Pleiotropic loci underlying bone mineral density and bone size identified by a bivariate genome-wide association analysis.

Aiming to identify pleiotropic genomic loci for bone mineral density and bone size, we performed a bivariate GWAS in five discovery samples and replicated in two large-scale samples. We identified 2 novel loci at 2q37.1 and 6q26. Our findings provide insight into common genetic architecture underlying both traits. INTRODUCTION: Bone mineral density (BMD) and bone size (BS) are two important factors that contribute to the development of osteoporosis and osteoporotic fracture. Both BMD and BS are highly heritable and they are genetically correlated. In this study, we aim to identify pleiotropic loci associated with BMD and BS. METHODS: We conducted a bivariate genome-wide association (GWA) analysis of hip BMD and hip BS in 6180 participants from 5 samples, followed by in silico replication in the UK Biobank study of BMD (N = 426,824) and the deCODE study of BS (N = 28,954), respectively. RESULTS: SNPs from 2 genomic loci were significant at the genome-wide significance (GWS) level (p lt; 5 × 10-8) in the discovery samples and were successfully replicated in the replication samples (2q37.1, lead SNP rs7575512, discovery p = 1.49 × 10-10, replication p = 0.05; 6q26, lead SNP rs1040724, discovery p = 1.95 × 10-8, replication p = 0.03). Functional annotations suggested functional relevance of the identified variants to bone development. CONCLUSION: Our findings provide insight into the common genetic architecture underlying BMD and BS, and enhance our understanding of the potential mechanism of osteoporosis fracture.

[Clinical application of hybrid surgery for the treatment of cerebral arteriovenous malformations].

Objective: To investigate the application of hybrid technique for the treatment of cerebral arteriovenous malformations (CAVMs) and to assess the value of hybrid technique. Methods: The cases of CAVMs treated in Qilu hospital and People's Hospital of Xinjiang Uygur Autonomous Region from July 2011 to July 2016 were analyzed retrospectively.Two modes of hybrid surgery, "angiographic diagnosis-craniotomy lesion resection or/and hematoma clearance-intraoperative angiography evaluation" and "angiographic diagnosis-intraoperative embolization-craniotomy lesion resection or/and hematoma clearance-intraoperative angiography evaluation" were applied for all the cases.We placed an aneurysm clip as marker in surgery field during real-time angiography.If CAVMs residues occurred during surgery, we re-resected the residue according to the guidance of the marker (clip) and DSA imaging. Intra-operative angiography evaluated the results of CAVMs resection one more time.Postoperatively, follow-up CT scan was performed for all the patients. Results: Of all the cases with CAVMs, there were 8 cases of scale Ⅰ, 13 cases of scale Ⅱ, 10 cases of scale Ⅲ and 6 cases of scale Ⅳ according to Spetzler-Martin Scale.There were 28 cases of acute hemorrhagic CAVMs and 9 cases of chronic hemorrhagic CAVMs or no-hemorrhagic CAVMs.Intra-operative angiography showed CAVMs residues in 6 cases of acute hemorrhagic CAVMs and only one in chronic group.About 18.92% residual rate of CAVMs were found for the first time intra-operative assessment angiography.With the guidance of intra-operative angiography and aneurysm clip as Marker, all residues of CAVMs were resected totally.Follow up CT showed the hematomas disappeared in all the cases of acute hemorrhagic cases.The cure rate of CAVMs with hybrid surgery was 100% according to the final intra-operative assessment angiography. Conclusions: (1)Hybrid surgery for the treatment of CAVMs in one session could evaluate the results of CAVMs resection and instruct the surgical procedure according to real-time angiography.This model could improve the treatment safety and efficacy for patients with CAVMs.(2)Patients with higher Spetzler-Martin Scale (Ⅲ-Ⅳ) who need intra-operative embolization and patients with hemorrhagic CAVMs are more suitable for hybrid surgery.

[Capsule endoscopy for Behcet's disease-treatment: five cases reports].

Behcet's disease (BD) is a chronic vascular inflammatory disease of unknown causes. It is called intestinal BD, when digestive tract is involved. To investigate small bowel feature of intestinal BD, we now report 5 intestinal BD cases undergone capsule endoscopy from December, 2010 to April, 2014 in Peking University People's Hospital. General information, clinical feature and endoscopic feature were presented, and literatures were reviewed. There were 3 male and 2 female patients. Age range was from 23 to 55 years old (median age 40 years old). Disease course was from 3 days to 28 years (median course 9 years). 4 patients were diagnosed as systemic BD, and the rest independent intestinal BD. 4 systemic BD patients all presented as recurrent oral aphthous as initial symptom and had history of vulvar ulcer and skin lesion. They all had gastrointestinal symptoms, including retrosternal pain (2 cases), hematochezia (3 cases), diarrhea (3 cases) and abdominal pain (2 cases). 1 patient had a history of fistula of ileocecal junction and underwent caecectomy. 5 patients all underwent whole digestive tract examination by endoscopy, including gastroscopy, colonoscopy and capsule endoscopy.Except of 1 normal result of colonoscopy, all endoscopy results revealed lesions. Capsule endoscopy results of all patients were abnormal. Types of small intestinal lesion were various, including ulceration, erosion, protrusion and vasculopathy. All digestive tract can be involved in BD patients. Capsule endoscopy can evaluate lesions throughout whole digestive tract, especially in small intestine. As a consequence, it is helpful to explain gastrointestinal symptom, increase early diagnostic rate. Intestinal BD (IBD) mainly involves small bowel, and ileum is the major involved segment, not only limited in ileocecum. The updated perspective of IBD lesion distribution will contribute to differential diagnosis between IBD and Crohn's disease.This is the first time to report capsule endoscopic feature of BD patients in China.

In vivo and in vitro models for the therapeutic targeting of Wnt signaling using a Tet-OΔN89ß-catenin system.

Although significant progress has been made in understanding the importance of Wnt signaling in the initiation of colorectal cancer, less is known about responses that accompany the reversal of oncogenic Wnt signaling. The aim of this study was to analyze in vivo and in vitro responses to an 'ideal' Wnt pathway inhibitor as a model for the therapeutic targeting of the pathway. A tetracycline-inducible transgenic mouse model expressing truncated ß-catenin (ΔN89ß-catenin) that exhibited a strong intestinal hyperplasia was analyzed during the removal of oncogenic ß-catenin expression both in 3D 'crypt culture' and in vivo. Oncogenic Wnt signaling was rapidly and completely reversed. The strongest inhibition of Wnt target gene expression occurred within 24 h of doxycycline removal at which time the target genes Ascl2, Axin2 and C-myc were downregulated to levels below that in the control intestine. In vitro, the small molecule Wnt inhibitor CCT036477 induced a response within 4 h of treatment. By 7 days following doxycycline withdrawal, gene expression, cell proliferation and tissue morphology were undistinguishable from control animals.In conclusion, these results demonstrate that the reversal of Wnt signaling by inhibitors should ideally be studied within hours of treatment. The reversible system described, involving medium throughput in vitro approaches and rapid in vivo responses, should allow the rapid advance of early stage compounds into efficacy models that are more usually considered later in the drug discovery pipeline.

Interactions between G-protein-coupled receptors and periplakin: a selective means to regulate G-protein activation.

A substantial number of G-protein-coupled receptor-interacting proteins have been identified initially by the use of yeast two-hybrid screens. Using the C-terminal tail of both opioid receptors and the melanin concentrating hormone receptor-1 as bait, the actin and intermediate filament-binding protein periplakin was isolated. In each case, the site of interaction is within helix VIII of the receptor and periplakin limits agonist-mediated G-protein activation potentially by competing with G-protein for this region of the receptor.

G protein-coupled receptor fusion proteins in drug discovery.

A wide range of peptides and polypeptides can be appended to either the N- or C-terminus of G protein-coupled receptors without disrupting substantially ligand binding and signal transduction. Following fusion of fluorescent proteins, reporter gene constructs or G protein alpha subunits to the C-terminal tail of a receptor high content and G protein activation assays can be employed to identify agonist ligands. Further modification of the receptor fusions to introduce enhanced levels of constitutive activity and to physically destabilise the protein allows antagonist/inverse agonists screens to be developed in parallel. Equivalent C-terminal addition of pairs of complementary, non-functional, polypeptide fragments allows the application of enzyme complementation techniques. Introduction of N-terminal tags to receptors has also allowed the introduction of novel assay techniques based on a pH-sensitive cyanine dye. These have the capacity to overcome certain limitations of GPCR-fluorescent protein fusions.

Extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases differentially regulate the lipopolysaccharide-mediated induction of inducible nitric oxide synthase and IL-12 in macrophages: Leishmania phosphoglycans subvert macrophage IL-12 production by targeting ERK MAP kinase.

Macrophage activation by cytokines or microbial products such as LPS results in the induction and release of several key immune effector molecules including NO and IL-12. These have been shown to play crucial roles in the development of immunity to intracellular pathogens such as Leishmania. The molecular mechanisms underlying the induction of these effector molecules are not fully understood. We now show that the extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases play differential roles in the regulation of LPS-stimulated inducible NO synthase and IL-12 gene expression. In macrophages, LPS stimulates the simultaneous activation of all three classes of MAP kinases, ERK, c-jun N-terminal kinase, and p38, albeit with differential activation kinetics. However, studies using inhibitors selective for ERK (PD98059) and p38 (SB203580) show that while p38 plays an essential role in the induction of inducible NO synthase, ERK MAP kinases play only a minor role in promoting NO generation. In contrast, while p38 promotes induction of IL-12 (p40) mRNA, ERK activation suppresses LPS-mediated IL-12 transcription. The biological relevance of these regulatory signals is demonstrated by our finding that Leishmania lipophosphoglycans, which promote parasite survival, act by stimulating ERK MAP kinase to inhibit macrophage IL-12 production. Thus, as ERK and p38 MAP kinases differentially regulate the induction of the macrophage effector molecules, inducible NO synthase and IL-12, these kinases are potential targets not only for the development of novel strategies to combat intracellular pathogens but also for therapeutic immunomodulation.

Altered immune responses and susceptibility to Leishmania major and Staphylococcus aureus infection in IL-18-deficient mice.

IL-18, formerly designated IFN-inducing factor, is a novel cytokine produced by activated macrophages. It synergizes with IL-12 in the induction of the development of Th1 cells and NK cells. To define the biological role of IL-18 in vivo, we have constructed a strain of mice lacking IL-18. Homozygous IL-18 knockout (-/-) mice are viable, fertile, and without evident histopathologic abnormalities. However, in contrast to the heterozygous (+/-) or wild-type (+/+) mice, which are highly resistant to the infection of the protozoan parasite Leishmania major, the IL-18-/- mice are uniformly susceptible. The infected IL-18-/- mice produced significantly lower levels of IFN-gamma and larger amounts of IL-4 compared with similarly infected +/- and +/+ mice. In contrast, when infected with the extracellular Gram-positive bacteria Staphylococcus aureus, the IL-18-/- mice developed markedly less septicemia than similarly infected wild-type (+/+) mice. However, the mutant mice developed significantly more severe septic arthritis than the control wild-type mice. This was accompanied by a reduction in the levels of Ag-induced splenic T cell proliferation, decreased IFN-gamma and TNF-alpha synthesis, but increased IL-4 production by the mutant mice compared with the wild-type mice. These results therefore provide direct evidence that IL-18 is not only essential for the host defense against intracellular infection, but it also plays a critical role in regulating the synthesis of inflammatory cytokines, and therefore could be an important target for therapeutic intervention.

Regulation of macrophage IL-12 synthesis by Leishmania phosphoglycans.

It is now generally accepted that IFN-gamma, secreted by Th1 cells, is the most potent cytokine leading to macrophage activation and host resistance against infection with the intracellular protozoan parasite Leishmania. It is also established that IL-12 is a critical cytokine involved in the differentiation and expansion of Th1 cells. Therefore, the ability of Leishmania parasites to actively suppress IL-12 production by host macrophages may be an important strategy for parasite survival. Here we report that a major parasite cell surface molecule, phosphoglycan (PG), of Leishmania could selectively inhibit the synthesis of IL-12(p40, p70) by activated murine macrophages. Furthermore, synthetic PG (sPG) was able to inhibit IL-12 release in a dose-dependent manner. Inhibition was dependent on the galactose(beta1-4)mannose(alpha1)-PO4 repeating units and not the glycophosphoinositol lipid anchor of lipophosphoglycan. At the concentration used, sPG had no effect on the release of TNF-alpha or IL-6 in activated macrophages. The inhibition of IL-12(p40) production was at the transcriptional level, but was not mediated through NF kappaB inhibition. These data demonstrate that PG may be an important molecule for the establishment and survival of the parasite in permissive hosts.

Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages.

We have previously reported that mice lacking inducible nitric oxide synthase (NOS2) developed enhanced Th1 cell responses. We now investigated the mechanism by which NO modulates Th1 cells differentiation. Peritoneal macrophages from NOS2-deficient mice infected with Leishmania major in vivo or stimulated with IFN-gamma or lipopolysaccharide (LPS) in vitro produced significantly higher levels of IL-12 than those from heterozygous or wild-type mice. A macrophage cell line, J774, produced significant amounts of IL-12 following activation with LPS, or LPS plus IFN-gamma. This could be markedly enhanced by the NOS inhibitor L-NG monomethyl arginine (L-NMMA), but profoundly inhibited by the NO-generating compound S-nitroso-N-acetyl-penicillamine (SNAP). The effect of NO in this system is selective, since SNAP enhanced and L-NMMA decreased TNF-alpha synthesis by LPS-activated J774 cells. The differential effect of NO on IL-12 and TNF-alpha is at the transcriptional level and is activation dependent. Since IL-12 is a major inducer of Th1 cells which produce IFN-gamma that can activate macrophages to produce IL-12, our data demonstrate that NO can be an inhibitor of this feedback loop, preventing the excessive amplification of Th1 cells which are implicated in a range of immunopathologies.

Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages.

Lipophosphoglycan (LPG) glycoconjugates from promastigotes of Leishmania were not able to induce the expression of the cytokine-inducible nitric oxide synthase (iNOS) by the murine macrophage cell line, J774. However, they synergize with interferon gamma to stimulate the macrophages to express high levels of iNOS. This synergistic effect was critically time-dependent. Preincubation of J774 cells with the LPG glycans 4-18 h before stimulation with interferon gamma resulted in a significant reduction in the expression of iNOS mRNA and of NO synthesis, compared with cells preincubated with culture medium alone. The regulatory effect on the induction of iNOS by LPG is located in the LPG phosphoglycan disaccharide backbone. Synthetic fragments of this backbone had a similar regulatory effect on NO synthesis. Further, the production of NO by activated macrophages in the present system was correlated directly with the leishmanicidal capacity of the cells. These data therefore demonstrate that LPG glycoconjugates have a profound effect on the survival of Leishmania parasites through their ability to regulate the expression of iNOS by macrophages.

The role of interleukin 12 and nitric oxide in the development of spontaneous autoimmune disease in MRL/MP-lpr/lpr mice.

MRL/MP-lpr/lpr (MRL/lpr) mice develop a spontaneous autoimmune disease. Serum from these mice contained significantly higher concentrations of nitrite/nitrate than serum from age-matched control MRL/MP-+/+ (MRL/+), BALB/c or CBA/6J mice. Spleen and peritoneal cells from MRL/lpr mice also produced significantly more nitric oxide (NO) than those from the control mice when cultured with interferon (IFN) gamma and lipopolysaccharide (LPS) in vitro. It is interesting to note that peritoneal cells from MRL/lpr mice also produced markedly higher concentrations of interleukin (IL) 12 than those from MRL/+ or BALB/c mice when cultured with same stimuli. It is striking that cells from MRL/lpr mice produced high concentrations of NO when cultured cells from MRL/+ or BALB/c mice. The enhanced NO synthesis induced by IFN-gamma/LPS was substantially inhibited by anti-IL-12 antibody. In addition, IL-12-induced NO production can also be markedly inhibited by anti-IFN-gamma antibody, but only weakly inhibited by anti-tumor necrosis factor alpha antibody. The effect of IL-12 on NO production was dependent on the presence of natural killer and possibly T cells. Serum from MRL/lpr mice contained significantly higher concentrations of IL-12 compared with those of MRL/+ or BALB/c control mice. Daily injection of recombinant IL-12 led to increased serum levels of IFN-gamma and NO metabolites, and accelerated glomerulonephritis in the young MRL/lpr mice (but not in the MRL/+ mice) compared with controls injected with phosphate-buffered saline alone. These data, together with previous finding that NO synthase inhibitors can ameliorate autoimmune disease in MRL/lpr mice, suggest that high capacity of such mice to produce IL-12 and their greater responsiveness to IL-12, leading to the production of high concentrations of NO, are important factors in this spontaneous model of autoimmune disease.

Altered immune responses in mice lacking inducible nitric oxide synthase.

Nitric oxide (NO) is important in many biological functions. It is generated from L-arginine by the enzyme NO synthase (NOS). The cytokine-inducible NOS (iNOS) is activated by several immunological stimuli, leading to the production of large quantities of NO which can be cytotoxic. To define the biological role of iNOS further, we generated iNOS mutant mice. These are viable, fertile and without evident histopathological abnormalities. However, in contrast to wild-type and heterozygous mice, which are highly resistant to the protozoa parasite Leishmania major infection, mutant mice are uniformly susceptible. The infected mutant mice developed a significantly stronger Th1 type of immune response than the wild-type or heterozygous mice. The mutant mice showed reduced nonspecific inflammatory response to carrageenin, and were resistant to lipopolysaccharide-induced mortality.

Seizures induced by intraventricular microinjection of ionized cobalt in the rat--a new experimental model of epilepsy.

A new animal model for epilepsy was successfully produced by microinjection of cobaltous chloride into the lateral cerebral ventricle of the rat. The median convulsive dose (CD50) and the median lethal dose (LD50) of CoCl2 was 0.45 microM/10 microliters (0.27-0.77 microM/10 microliters) and 1.07 microM/10 microliters (0.73-1.57 microM/10 microliters), respectively. The behavioral changes, electrocorticogram (ECoG), and the action of 5 classical anticonvulsants were studied using this new model. Seizures induced by cobaltous chloride are clinically similar to those produced by systemic administration of kainic acid and amygdala kindling. These are characterized by staring spells, wet dog shakes, mild convulsive movements, and stereotyped convulsions. ECoG findings demonstrated a unique epileptic burst during the wet dog shakes. Generalized epileptiform discharges were seen during typical seizures. The burst of spikes first occurred in the opposite temporal and frontal regions; and then became generalized. Among the 5 anticonvulsants studied, phenobarbital (30 mg/kg) and nitrazepam (3 mg/kg) completely antagonized the seizures; carbamazepine showed a moderate effect; and phenytoin as well as sodium valproate showed little effect. It is postulated that the seizures induced by cobaltous chloride may originate in the limbic system; and that cobalt ions are responsible for the seizure-inducing action. The mechanism remains to be investigated.