[Pathogenesis of glucocorticoid-induced osteoporosis based on label-free mass proteomics].

OBJECTIVE: To explore pathogenesis of glucocortocoid-induced osteoporosis(GIOP) based on label-free mass proteomics. METHODS: Twevle female Sprague-Dawley(SD) rats were randomly divided into two groups, named as sham group and GIOP group. After one-week adaptive feeding, the rats of GIOP group were administered with dexamethasone via intramuscular injection according to 2.5 mg/kg weighting, while the rats of sham group were administered with the same amount of saline, twice a week. The tibias of each group were collected after 8-week modeling and made pathological sections to confirm the success of modeling. Three samples of each group were picked up to perform label-free mass proteomics. After quality control, differentially expressed proteins were identified according to qualitative and quantitative analyses. Then gene ontology(GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, cluster analysis as well as protein-protein interaction analysis were performed using bioinformatics analysis. RESULTS: Compared with sham group, the structure of bone trabecular in GIOP group showed abnormal arrangement, uneven distribution and obvious fragmentation, which could demonstrate successful modeling. A total of 47 differentially expressed proteins (DEPs) were identified including 20 up-regulated and 27 down-regulated proteins. The expression of protein nucleophosmin 1(NPM1), adipocyte plasma membrane associated protein (APMAP), cytochromec oxidase subunit 6A1 (COX6A1) and tartrate-resistant acid phosphatase (ACP5) showed a significant difference between two groups. KEGG results showed DEPs were enriched on metabolism-related pathways, immune-related pathways and AMP-activated kinase (AMPK) signaling pathway. CONCLUSION: Protein NPM1, APMAP, COX6A1 and ACP5 showed a close relationship with pathogenesis of GIOP, which could serve as potential biomarkers of GIOP. AMPK signaling pathway played an important role in the occurrence and development of GIOP, which could be regarded as potential signaling pathway to treatment GIOP.

Nucleophosmin contributes to vascular inflammation and endothelial dysfunction in atherosclerosis progression.

OBJECTIVE: It is unclear whether nucleophosmin (NPM) participates in cardiovascular disease. The present study aimed to investigate the role and underlying mechanisms of NPM in atherosclerosis. METHODS: Levels and location of NPM in human carotid atherosclerotic plaques and healthy controls were detected by real-time polymerase chain reaction, immunoblots, and immunofluorescence. Atherosclerotic prone ApoE-/- mice were fed with a Western diet for 16 weeks as an in vivo model. Human primary umbilical vein endothelial cells (HUVECs) were cultured as an in vitro model. RESULTS: Compared with controls, we found that NPM levels in human carotid atherosclerotic plaques were more than twice as high as in normal arteries, which mainly localized in endothelial cells. In vivo, adenovirus-containing NPM small hairpin RNA attenuated atherosclerotic lesion and promoted plaque stabilization in ApoE-/- mice fed a Western diet by reducing vascular inflammation, maintaining endothelial function, and decreasing macrophage infiltration. Furthermore, NPM knockdown decreased nuclear factor-κB (NF-κB) p65 phosphorylation. In cultured HUVECs, palmitic acid increased the protein levels of NPM and induced the expression of inflammatory cytokines and monocyte adhesion, whereas NPM knockdown attenuated this effect. In HUVECs, NPM protein physically interacted with NF-κB p65 subunit and promoted its nuclear transposition. NPM also increased the transcriptional activity of NF-κB p65 promoter and enhance its binding to target genes, including interleukin-1ß, interleukin-6, intercellular adhesion molecule-1, and E-selectin. CONCLUSIONS: These data provide novel evidence that NPM promotes atherosclerosis by inducing vascular inflammation and endothelial dysfunction through the NF-κB signaling pathway and suggest that NPM may be a promising target for atherosclerosis prevention and treatment.

The phosphatidic acid phosphatase lipin-1 facilitates inflammation-driven colon carcinogenesis.

Colon cancer is a devastating illness that is associated with gut inflammation. Here, we explored the possible role of lipin-1, a phosphatidic acid phosphatase, in the development of colitis-associated tumorigenesis. Azoxymethane and dextran sodium sulfate-treated (DSS-treated) animals deficient in lipin-1 harbored fewer tumors and carcinomas than WT animals due to decreased cellular proliferation, lower expression of antiapoptotic and protumorigenic factors, and a reduced infiltration of macrophages in colon tumors. They also displayed increased resistance to DSS-induced colitis by producing less proinflammatory cytokines and experiencing less immune infiltration. Lipin-1-deficient macrophages from the colon were less activated and displayed lower phosphatidic acid phosphatase activity than WT macrophages isolated from DSS-treated animals. Transference of WT macrophages into lipin-1-deficient animals was sufficient to increase colitis burden. Furthermore, treatment of lipin-1-deficient mice with IL-23 exacerbated colon inflammation. Analysis of human databases from colon cancer and ulcerative colitis patients showed that lipin-1 expression is increased in those disorders and correlates with the expression of the proinflammatory markers CXCL1 and CXCL2. And finally, clinically, LPIN1 expression had prognostic value in inflammatory and stem-cell subtypes of colon cancers. Collectively, these data demonstrate that lipin-1 is a critical regulator of intestinal inflammation and inflammation-driven colon cancer development.

Development of surface modified biodegradable polymeric nanoparticles to deliver GSE24.2 peptide to cells: a promising approach for the treatment of defective telomerase disorders.

The aim of the present study was to develop a novel strategy to deliver intracellularly the peptide GSE24.2 for the treatment of Dyskeratosis congenita (DC) and other defective telomerase disorders. For this purpose, biodegradable polymeric nanoparticles using poly(lactic-co-glycolic acid) (PLGA NPs) or poly(lactic-co-glycolic acid)-poly ethylene glycol (PLGA-PEG NPs) attached to either polycations or cell-penetrating peptides (CPPs) were prepared in order to increase their cellular uptake. The particles exhibited an adequate size and zeta potential, with good peptide loading and a biphasic pattern obtained in the in vitro release assay, showing an initial burst release and a later sustained release. GSE24.2 structural integrity after encapsulation was assessed using SDS-PAGE, revealing an unaltered peptide after the NPs elaboration. According to the cytotoxicity results, cell viability was not affected by uncoated polymeric NPs, but the incorporation of surface modifiers slightly decreased the viability of cells. The intracellular uptake exhibited a remarkable improvement of the internalization, when the NPs were conjugated to the CPPs. Finally, the bioactivity, addressed by measuring DNA damage rescue and telomerase reactivation, showed that some formulations had the lowest cytotoxicity and highest biological activity. These results proved that GSE24.2-loaded NPs could be delivered to cells, and therefore, become an effective approach for the treatment of DC and other defective telomerase syndromes.

RanBP9 overexpression down-regulates phospho-cofilin, causes early synaptic deficits and impaired learning, and accelerates accumulation of amyloid plaques in the mouse brain.

Loss of synaptic proteins and functional synapses in the brains of patients with Alzheimer's disease (AD) as well as transgenic mouse models expressing amyloid-ß protein precursor is now well established. However, the earliest age at which such loss of synapses occurs, and whether known markers of AD progression accelerate functional deficits is completely unknown. We previously showed that RanBP9 overexpression leads to enhanced amyloid plaque burden in a mouse model of AD. In this study, we found significant reductions in the levels of synaptophysin and spinophilin, compared with wild-type controls, in both the cortex and the hippocampus of 5- and 6-month old but not 3- or 4-month old APΔE9/RanBP9 triple transgenic mice, and not in APΔE9 double transgenic mice, nor in RanBP9 single transgenic mice. Interestingly, amyloid plaque burden was also increased in the APΔE9/RanBP9 mice at 5-6 months. Consistent with these results, we found significant deficits in learning and memory in the APΔE9/RanBP9 mice at 5 and 6 month. These data suggest that increased amyloid plaques and accelerated learning and memory deficits and loss of synaptic proteins induced by RanBP9 are correlated. Most importantly, APΔE9/RanBP9 mice also showed significantly reduced levels of the phosphorylated form of cofilin in the hippocampus. Taken together these data suggest that RanBP9 overexpression down-regulates cofilin, causes early synaptic deficits and impaired learning, and accelerates accumulation of amyloid plaques in the mouse brain.

H1152 promotes the degradation of polyglutamine-expanded ataxin-3 or ataxin-7 independently of its ROCK-inhibiting effect and ameliorates mutant ataxin-3-induced neurodegeneration in the SCA3 transgenic mouse.

Spinocerebellar ataxia type 3 (SCA3) caused by polyglutamine-expanded ataxin-3 is the most prevalent subtype of spinocerebellar ataxias. A compound, which decreases protein level of mutant ataxin-3 in SCA3 affected CNS regions, should be a promising therapeutic agent for SCA3. SCA3 and Huntington's disease (HD) belong to a family of polyglutamine neurodegenerative diseases. Rho-kinase (ROCK) inhibitor Y27632 reduced brain level of polyglutamine-expanded huntingtin in HD transgenic mouse. Therefore, we tested the possibility that ROCK inhibitors, Y27632, H1152 and GSK429286, downregulate protein expression of polyglutamine-expanded ataxin-3-Q79. Y27632 or H1152 reduced protein level of HA-tagged ataxin-3-Q79 (ATX-3-Q79HA) expressed in HEK 293 cells. Compared to Y27632, H1152 decreased ATX-3-Q79HA protein level with a significantly more potency and efficacy. H1152 also reduced protein level of HA-tagged polyglutamine-expanded ataxin-7-Q52 (ATX-7-Q52HA), which causes spinocerebellar ataxia type 7 (SCA7). H1152 decreased ATX-3-Q79HA or ATX-7-Q52HA protein level in vitro by augmenting proteasome activity and promoting ATX-3-Q79HA or ATX-7-Q52HA degradation. GSK429286, which is structurally different from H1152 but equally inhibits ROCK, failed to affect protein level of ATX-3-Q79HA or ATX-7-Q52HA. Furthermore, shRNA-mediated suppression of ROCK1 or ROCK2 expression in 293 cells did not affect protein level of ATX-3-Q79HA or ATX-7-Q52HA and H1152 reduction of ATX-3-Q79HA. Daily intraperitoneal administration of H1152 significantly decreased protein level of ATX-3-Q79HA in the cerebellum, pontine nuclei and spinal cord of SCA3 transgenic mice. H1152 also ameliorated pontine neuronal death and neurological phenotype of SCA3 transgenic mice. Our results suggest that H1152 might be an effective therapeutic agent for SCA3 or SCA7.

Long-term administration of Wilms tumor-1 peptide vaccine in combination with gemcitabine causes severe local skin inflammation at injection sites.

The skin toxicity of vaccine therapy at injection sites is generally limited to Grades 1-2 due to the nature of their function. We experienced two cases of severe and prolonged local adverse effects in 25 patients following a Phase I study of gemcitabine and Wilms tumor-1 peptide vaccine mixed with incomplete Freund's adjuvant for inoperable pancreatic or biliary tract cancer. These patients requested to continue the treatment after the study period; however, in the course of compassionate use, they developed unacceptable local skin reactions and terminated their vaccine treatment. One patient (human leukocyte antigen, A0201, 3 mg) developed Grade 3 ulceration at the 10th vaccination and another (human leukocyte antigen, A2402, 1 mg) developed Grade 2 indulation and fibrosis at the 16th vaccination. Skin toxicity occurred at 6.4-8.4 months and continued for several months after the final vaccination during gemcitabine treatment. In these cases, activation or induction of Wilms tumor-1-specific T lymphocytes was not apparent in the peripheral blood despite their severe local reactions. Therefore, we need to monitor patients for late-onset, severe and long-lasting skin reactions at injection sites in Wilms tumor-1 cancer vaccine therapy, particularly for combination treatment with gemcitabine.

Replication study of 10 genetic polymorphisms associated with coronary heart disease in a specific high-risk population with familial hypercholesterolemia.

AIMS: Recent large association studies have revealed associations between genetic polymorphisms and myocardial infarction and coronary heart disease (CHD). We performed a replication study of 10 polymorphisms and CHD in a population with familial hypercholesterolemia (FH), individuals at extreme risk of CHD. METHODS AND RESULTS: We genotyped 10 polymorphisms in 2145 FH patients and studied the association between these polymorphisms and CHD in Cox proportional hazards models. We confirmed the associations between four polymorphisms and CHD, the rs1151640 polymorphism in the olfactory receptor family 13 subfamily G member 1 (OR13G1) gene (HR 1.14, 95% CI 1.01-1.28, P = 0.03), the rs11881940 polymorphism in the heterogeneous nuclear ribonucleoprotein U-like 1 (HNRPUL1) gene (HR 1.27, 95% CI 1.07-1.51, P = 0.007), the rs3746731 polymorphism in the complement component 1 q subcomponent receptor 1 (CD93) gene (HR 1.26, 95% CI 1.06-1.49, P = 0.01), and the rs10757274 polymorphism near the cyclin-dependent kinase N2A and N2B (CDKN2A and CDKN2B) genes (HR 1.39, 95% CI 1.15-1.69, P < 0.001). CONCLUSION: We confirmed previously found associations between four polymorphisms and CHD, but refuted associations for six other polymorphisms in our large FH population. These findings stress the importance of replication before genetic information can be implemented in the prediction of CHD.

Decreased immunoglobulin class switching in Nijmegen Breakage syndrome due to the DNA repair defect.

Nijmegen breakage syndrome (NBS) is a rare chromosomal-instability syndrome associated with defective DNA repair. Approximately 90% of NBS patients are homozygous for a truncating mutation of the NBS1 gene. As development of the immune system relies on recombination, which involves repair of DNA breaks, one might predict that mutations in the NBS1 gene could cause immunodeficiency. We immunologically investigated the world's largest series of NBS patients (n = 74), confirmed immunodeficiency, and found a discrepancy between relatively normal IgM concentrations, and decreased IgG and IgA concentrations. In addition, a significant relation between low IgA and low IgG levels was found. These data are compatible with a defective class switching in NBS and can be explained by a role of the NBS1 protein in DNA repair, signal transduction, cell cycle regulation or apoptosis.

Patch test reactions to mite antigens: a GERDA multicentre study. Groupe d'Etudes et de Recherches en Dermato-Allergie.

We performed patch tests with Dermatophagoides pteronyssinus (Dp) antigens from 2 different sources in 355 non-randomly selected patients with atopic dermatitis (AD) and 398 subjects of a control group. The study demonstrated that contact sensitization to mites occurred in an appreciable % of AD cases (20.8%), using commonly available assay products. The differences recorded between the 2 materials tested were related to the concentration of P1 antigen. Non-atopic patients rarely showed positive reactions to Dp (0.75%), when strict criteria for readings were applied and if 2 readings were performed. Patients with positive patch tests did not necessarily show positive immediate skin tests. It would be useful to carry out tests systematically in atopic patients, even if it is not yet known what modern treatment would be best for the patient. Laboratories still do not provide standardized house dust mite preparations--measuring and codifying their biological activity--for use in patch tests. It is to be hoped that the extension of this type of test will lead to the production of better test materials, in syringes with homogeneous dispersion and concentration.