Clearance of VWF by hepatic macrophages is critical for the protective effect of ADAMTS13 in sickle cell anemia mice.

ABSTRACT: Although it is caused by a single-nucleotide mutation in the ß-globin gene, sickle cell anemia (SCA) is a systemic disease with complex, incompletely elucidated pathologies. The mononuclear phagocyte system plays critical roles in SCA pathophysiology. However, how heterogeneous populations of hepatic macrophages contribute to SCA remains unclear. Using a combination of single-cell RNA sequencing and spatial transcriptomics via multiplexed error-robust fluorescence in situ hybridization, we identified distinct macrophage populations with diversified origins and biological functions in SCA mouse liver. We previously found that administering the von Willebrand factor (VWF)-cleaving protease ADAMTS13 alleviated vaso-occlusive episode in mice with SCA. Here, we discovered that the ADAMTS13-cleaved VWF was cleared from the circulation by a Clec4f+Marcohigh macrophage subset in a desialylation-dependent manner in the liver. In addition, sickle erythrocytes were phagocytized predominantly by Clec4f+Marcohigh macrophages. Depletion of macrophages not only abolished the protective effect of ADAMTS13 but exacerbated vaso-occlusive episode in mice with SCA. Furthermore, promoting macrophage-mediated VWF clearance reduced vaso-occlusion in SCA mice. Our study demonstrates that hepatic macrophages are important in the pathogenesis of SCA, and efficient clearance of VWF by hepatic macrophages is critical for the protective effect of ADAMTS13 in SCA mice.

Deletion of Talin1 in Myeloid Cells Facilitates Atherosclerosis in Mice.

BACKGROUND: Integrin-regulated monocyte recruitment and cellular responses of monocyte-derived macrophages are critical for the pathogenesis of atherosclerosis. In the canonical model, talin1 controls ligand binding to integrins, a prerequisite for integrins to mediate leukocyte recruitment and induce immune responses. However, the role of talin1 in the development of atherosclerosis has not been studied. Our study investigated how talin1 in myeloid cells regulates the progression of atherosclerosis. METHODS: On an Apoe-/- background, myeloid talin1-deficient mice and the control mice were fed with a high-fat diet for 8 or 12 weeks to induce atherosclerosis. The atherosclerosis development in the aorta and monocyte recruitment into atherosclerotic lesions were analyzed. RESULTS: Myeloid talin1 deletion facilitated the formation of atherosclerotic lesions and macrophage deposition in lesions. Talin1 deletion abolished integrin ß2-mediated adhesion of monocytes but did not impair integrin α4ß1-dependent cell adhesion in a flow adhesion assay. Strikingly, talin1 deletion did not prevent Mn2+- or chemokine-induced activation of integrin α4ß1 to the high-affinity state for ligands. In an in vivo competitive homing assay, monocyte infiltration into inflamed tissues was prohibited by antibodies to integrin α4ß1 but was not affected by talin1 deletion or antibodies to integrin ß2. Furthermore, quantitative polymerase chain reaction and ELISA analysis showed that macrophages produced cytokines to promote inflammation and the proliferation of smooth muscle cells. Ligand binding to integrin ß3 inhibited cytokine generation in macrophages, although talin1 deletion abolished the negative effects of integrin ß3. CONCLUSIONS: Integrin α4ß1 controls monocyte recruitment during atherosclerosis. Talin1 is dispensable for integrin α4ß1 activation to the high-affinity state and integrin α4ß1-mediated monocyte recruitment. Yet, talin1 is required for integrin ß3 to inhibit the production of inflammatory cytokines in macrophages. Thus, intact monocyte recruitment and elevated inflammatory responses cause enhanced atherosclerosis in talin1-deficient mice. Our study provides novel insights into the roles of myeloid talin1 and integrins in the progression of atherosclerosis.

Endothelial VWF is critical for the pathogenesis of vaso-occlusive episode in a mouse model of sickle cell disease.

Vaso-occlusive episode (VOE) is a common and critical complication of sickle cell disease (SCD). Its pathogenesis is incompletely understood. von Willebrand factor (VWF), a multimeric plasma hemostatic protein synthesized and secreted by endothelial cells and platelets, is increased during a VOE. However, whether and how VWF contributes to the pathogenesis of VOE is not fully understood. In this study, we found increased VWF levels during tumor necrosis factor (TNF)-induced VOE in a humanized mouse model of SCD. Deletion of endothelial VWF decreased hemolysis, vascular occlusion, and organ damage caused by TNF-induced VOE in SCD mice. Moreover, administering ADAMTS13, the VWF-cleaving plasma protease, reduced plasma VWF levels, decreased inflammation and vaso-occlusion, and alleviated organ damage during VOE. These data suggest that promoting VWF cleavage via ADAMTS13 may be an effective treatment for reducing hemolysis, inflammation, and vaso-occlusion during VOE.

Deletion of platelet CLEC-2 decreases GPIbα-mediated integrin αIIbß3 activation and decreases thrombosis in TTP.

Microvascular thrombosis in patients with thrombotic thrombocytopenic purpura (TTP) is initiated by GPIbα-mediated platelet binding to von Willebrand factor (VWF). Binding of VWF to GPIbα causes activation of the platelet surface integrin αIIbß3. However, the mechanism of GPIbα-initiated activation of αIIbß3 and its clinical importance for microvascular thrombosis remain elusive. Deletion of platelet C-type lectin-like receptor 2 (CLEC-2) did not prevent VWF binding to platelets but specifically inhibited platelet aggregation induced by VWF binding in mice. Deletion of platelet CLEC-2 also inhibited αIIbß3 activation induced by the binding of VWF to GPIbα. Using a mouse model of TTP, which was created by infusion of anti-mouse ADAMTS13 monoclonal antibodies followed by infusion of VWF, we found that deletion of platelet CLEC-2 decreased pulmonary arterial thrombosis and the severity of thrombocytopenia. Importantly, prophylactic oral administration of aspirin, an inhibitor of platelet activation, and therapeutic treatment of the TTP mice with eptifibatide, an integrin αIIbß3 antagonist, reduced pulmonary arterial thrombosis in the TTP mouse model. Our observations demonstrate that GPIbα-mediated activation of integrin αIIbß3 plays an important role in the formation of thrombosis in TTP. These observations suggest that prevention of platelet activation with aspirin may reduce the risk for thrombosis in patients with TTP.

Heightened activation of embryonic megakaryocytes causes aneurysms in the developing brain of mice lacking podoplanin.

During early embryonic development in mammals, including humans and mice, megakaryocytes (Mks) first originate from primitive hematopoiesis in the yolk sac. These embryonic Mks (eMks) circulate in the vasculature with unclear function. Herein, we report that podoplanin (PDPN), the ligand of C-type lectin-like receptor (CLEC-2) on Mks/platelets, is temporarily expressed in neural tissue during midgestation in mice. Loss of PDPN or CLEC-2 resulted in aneurysms and spontaneous hemorrhage, specifically in the lower diencephalon during midgestation. Surprisingly, more eMks/platelets had enhanced granule release and localized to the lower diencephalon in mutant mouse embryos than in wild-type littermates before hemorrhage. We found that PDPN counteracted the collagen-1-induced secretion of angiopoietin-1 from fetal Mks, which coincided with enhanced TIE-2 activation in aneurysm-like sprouts of PDPN-deficient embryos. Blocking platelet activation prevented the PDPN-deficient embryo from developing vascular defects. Our data reveal a new role for PDPN in regulating eMk function during midgestation.

Sialylation on O-glycans protects platelets from clearance by liver Kupffer cells.

Most platelet membrane proteins are modified by mucin-type core 1-derived glycans (O-glycans). However, the biological importance of O-glycans in platelet clearance is unclear. Here, we generated mice with a hematopoietic cell-specific loss of O-glycans (HC C1galt1-/- ). These mice lack O-glycans on platelets and exhibit reduced peripheral platelet numbers. Platelets from HC C1galt1-/- mice show reduced levels of α-2,3-linked sialic acids and increased accumulation in the liver relative to wild-type platelets. The preferential accumulation of HC C1galt1-/- platelets in the liver was reduced in mice lacking the hepatic asialoglycoprotein receptor [Ashwell-Morell receptor (AMR)]. However, we found that Kupffer cells are the primary cells phagocytosing HC C1galt1-/- platelets in the liver. Our results demonstrate that hepatic AMR promotes preferential adherence to and phagocytosis of desialylated and/or HC C1galt1-/- platelets by the Kupffer cell through its C-type lectin receptor CLEC4F. These findings provide insights into an essential role for core 1 O-glycosylation of platelets in their clearance in the liver.

Podoplanin maintains high endothelial venule integrity by interacting with platelet CLEC-2.

Circulating lymphocytes continuously enter lymph nodes for immune surveillance through specialized blood vessels named high endothelial venules, a process that increases markedly during immune responses. How high endothelial venules (HEVs) permit lymphocyte transmigration while maintaining vascular integrity is unknown. Here we report a role for the transmembrane O-glycoprotein podoplanin (PDPN, also known as gp38 and T1α) in maintaining HEV barrier function. Mice with postnatal deletion of Pdpn lost HEV integrity and exhibited spontaneous bleeding in mucosal lymph nodes, and bleeding in the draining peripheral lymph nodes after immunization. Blocking lymphocyte homing rescued bleeding, indicating that PDPN is required to protect the barrier function of HEVs during lymphocyte trafficking. Further analyses demonstrated that PDPN expressed on fibroblastic reticular cells, which surround HEVs, functions as an activating ligand for platelet C-type lectin-like receptor 2 (CLEC-2, also known as CLEC1B). Mice lacking fibroblastic reticular cell PDPN or platelet CLEC-2 exhibited significantly reduced levels of VE-cadherin (also known as CDH5), which is essential for overall vascular integrity, on HEVs. Infusion of wild-type platelets restored HEV integrity in Clec-2-deficient mice. Activation of CLEC-2 induced release of sphingosine-1-phosphate from platelets, which promoted expression of VE-cadherin on HEVs ex vivo. Furthermore, draining peripheral lymph nodes of immunized mice lacking sphingosine-1-phosphate had impaired HEV integrity similar to Pdpn- and Clec-2-deficient mice. These data demonstrate that local sphingosine-1-phosphate release after PDPN-CLEC-2-mediated platelet activation is critical for HEV integrity during immune responses.

Loss of mucin-type O-glycans impairs the integrity of the glomerular filtration barrier in the mouse kidney.

The kidney's filtration activity is essential for removing toxins and waste products from the body. The vascular endothelial cells of the glomerulus are fenestrated, flattened, and surrounded by podocytes, specialized cells that support glomerular endothelial cells. Mucin-type core 1-derived O-glycans (O-glycans) are highly expressed on both glomerular capillary endothelial cells and their supporting podocytes, but their biological role is unclear. Biosynthesis of core 1-derived O-glycans is catalyzed by the glycosyltransferase core 1 ß1,3-galactosyltransferase (C1galt1). Here we report that neonatal or adult mice with inducible deletion of C1galt1 (iC1galt1-/-) exhibit spontaneous proteinuria and rapidly progressing glomerulosclerosis. Ultrastructural analysis of the glomerular filtration barrier components revealed that loss of O-glycans results in altered podocyte foot processes. Further analysis indicated that O-glycan is essential for the normal signaling function of podocalyxin, a podocyte foot process-associated glycoprotein. Our results reveal a new function of O-glycosylation in the integrity of the glomerular filtration barrier.

Defective Intestinal Mucin-Type O-Glycosylation Causes Spontaneous Colitis-Associated Cancer in Mice.

BACKGROUND & AIMS: Core 1- and core 3-derived mucin-type O-linked oligosaccharides (O-glycans) are major components of the colonic mucus layer. Defective forms of colonic O-glycans, such as the Thomsen-nouveau (Tn) antigen, frequently are observed in patients with ulcerative colitis and colorectal cancer, but it is not clear if they contribute to their pathogenesis. We investigated whether and how impaired O-glycosylation contributes to the development of colitis-associated colorectal cancer using mice lacking intestinal core 1- and core 3-derived O-glycans. METHODS: We generated mice that lack core 1- and core 3-derived intestinal O-glycans (DKO mice) and analyzed them, along with mice that singly lack intestinal epithelial core 1 O-glycans (IEC C1galt1(-/-) mice) or core 3 O-glycans (C3Gnt(-/-) mice). Intestinal tissues were collected at different time points and analyzed for levels of mucin and Tn antigen, development of colitis, and tumor formation using imaging, quantitative polymerase chain reaction, immunoblot, and enzyme-linked immunosorbent assay techniques. We also used cellular and genetic approaches, as well as intestinal microbiota depletion, to identify inflammatory mediators and pathways that contribute to disease in DKO and wild-type littermates (controls). RESULTS: Intestinal tissues from DKO mice contained higher levels of Tn antigen and had more severe spontaneous chronic colitis than tissues from IEC C1galt1(-/-) mice, whereas spontaneous colitis was absent in C3GnT(-/-) and control mice. IEC C1galt1(-/-) mice and DKO mice developed spontaneous colorectal tumors, although the onset of tumors in the DKO mice occurred earlier (age, 8-9 months) than that in IEC C1galt1(-/-) mice (15 months old). Antibiotic depletion of the microbiota did not cause loss of Tn antigen but did reduce the development of colitis and cancer formation in DKO mice. Colon tissues from DKO mice, but not control mice, contained active forms of caspase 1 and increased caspase 11, which were reduced after antibiotic administration. Supernatants from colon tissues of DKO mice contained increased levels of interleukin-1ß and interleukin-18, compared with those from control mice. Disruption of the caspase 1 and caspase 11 genes in DKO mice (DKO/Casp1/11(-/-) mice) decreased the development of colitis and cancer, characterized by reduced colonic thickening, hyperplasia, inflammatory infiltrate, and tumors compared with DKO mice. CONCLUSIONS: Impaired expression of O-glycans causes colonic mucus barrier breach and subsequent microbiota-mediated activation of caspase 1-dependent inflammasomes in colonic epithelial cells of mice. These processes could contribute to colitis-associated colon cancer in humans.

Loss of Core 1-derived O-Glycans Decreases Breast Cancer Development in Mice.

Mucin-type core 1-derived O-glycans, one of the major types of O-glycans, are highly expressed in mammary gland epithelium. Abnormal O-glycans such as Tn antigen are found in over 90% of breast cancers; however, the in vivo role of these aberrant O-glycans in the etiology of breast cancer is unclear. We generated mice with mammary epithelial specific deletion of core 1-derived O-glycans. By crossing with two spontaneous mouse breast cancer models, we determined that loss of core 1-derived O-glycans delays the onset and progression of breast cancer development. Deficiency of core 1 O-glycosylation impaired the localization of Muc1, a major O-glycoprotein, on the apical surfaces of mammary epithelium. Signaling mediated by Muc1, which is critical for breast cancer development, was also defective in the absence of core 1 O-glycans. This study reveals an unexpected role of core 1-derived O-glycans in breast cancer development in mice.

Endothelial cell O-glycan deficiency causes blood/lymphatic misconnections and consequent fatty liver disease in mice.

Mucin-type O-glycans (O-glycans) are highly expressed in vascular ECs. However, it is not known whether they are important for vascular development. To investigate the roles of EC O-glycans, we generated mice lacking T-synthase, a glycosyltransferase encoded by the gene C1galt1 that is critical for the biosynthesis of core 1-derived O-glycans, in ECs and hematopoietic cells (termed here EHC T-syn(-/-) mice). EHC T-syn(-/-) mice exhibited embryonic and neonatal lethality associated with disorganized and blood-filled lymphatic vessels. Bone marrow transplantation and EC C1galt1 transgene rescue demonstrated that lymphangiogenesis specifically requires EC O-glycans, and intestinal lymphatic microvessels in EHC T-syn(-/-) mice expressed a mosaic of blood and lymphatic EC markers. The level of O-glycoprotein podoplanin was significantly reduced in EHC T-syn(-/-) lymphatics, and podoplanin-deficient mice developed blood-filled lymphatics resembling EHC T-syn(-/-) defects. In addition, postnatal inactivation of C1galt1 caused blood/lymphatic vessel misconnections that were similar to the vascular defects in the EHC T-syn(-/-) mice. One consequence of eliminating T-synthase in ECs and hematopoietic cells was that the EHC T-syn(-/-) pups developed fatty liver disease, because of direct chylomicron deposition via misconnected portal vein and intestinal lymphatic systems. Our studies therefore demonstrate that EC O-glycans control the separation of blood and lymphatic vessels during embryonic and postnatal development, in part by regulating podoplanin expression.

Kupffer cell receptor CLEC4F is important for the destruction of desialylated platelets in mice.

The liver has recently been identified as a major organ for destruction of desialylated platelets. However, the underlying mechanism remains unclear. Kupffer cells, which are professional phagocytic cells in the liver, comprise the largest population of resident tissue macrophages in the body. Kupffer cells express a C-type lectin receptor, CLEC4F, that recognizes desialylated glycans with an unclear in vivo role in mediating platelet destruction. In this study, we generated a CLEC4F-deficient mouse model (Clec4f-/-) and found that CLEC4F was specifically expressed by Kupffer cells. Using the Clec4f-/- mice and a newly generated platelet-specific reporter mouse line, we revealed a critical role for CLEC4F on Kupffer cells in mediating destruction of desialylated platelets in the liver in vivo. Platelet clearance experiments and ultrastructural analysis revealed that desialylated platelets were phagocytized predominantly by Kupffer cells in a CLEC4F-dependent manner in mice. Collectively, these findings identify CLEC4F as a Kupffer cell receptor important for the destruction of desialylated platelets induced by bacteria-derived neuraminidases, which provide new insights into the pathogenesis of thrombocytopenia in disease conditions such as sepsis.

Core 1-derived mucin-type O-glycosylation protects against spontaneous gastritis and gastric cancer.

Core 1-derived mucin-type O-glycans (O-glycans) are a major component of gastric mucus with an unclear role. To address this, we generated mice lacking gastric epithelial O-glycans (GEC C1galt1-/-). GEC C1galt1-/- mice exhibited spontaneous gastritis that progressed to adenocarcinoma with ∼80% penetrance by 1 yr. GEC C1galt1-/- gastric epithelium exhibited defective expression of a major mucus forming O-glycoprotein Muc5AC relative to WT controls, which was associated with impaired gastric acid homeostasis. Inflammation and tumorigenesis in GEC C1galt1-/- stomach were concurrent with activation of caspases 1 and 11 (Casp1/11)-dependent inflammasome. GEC C1galt1-/- mice genetically lacking Casp1/11 had reduced gastritis and gastric cancer progression. Notably, expression of Tn antigen, a truncated form of O-glycan, and CASP1 activation was associated with tumor progression in gastric cancer patients. These results reveal a critical role of O-glycosylation in gastric homeostasis and the protection of the gastric mucosa from Casp1-mediated gastric inflammation and cancer.

Proximal colon-derived O-glycosylated mucus encapsulates and modulates the microbiota.

Colon mucus segregates the intestinal microbiota from host tissues, but how it organizes to function throughout the colon is unclear. In mice, we found that colon mucus consists of two distinct O-glycosylated entities of Muc2: a major form produced by the proximal colon, which encapsulates the fecal material including the microbiota, and a minor form derived from the distal colon, which adheres to the major form. The microbiota directs its own encapsulation by inducing Muc2 production from proximal colon goblet cells. In turn, O-glycans on proximal colon-derived Muc2 modulate the structure and function of the microbiota as well as transcription in the colon mucosa. Our work shows how proximal colon control of mucin production is an important element in the regulation of host-microbiota symbiosis.

P-selectin glycoprotein ligand-1 is highly expressed on Ly-6Chi monocytes and a major determinant for Ly-6Chi monocyte recruitment to sites of atherosclerosis in mice.

BACKGROUND: Ly-6C(hi) monocytes are key contributors to atherosclerosis in mice. However, the manner in which Ly-6C(hi) monocytes selectively accumulate in atherosclerotic lesions is largely unknown. Monocyte homing to sites of atherosclerosis is primarily initiated by rolling on P- and E-selectin expressed on endothelium. We hypothesize that P-selectin glycoprotein ligand-1 (PSGL-1), the common ligand of P- and E-selectin on leukocytes, contributes to the preferential homing of Ly-6C(hi) monocytes to atherosclerotic lesions. METHODS AND RESULTS: To test this hypothesis, we examined the expression and function of PSGL-1 on Ly-6C(hi) and Ly-6C(lo) monocytes from wild-type mice, ApoE(-/-) mice, and mice lacking both ApoE and PSGL-1 genes (ApoE(-/-)/PSGL-1(-/-)). We found that Ly-6C(hi) monocytes expressed a higher level of PSGL-1 and had enhanced binding to fluid-phase P- and E-selectin compared with Ly-6C(lo) monocytes. Under in vitro flow conditions, more Ly-6C(hi) monocytes rolled on P-, E-, and L-selectin at slower velocities than Ly-6C(lo) cells. In an ex vivo perfused carotid artery model, Ly-6C(hi) monocytes interacted preferentially with atherosclerotic endothelium compared with Ly-6C(lo) monocytes in a PSGL-1-dependent manner. In vivo, ApoE(-/-) mice lacking PSGL-1 had impaired Ly-6C(hi) monocyte recruitment to atherosclerotic lesions. Moreover, ApoE(-/-)/PSGL-1(-/-) mice exhibited significantly reduced monocyte infiltration in wire injury-induced neointima and in atherosclerotic lesions. ApoE(-/-)/PSGL-1(-/-) mice also developed smaller neointima and atherosclerotic plaques. CONCLUSIONS: These data indicate that PSGL-1 is a new marker for Ly-6C(hi) monocytes and a major determinant for Ly-6C(hi) cell recruitment to sites of atherosclerosis in mice.

Dclk1 in tuft cells promotes inflammation-driven epithelial restitution and mitigates chronic colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by defective intestinal barrier integrity toward the microbiota and epithelial damage. Double cortin-like kinase 1 (Dclk1), a marker of intestinal tuft cells, can regulate tissue regenerative responses, but its role in epithelial repair during bacterial-dependent chronic colitis is unclear. We addressed this question using our recently developed mouse model of spontaneous microbiota-dependent colitis induced by mucin-type O-glycan deficiency (DKO), which recapitulates most features of human UC. We generated DKO mice lacking intestinal epithelial Dclk1 (DKO;Dclk1ΔIEC) and analyzed colitis onset and severity using clinical and histologic indices, immune responses by qPCR and immunostaining, and epithelial responses using proliferation markers and organoid culture. We found 3-4-week-old DKO;Dclk1ΔIEC mice developed worsened spontaneous colitis characterized by reduced body weight, loose stool, severe colon thickening, epithelial lesions, and inflammatory cell infiltrates compared with DKO mice. The primary defect was an impaired epithelial proliferative response during inflammation. Dclk1 deficiency also reduced inflammation-induced proliferation and growth of colon organoids ex vivo. Mechanistically, Dclk1 expression was important for inflammation-induced Cox2 expression and prostaglandin E2 (PGE2) production in vivo, and PGE2 rescued proliferative defects in Dclk1-deficient colonic organoids. Although tuft cells were expanded in both DKO and DKO;Dclk1ΔIEC relative to WT mice, loss of Dclk1 was associated with reduced tuft cell activation (i.e., proliferation) during inflammation. Similar results were found in DKO vs. DKO;Dclk1ΔIEC mice at 3-6 months of age. Our results support that tuft cells, via Dclk1, are important responders to bacterial-induced colitis by enhancing epithelial repair responses, which in turn limits bacterial infiltration into the mucosa.

Site-1 protease deficiency causes human skeletal dysplasia due to defective inter-organelle protein trafficking.

Site-1 protease (S1P), encoded by MBTPS1, is a serine protease in the Golgi. S1P regulates lipogenesis, endoplasmic reticulum (ER) function, and lysosome biogenesis in mice and in cultured cells. However, how S1P differentially regulates these diverse functions in humans has been unclear. In addition, no human disease with S1P deficiency has been identified. Here, we report a pediatric patient with an amorphic and a severely hypomorphic mutation in MBTPS1. The unique combination of these mutations results in a frequency of functional MBTPS1 transcripts of approximately 1%, a finding that is associated with skeletal dysplasia and elevated blood lysosomal enzymes. We found that the residually expressed S1P is sufficient for lipid homeostasis but not for ER and lysosomal functions, especially in chondrocytes. The defective S1P function specifically impairs activation of the ER stress transducer BBF2H7, leading to ER retention of collagen in chondrocytes. S1P deficiency also causes abnormal secretion of lysosomal enzymes due to partial impairment of mannose-6-phosphate-dependent delivery to lysosomes. Collectively, these abnormalities lead to apoptosis of chondrocytes and lysosomal enzyme-mediated degradation of the bone matrix. Correction of an MBTPS1 variant or reduction of ER stress mitigated collagen-trafficking defects. These results define a new congenital human skeletal disorder and, more importantly, reveal that S1P is particularly required for skeletal development in humans. Our findings may also lead to new therapies for other genetic skeletal diseases, as ER dysfunction is common in these disorders.

Characterizing the Influence of Preload Dosing on Percent Signal Recovery (PSR) and Cerebral Blood Volume (CBV) Measurements in a Patient Population With High-Grade Glioma Using Dynamic Susceptibility Contrast MRI.

With DSC-MRI, contrast agent leakage effects in brain tumors can either be leveraged for percent signal recovery (PSR) measurements or be adequately resolved for accurate relative cerebral blood volume (rCBV) measurements. Leakage effects can be dimished by administration of a preload dose before imaging and/or specific postprocessing steps. This study compares the consistency of both PSR and rCBV measurements as a function of varying preload doses in a retrospective analysis of 14 subjects with high-grade gliomas. The scans consisted of 6 DSC-MRI scans during 6 sequential bolus injections (0.05 mmol/kg). Mean PSR was calculated for tumor and normal-appearing white matter regions of interest. DSC-MRI data were corrected for leakage effects before computing mean tumor rCBV. Statistical differences were seen across varying preloads for tumor PSR (P value = 4.57E-24). Tumor rCBV values did not exhibit statistically significant differences across preloads (P value = .14) and were found to be highly consistent for clinically relevant preloads (intraclass correlation coefficient = 0.93). For a 0.05 mmol/kg injection bolus and pulse sequence parameters used, the highest PSR contrast between normal-appearing white matter and tumor occurs when no preload is used. This suggests that studies using PSR as a biomarker should acquire DSC-MRI data without preload. The finding that leakage-corrected rCBV values do not depend on the presence or dose of preload contradicts that of previous studies with dissimilar acquisition protocols. This further confirms the sensitivity of rCBV to preload dosing schemes and pulse sequence parameters and highlights the importance of standardization efforts for achieving multisite rCBV consistency.

Executive function and functional and structural brain differences in middle-age adults with autism spectrum disorder.

There is a rapidly growing group of aging adults with autism spectrum disorder (ASD) who may have unique needs, yet cognitive and brain function in older adults with ASD is understudied. We combined functional and structural neuroimaging and neuropsychological tests to examine differences between middle-aged men with ASD and matched neurotypical (NT) men. Participants (ASD, n = 16; NT, n = 17) aged 40-64 years were well-matched according to age, IQ (range: 83-131), and education (range: 9-20 years). Middle-age adults with ASD made more errors on an executive function task (Wisconsin Card Sorting Test) but performed similarly to NT adults on tests of delayed verbal memory (Rey Auditory Verbal Learning Test) and local visual search (Embedded Figures Task). Independent component analysis of a functional MRI working memory task (n-back) completed by most participants (ASD = 14, NT = 17) showed decreased engagement of a cortico-striatal-thalamic-cortical neural network in older adults with ASD. Structurally, older adults with ASD had reduced bilateral hippocampal volumes, as measured by FreeSurfer. Findings expand our understanding of ASD as a lifelong condition with persistent cognitive and functional and structural brain differences evident at middle-age. Autism Res 2017, 10: 1945-1959. © 2017 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: We compared cognitive abilities and brain measures between 16 middle-age men with high-functioning autism spectrum disorder (ASD) and 17 typical middle-age men to better understand how aging affects an older group of adults with ASD. Men with ASD made more errors on a test involving flexible thinking, had less activity in a flexible thinking brain network, and had smaller volume of a brain structure related to memory than typical men. We will follow these older adults over time to determine if aging changes are greater for individuals with ASD.

Loss of intestinal core 1-derived O-glycans causes spontaneous colitis in mice.

Mucin-type O-linked oligosaccharides (O-glycans) are primary components of the intestinal mucins that form the mucus gel layer overlying the gut epithelium. Impaired expression of intestinal O-glycans has been observed in patients with ulcerative colitis (UC), but its role in the etiology of this disease is unknown. Here, we report that mice with intestinal epithelial cell-specific deficiency of core 1-derived O-glycans, the predominant form of O-glycans, developed spontaneous colitis that resembled human UC, including massive myeloid infiltrates and crypt abscesses. The colitis manifested in these mice was also characterized by TNF-producing myeloid infiltrates in colon mucosa in the absence of lymphocytes, supporting an essential role for myeloid cells in colitis initiation. Furthermore, induced deletion of intestinal core 1-derived O-glycans caused spontaneous colitis in adult mice. These data indicate a causal role for the loss of core 1-derived O-glycans in colitis. Finally, we detected a biosynthetic intermediate typically exposed in the absence of core 1 O-glycan, Tn antigen, in the colon epithelium of a subset of UC patients. Somatic mutations in the X-linked gene that encodes core 1 ß1,3-galactosyltransferase-specific chaperone 1 (C1GALT1C1, also known as Cosmc), which is essential for core 1 O-glycosylation, were found in Tn-positive epithelia. These data suggest what we believe to be a new molecular mechanism for the pathogenesis of UC.