer diagnosis.

Characterizing Diagnostic Search Patterns in Digital Breast Pathology: Scanners and Drillers.

Following a baseline demographic survey, 87 pathologists interpreted 240 digital whole slide images of breast biopsy specimens representing a range of diagnostic categories from benign to atypia, ductal carcinoma in situ, and invasive cancer. A web-based viewer recorded pathologists' behaviors while interpreting a subset of 60 randomly selected and randomly ordered slides. To characterize diagnostic search patterns, we used the viewport location, time stamp, and zoom level data to calculate four variables: average zoom level, maximum zoom level, zoom level variance, and scanning percentage. Two distinct search strategies were confirmed: scanning is characterized by panning at a constant zoom level, while drilling involves zooming in and out at various locations. Statistical analysis was applied to examine the associations of different visual interpretive strategies with pathologist characteristics, diagnostic accuracy, and efficiency. We found that females scanned more than males, and age was positively correlated with scanning percentage, while the facility size was negatively correlated. Throughout 60 cases, the scanning percentage and total interpretation time per slide decreased, and these two variables were positively correlated. The scanning percentage was not predictive of diagnostic accuracy. Increasing average zoom level, maximum zoom level, and zoom variance were correlated with over-interpretation.

CD13 restricts TLR4 endocytic signal transduction in inflammation.

Dysregulation of the innate immune response underlies numerous pathological conditions. The TLR4 is the prototypical sensor of infection or injury that orchestrates the innate response via sequential activation of both cell surface and endocytic signaling pathways that trigger distinct downstream consequences. CD14 binds and delivers LPS to TLR4 and has been identified as a positive regulator of TLR4 signal transduction. It is logical that negative regulators of this process also exist to maintain the critical balance required for fighting infection, healing damaged tissue, and resolving inflammation. We showed that CD13 negatively modulates receptor-mediated Ag uptake in dendritic cells to control T cell activation in adaptive immunity. In this study, we report that myeloid CD13 governs internalization of TLR4 and subsequent innate signaling cascades, activating IRF-3 independently of CD14. CD13 is cointernalized with TLR4, CD14, and dynamin into Rab5(+) early endosomes upon LPS treatment. Importantly, in response to TLR4 ligands HMGB1 and LPS, p-IRF-3 activation and transcription of its target genes are enhanced in CD13(KO) dendritic cells, whereas TLR4 surface signaling remains unaffected, resulting in a skewed inflammatory response. This finding is physiologically relevant as ischemic injury in vivo provoked identical TLR4 responses. Finally, CD13(KO) mice showed significantly enhanced IFNß-mediated signal transduction via JAK-STAT, escalating inducible NO synthase transcription levels and promoting accumulation of oxidative stress mediators and tissue injury. Mechanistically, inflammatory activation of macrophages upregulates CD13 expression and CD13 and TLR4 coimmunoprecipitate. Therefore, CD13 negatively regulates TLR4 signaling, thereby balancing the innate response by maintaining the inflammatory equilibrium critical to innate immune regulation.

Measuring Symmetry in Children With Cleft Lip. Part 3: Quantifying Nasal Symmetry and Nasal Normalcy Before and After Unilateral Cleft Lip Repair.

OBJECTIVE: The purpose of this project was to develop objective computer-based methods to measure nasal asymmetry and abnormality in children undergoing treatment of unilateral cleft lip (UCL) and to determine the correlation of these measures to clinical expectations. PARTICIPANTS: Thirty infants with UCL undergoing cleft lip repair; 27 children with UCL aged 8 to 10 years who had previously undergone cleft lip repair; 3 control infants; 3 control children aged 8 to 10 years. INTERVENTIONS: To measure nasal symmetry, we used a process of depth mapping and calculated the Depth Area Difference. To measure abnormality, we used the reconstruction error from Principle Component Analysis (PCA) that was based upon characteristics of a dataset of over 2000 images of normal control subjects. MAIN OUTCOME MEASURES: Depth Area Difference and PCA Reconstruction Error for cleft type, changes with surgery, and individual subjects ranked according to cleft severity were assessed. RESULTS: Significant differences in Depth Area Difference and PCA Reconstruction Error were found between cleft types and found before and after surgery. Nasal symmetry and normalcy scores for infants with UCL approached those of controls after surgery, and there was a strong correlation with ranked cleft severity. For older children, measures of nasal symmetry and abnormality were better than infants prior to repair but worse than infants following UCL repair. CONCLUSIONS: Our computer-based 3D analysis of nasal symmetry and normalcy correlated with clinical expectations. Automated processing made measurement convenient. Use of these measures may help to objectively measure cleft severity and treatment outcome.

Prostate-specific membrane antigen (PSMA)-mediated laminin proteolysis generates a pro-angiogenic peptide.

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase expressed in a number of tissues. PSMA participates in various biological functions depending on the substrate available in the particular tissue; in the brain, PSMA cleaves the abundant neuropeptide N-acetyl-aspartyl-glutamate to regulate release of key neurotransmitters, while intestinal PSMA cleaves polyglutamated peptides to supply dietary folate. PSMA expression is also progressively upregulated in prostate cancer where it correlates with tumor progression as well as in tumor vasculature, where it regulates angiogenesis. The previous research determined that PSMA cleavage of small peptides generated via matrix metalloprotease-mediated proteolysis of the extracellular matrix protein laminin potently activated endothelial cells, integrin signaling and angiogenesis, although the specific peptide substrates were not identified. Herein, using enzymatic analyses and LC/MS, we unequivocally demonstrate that several laminin-derived peptides containing carboxy-terminal glutamate moieties (LQE, IEE, LNE) are bona fide substrates for PSMA. Subsequently, the peptide products were tested for their effects on angiogenesis in various models. We report that LQ, the dipeptide product of PSMA cleavage of LQE, efficiently activates endothelial cells in vitro and enhances angiogenesis in vivo. Importantly, LQE is not cleaved by an inactive PSMA enzyme containing an active site mutation (E424S). Endothelial cell activation by LQ was dependent on integrin beta-1-induced activation of focal adhesion kinase. These results characterize a novel PSMA substrate, provide a functional rationale for the upregulation of PSMA in cancer cells and tumor vasculature and suggest that inhibition of PSMA could lead to the development of new angiogenic therapies.

Region of interest identification and diagnostic agreement in breast pathology.

A pathologist's accurate interpretation relies on identifying relevant histopathological features. Little is known about the precise relationship between feature identification and diagnostic decision making. We hypothesized that greater overlap between a pathologist's selected diagnostic region of interest (ROI) and a consensus derived ROI is associated with higher diagnostic accuracy. We developed breast biopsy test cases that included atypical ductal hyperplasia (n=80); ductal carcinoma in situ (n=78); and invasive breast cancer (n=22). Benign cases were excluded due to the absence of specific abnormalities. Three experienced breast pathologists conducted an independent review of the 180 digital whole slide images, established a reference consensus diagnosis and marked one or more diagnostic ROIs for each case. Forty-four participating pathologists independently diagnosed and marked ROIs on the images. Participant diagnoses and ROI were compared with consensus reference diagnoses and ROI. Regression models tested whether percent overlap between participant ROI and consensus reference ROI predicted diagnostic accuracy. Each of the 44 participants interpreted 39-50 cases for a total of 1972 individual diagnoses. Percent ROI overlap with the expert reference ROI was higher in pathologists who self-reported academic affiliation (69 vs 65%, P=0.002). Percent overlap between participants' ROI and consensus reference ROI was then classified into ordinal categories: 0, 1-33, 34-65, 66-99 and 100% overlap. For each incremental change in the ordinal percent ROI overlap, diagnostic agreement increased by 60% (OR 1.6, 95% CI (1.5-1.7), P<0.001) and the association remained significant even after adjustment for other covariates. The magnitude of the association between ROI overlap and diagnostic agreement increased with increasing diagnostic severity. The findings indicate that pathologists are more likely to converge with an expert reference diagnosis when they identify an overlapping diagnostic image region, suggesting that future computer-aided detection systems that highlight potential diagnostic regions could be a helpful tool to improve accuracy and education.

Localization of Diagnostically Relevant Regions of Interest in Whole Slide Images: a Comparative Study.

Whole slide digital imaging technology enables researchers to study pathologists' interpretive behavior as they view digital slides and gain new understanding of the diagnostic medical decision-making process. In this study, we propose a simple yet important analysis to extract diagnostically relevant regions of interest (ROIs) from tracking records using only pathologists' actions as they viewed biopsy specimens in the whole slide digital imaging format (zooming, panning, and fixating). We use these extracted regions in a visual bag-of-words model based on color and texture features to predict diagnostically relevant ROIs on whole slide images. Using a logistic regression classifier in a cross-validation setting on 240 digital breast biopsy slides and viewport tracking logs of three expert pathologists, we produce probability maps that show 74 % overlap with the actual regions at which pathologists looked. We compare different bag-of-words models by changing dictionary size, visual word definition (patches vs. superpixels), and training data (automatically extracted ROIs vs. manually marked ROIs). This study is a first step in understanding the scanning behaviors of pathologists and the underlying reasons for diagnostic errors.

Measuring Symmetry in Children With Cleft Lip. Part 2: Quantification of Nasolabial Symmetry Before and After Cleft Lip Repair.

OBJECTIVE: The first part of this study validated an automated computer-based method of identifying the three-dimensional midfacial plane in children with unrepaired cleft lip. The purpose of this second part is to develop computer-based methods to quantify symmetry and to determine the correlation of these measures to clinical expectations. PARTICIPANTS: A total of 35 infants with unrepaired unilateral cleft lip and 14 infant controls. INTERVENTIONS: Six computer-based methods of quantifying symmetry were developed and applied to the three-dimensional images of infants with unilateral cleft lip before and after cleft lip repair and to those of controls. MAIN OUTCOME MEASURE: Symmetry scores for cleft type, changes with surgery, and individual subjects ranked according to cleft severity were assessed. RESULTS: Significant differences in symmetry scores were found between cleft types and found before and after surgery. Symmetry scores for infants with unilateral cleft lip approached those of controls after surgery, and there was a strong correlation with ranked cleft severity. CONCLUSIONS: Our computer-based three-dimensional analysis of nasolabial symmetry correlated with clinical expectations. Automated processing made measurement convenient. Use of these measures may help to objectively measure cleft severity and treatment outcome.

Measuring Symmetry in Children With Unrepaired Cleft Lip: Defining a Standard for the Three-Dimensional Midfacial Reference Plane.

OBJECTIVE: Quantitative measures of facial form to evaluate treatment outcomes for cleft lip (CL) are currently limited. Computer-based analysis of three-dimensional (3D) images provides an opportunity for efficient and objective analysis. The purpose of this study was to define a computer-based standard of identifying the 3D midfacial reference plane of the face in children with unrepaired cleft lip for measurement of facial symmetry. PARTICIPANTS: The 3D images of 50 subjects (35 with unilateral CL, 10 with bilateral CL, five controls) were included in this study. INTERVENTIONS: Five methods of defining a midfacial plane were applied to each image, including two human-based (Direct Placement, Manual Landmark) and three computer-based (Mirror, Deformation, Learning) methods. MAIN OUTCOME MEASURE: Six blinded raters (three cleft surgeons, two craniofacial pediatricians, and one craniofacial researcher) independently ranked and rated the accuracy of the defined planes. RESULTS: Among computer-based methods, the Deformation method performed significantly better than the others. Although human-based methods performed best, there was no significant difference compared with the Deformation method. The average correlation coefficient among raters was .4; however, it was .7 and .9 when the angular difference between planes was greater than 6° and 8°, respectively. CONCLUSIONS: Raters can agree on the 3D midfacial reference plane in children with unrepaired CL using digital surface mesh. The Deformation method performed best among computer-based methods evaluated and can be considered a useful tool to carry out automated measurements of facial symmetry in children with unrepaired cleft lip.

Antitumor Activity of a Novel Sphingosine-1-Phosphate 2 Antagonist, AB1, in Neuroblastoma.

The bioactive lipid sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) play critical roles in many pathologic processes, including cancer. The S1P axis has become a bona fide therapeutic target in cancer. JTE-013 [N-​(2,​6-​dichloro-​4-​pyridinyl)-​2-​[1,​3-​dimethyl-​4-​(1-​methylethyl)-​1H-​pyrazolo[3,​4-​b]pyridin-​6-​yl]-​hydrazinecarboxamide], a known S1P2 antagonist, suffers from instability in vivo. Structurally modified, more potent, and stable S1P2 inhibitors would be desirable pharmacological tools. One of the JTE-013 derivatives, AB1 [N-(1H-4-isopropyl-1-allyl-3-methylpyrazolo[3,4-b]pyridine-6-yl)-amino-N'-(2,6-dichloropyridine-4-yl) urea], exhibited improved S1P2 antagonism compared with JTE-013. Intravenous pharmacokinetics indicated enhanced stability or slower clearance of AB1 in vivo. Migration assays in glioblastoma showed that AB1 was slightly more effective than JTE-013 in blocking S1P2-mediated inhibition of cell migration. Functional studies in the neuroblastoma (NB) cell line SK-N-AS showed that AB1 displayed potency at least equivalent to JTE-013 in affecting signaling molecules downstream of S1P2. Similarly, AB1 inhibition of the growth of SK-N-AS tumor xenografts was improved compared with JTE-013. Cell viability assays excluded that this enhanced AB1 effect is caused by inhibition of cancer cell survival. Both JTE-013 and AB1 trended to inhibit (C-C motif) ligand 2 expression and were able to significantly inhibit subsequent tumor-associated macrophage infiltration in NB xenografts. Interestingly, AB1 was more effective than JTE-013 in inhibiting the expression of the profibrotic mediator connective tissue growth factor. The terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling assay and cleaved caspase-3 detection further demonstrated that apoptosis was increased in AB1-treated NB xenografts compared with JTE-013. Overall, the modification of JTE-013 to produce the AB1 compound improved potency, intravenous pharmacokinetics, cellular activity, and antitumor activity in NB and may have enhanced clinical and experimental applicability.

CD13 regulates anchorage and differentiation of the skeletal muscle satellite stem cell population in ischemic injury.

CD13 is a multifunctional cell surface molecule that regulates inflammatory and angiogenic mechanisms in vitro, but its contribution to these processes in vivo or potential roles in stem cell biology remains unexplored. We investigated the impact of loss of CD13 on a model of ischemic skeletal muscle injury that involves angiogenesis, inflammation, and stem cell mobilization. Consistent with its role as an inflammatory adhesion molecule, lack of CD13 altered myeloid trafficking in the injured muscle, resulting in cytokine profiles skewed toward a prohealing environment. Despite this healing-favorable context, CD13(KO) animals showed significantly impaired limb perfusion with increased necrosis, fibrosis, and lipid accumulation. Capillary density was correspondingly decreased, implicating CD13 in skeletal muscle angiogenesis. The number of CD45-/Sca1-/α7-integrin+/ß1-integrin+ satellite cells was markedly diminished in injured CD13(KO) muscles and adhesion of isolated CD13(KO) satellite cells was impaired while their differentiation was accelerated. Bone marrow transplantation studies showed contributions from both host and donor cells to wound healing. Importantly, CD13 was coexpressed with Pax7 on isolated muscle-resident satellite cells. Finally, phosphorylated-focal adhesion kinase and ERK levels were reduced in injured CD13(KO) muscles, consistent with CD13 regulating satellite cell adhesion, potentially contributing to the maintenance and renewal of the satellite stem cell pool and facilitating skeletal muscle regeneration.

Tyrosine phosphorylation of CD13 regulates inflammatory cell-cell adhesion and monocyte trafficking.

CD13 is a large cell surface peptidase expressed on the monocytes and activated endothelial cells that is important for homing to and resolving the damaged tissue at sites of injury. We showed previously that cross-linking of human monocytic CD13 with activating Abs induces strong adhesion to endothelial cells in a tyrosine kinase- and microtubule-dependent manner. In the current study, we examined the molecular mechanisms underlying these observations in vitro and in vivo. We found that cross-linking of CD13 on U937 monocytic cells induced phosphorylation of a number of proteins, including Src, FAK, and ERK, and inhibition of these abrogated CD13-dependent adhesion. We found that CD13 itself was phosphorylated in a Src-dependent manner, which was an unexpected finding because its 7-aa cytoplasmic tail was assumed to be inert. Furthermore, CD13 was constitutively associated with the scaffolding protein IQGAP1, and CD13 cross-linking induced complex formation with the actin-binding protein α-actinin, linking membrane-bound CD13 to the cytoskeleton, further supporting CD13 as an inflammatory adhesion molecule. Mechanistically, mutation of the conserved CD13 cytoplasmic tyrosine to phenylalanine abrogated adhesion; Src, FAK, and ERK phosphorylation; and cytoskeletal alterations upon Ab cross-linking. Finally, CD13 was phosphorylated in isolated murine inflammatory peritoneal exudate cells, and adoptive transfer of monocytic cell lines engineered to express the mutant CD13 were severely impaired in their ability to migrate into the inflamed peritoneum, confirming that CD13 phosphorylation is relevant to inflammatory cell trafficking in vivo. Therefore, this study identifies CD13 as a novel, direct activator of intracellular signaling pathways in pathophysiological conditions.

Molecular mechanisms regulating CD13-mediated adhesion.

CD13/Aminopeptidase N is a transmembrane metalloproteinase that is expressed in many tissues where it regulates various cellular functions. In inflammation, CD13 is expressed on myeloid cells, is up-regulated on endothelial cells at sites of inflammation and mediates monocyte/endothelial adhesion by homotypic interactions. In animal models the lack of CD13 alters the profiles of infiltrating inflammatory cells at sites of ischaemic injury. Here, we found that CD13 expression is enriched specifically on the pro-inflammatory subset of monocytes, suggesting that CD13 may regulate trafficking and function of specific subsets of immune cells. To further dissect the mechanisms regulating CD13-dependent trafficking we used the murine model of thioglycollate-induced sterile peritonitis. Peritoneal monocytes, macrophages and dendritic cells were significantly decreased in inflammatory exudates from global CD13(KO) animals when compared with wild-type controls. Furthermore, adoptive transfer of wild-type and CD13(KO) primary myeloid cells, or wild-type myeloid cells pre-treated with CD13-blocking antibodies into thioglycollate-challenged wild-type recipients demonstrated fewer CD13(KO) or treated cells in the lavage, suggesting that CD13 expression confers a competitive advantage in trafficking. Similarly, both wild-type and CD13(KO) cells were reduced in infiltrates in CD13(KO) recipients, confirming that both monocytic and endothelial CD13 contribute to trafficking. Finally, murine monocyte cell lines expressing mouse/human chimeric CD13 molecules demonstrated that the C-terminal domain of the protein mediates CD13 adhesion. Therefore, this work verifies that the altered inflammatory trafficking in CD13(KO) mice is the result of aberrant myeloid cell subset trafficking and further defines the molecular mechanisms underlying this regulation.