Association of Polymorphisms of the CHI3L1 Gene with Asthma and Atopy: A Populations-Based Study of 6514 Danish Adults

Background

YKL-40 is a chitinase-like glycoprotein encoded by the chitinase 3-like 1 gene, CHI3L1, localized at chromosome 1q32.1. Increased levels of serum YKL-40 have been reported to be a biomarker for asthma and a reduced lung function. Interestingly, the C-allele of the -131 C→G (rs4950928) polymorphism of CHI3L1 has been shown to associate with bronchial hyperresponsiveness and reduced lung function suggesting that variations in CHI3L1 may influence risk of asthma. The objective of the present study was to investigate the association of common variation in the CHI3L1 locus with asthma, atopy and lung function in a large population-based sample of adults.

Methods/Principal Findings

Eleven single nucleotide polymorphisms (SNPs) of CHI3L1 including rs4950928 were genotyped in 6514 individuals. Asthma was defined as self-reported history of physician-diagnosed asthma. Total IgE and specific IgE to inhalant allergens were measured on serum samples. Lung function was measured by spirometry. Homozygosity of the rs4950928 G allele as compared to homozygosity of the C allele was associated with self-reported physician diagnosed asthma (OR 1.5 (95% CI, 1.00–2.26)) and with prevalence of atopic asthma (OR 1.93 (95% CI, 1.21–3.07)) after adjustment for age, sex, smoking status, socio-economic class and BMI. Carriers of rs883125 G allele had a significantly lower prevalence of atopy (OR 0.82 (CI, 0.72; 0.94)) as compared to homozygosity of the C allele. None of the SNPs examined were significantly associated with FEV1. However, two SNPs (rs10399931and rs4950930) appeared to be significantly associated with FEV₁/FVC-ratio. Subgroup analyses of never-smokers did not consistently influence the associations in an either positively og negatively way.

Conclusions

In contrast to previous studies, the rs4950928 G allele, and not the C allele, was found to be associated with asthma. A few other SNPs of the CHI3L1 was found to be significantly associated with atopy and FEV1/FVC ratio, respectively. Thus, more studies seem warranted to establish the role of CHI3L1 gene in asthma and atopy.     

Metastasis-related Plasma Membrane Proteins of Human Breast Cancer Cells Identified by Comparative Quantitative Mass Spectrometry

The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multistep process. The cancer cell proteins and plasma membrane proteins in particular involved in this process are poorly defined, and a study of the very early events of the metastatic process using clinical samples or in vitro assays is not feasible. We have used a unique model system consisting of two isogenic human breast cancer cell lines that are equally tumorigenic in mice; but although one gives rise to metastasis, the other disseminates single cells that remain dormant at distant organs. Membrane purification and comparative quantitative LC-MS/MS proteomics identified 13 membrane proteins that were expressed at higher levels and three that were underexpressed in the metastatic compared with the non-metastatic cell line from a total of 1919 identified protein entries. Among the proteins were ecto-5′-nucleotidase (CD73), NDRG1, integrin β1, CD44, CD74, and major histocompatibility complex class II proteins. The altered expression levels of proteins identified by LC-MS/MS were validated using flow cytometry, Western blotting, and immunocyto- and immunohistochemistry. Analysis of clinical breast cancer biopsies demonstrated a significant correlation between high ecto-5′-nucleotidase and integrin β1 expression and poor outcome, measured as tumor spread or distant recurrence within a 10-year follow-up. Further the tissue analysis suggested that NDRG1, HLA-DRα, HLA-DRβ, and CD74 were associated with the ER⁻/PR⁻ phenotype represented by the two cell lines. The study demonstrates a quantitative and comparative proteomics strategy to identify clinically relevant key molecules in the early events of metastasis, some of which may prove to be potential targets for cancer therapy.Breast cancer is the most common malignant disease among women in Western countries, occurring in approximately one in 11 women (1). In this disease, malignant cells often disseminate to regional lymph nodes and establish distant metastases, preferentially in the bone, lung, and liver, resulting in poor outcome and high mortality (2, 3).Metastases are established through a complex set of events that is yet not fully understood but requires detachment of single cells from the primary tumor, penetration of the tissue matrix, and migration of these cells to distant locations where they induce angiogenesis and undergo expansive growth (4). Some disseminated cancer cells seem to be capable of maintaining dormancy in distant organs without establishing metastases but may suddenly become activated many years after resection of the primary tumor (5). The dormancy may be caused by environmental signals, either lack of those inducing differentiation or the presence of signals stimulating growth arrest. Cellular factors and changes in the microenvironment, such as inflammation or a change in hormonal status, might eventually induce proliferation, differentiation, and subsequent metastatic growth, whereas other disseminated cancer cells remain dormant for a lifetime (6).Traditional models of metastasis suggest that a subpopulation of cells in the primary tumor acquire metastatic capacity late in tumorigenesis, but gene expression profiles and cellular studies have recently provided evidence for a possible alternative model that suggests the metastatic capacity is acquired early in tumorigenesis (7). Stem cell populations have been identified in a range of hematopoietic and solid tumors and might represent the cells of origin for these tumors but might also be responsible for metastasis (8). Although a preserved genetic signature between the primary tumor and the metastasis has been found, other studies provide evidence of a gradual acquisition of genomic changes because distant metastases may not uniformly share mutations and often differ extensively from the primary tumor, reflecting the extent of genetic instability of breast cancer (9, 10). Only few studies provide proteomic characteristics of metastatic versus primary tumor of breast cancer because of the difficulties of obtaining high quality human tumor samples with full clinical histories and the absence of directly relevant in vitro assays (11, 12).The two isogenic cell lines M-4A4 and NM-2C5, which were derived from the MDA-MB-435 cell line and originated from a highly aggressive human invasive ductal carcinoma, provide an interesting model of the metastatic process (13). M-4A4 and NM-2C5, when inoculated into the mammary fat pad of nude mice, showed equal tumorigeneity, but although M-4A4 established easily detectable metastases restricted to lymph nodes and lungs, NM-2C5 cells disseminated to distal organs, but the cells remained dormant and did not establish metastasis (14). There is an ongoing debate on whether the parent cell line MDA-MB-435 can be defined as a breast cancer cell line because it, along with breast- and epithelia-specific markers, also expresses melanoma-specific genes (15). However, MDA-MB-435 can be induced to express breast differentiation-specific proteins and secrete milk lipids as observed in other well established breast cancer cell lines and has therefore been considered as an excellent model of a highly malignant and dedifferentiated breast cancer (16). Regardless of this debate, our model system remains valuable in the context of cancer metastasis, but the results should, as always when using cell line models, be supported by studies of clinically relevant human tissue specimens.M-4A4 and NM-2C5 have been extensively compared using gene expression analysis identifying a panel of differentially expressed genes (13, 17–20). However, because the proteome is so much more complex than the genome, similar studies at the protein level with special focus on plasma membrane proteins may add valuable biological insight and identify cell surface molecules that might be targeted with drugs or antibodies to inhibit the metastatic process.Comparative quantitative proteomics using stable isotope labeling with amino acids in cell culture (SILAC)¹ and LC-MS/MS allows a study of proteins with quantitatively different expression levels on metastasizing versus non-metastasizing cells. We used this technique to identify a panel of plasma membrane proteins showing altered expression in cells capable of forming metastasis. Validation studies at the protein and RNA expression level of the cell lines indicate that several of the identified proteins may be important for establishing metastasis in distant organs and thus have potential in target-specific therapy. Therefore, to further evaluate the clinical relevance of a selected number of the candidates identified by our analysis, their expression levels were evaluated in a panel of primary breast cancer biopsies and corresponding axillary lymph node metastasis from patients with known clinical outcomes. The results demonstrated the power of this systematic stepwise strategy for identifying targets of potential clinical value.     

Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus

Due to the recent development of a cell culture model, hepatitis C virus (HCV) can be efficiently propagated in cell culture. This allowed us to reinvestigate the subcellular localization of HCV structural proteins in the context of an infectious cycle. In agreement with previous reports, confocal immunofluorescence analysis of the subcellular localization of HCV structural proteins indicated that, in infected cells, the glycoprotein heterodimer is retained in the endoplasmic reticulum. However, in contrast to other studies, the glycoprotein heterodimer did not accumulate in other intracellular compartments or at the plasma membrane. As previously reported, an association between the capsid protein and lipid droplets was also observed. In addition, a fraction of labeling was consistent with the capsid protein being localized in a membranous compartment that is associated with the lipid droplets. However, in contrast to previous reports, the capsid protein was not found in the nucleus or in association with mitochondria or other well-defined intracellular compartments. Surprisingly, no colocalization was observed between the glycoprotein heterodimer and the capsid protein in infected cells. Electron microscopy analyses allowed us to identify a membrane alteration similar to the previously reported "membranous web." However, no virus-like particles were found in this type of structure. In addition, dense elements compatible with the size and shape of a viral particle were seldom observed in infected cells. In conclusion, the cell culture system for HCV allowed us for the first time to characterize the subcellular localization of HCV structural proteins in the context an infectious cycle.     

The glutamate/GABA-glutamine cycle: aspects of transport, neurotransmitter homeostasis and ammonia transfer

Neurons are metabolically handicapped in the sense that they are not able to perform de novo synthesis of neurotransmitter glutamate and gamma-aminobutyric acid (GABA) from glucose. A metabolite shuttle known as the glutamate/GABA-glutamine cycle describes the release of neurotransmitter glutamate or GABA from neurons and subsequent uptake into astrocytes. In return, astrocytes release glutamine to be taken up into neurons for use as neurotransmitter precursor. In this review, the basic properties of the glutamate/GABA-glutamine cycle will be discussed, including aspects of transport and metabolism. Discussions of stoichiometry, the relative role of glutamate vs. GABA and pathological conditions affecting the glutamate/GABA-glutamine cycling are presented. Furthermore, a section is devoted to the accompanying ammonia homeostasis of the glutamate/GABA-glutamine cycle, examining the possible means of intercellular transfer of ammonia produced in neurons (when glutamine is deamidated to glutamate) and utilized in astrocytes (for amidation of glutamate) when the glutamate/GABA-glutamine cycle is operating. A main objective of this review is to endorse the view that the glutamate/GABA-glutamine cycle must be seen as a bi-directional transfer of not only carbon units but also nitrogen units.     

Early Life Disease Programming during the Preconception and Prenatal Period: Making the Link between Stressful Life Events and Type-1 Diabetes

Background

To assess the risk of developing Type-1 diabetes among children who were exposed to maternal bereavement during the prenatal or 1-year preconception period.

Methods

We identified N = 1,548,746 singleton births born in Denmark between January 1^st 1979 through December 31^st 2004, and their next of kin. Altogether, 39,857 children were exposed to bereavement during their prenatal life. The main outcome of interest was hospitalization for type-1 diabetes (ICD 8: 249; ICD 10: E10).

Results

We found the strongest association for type-1 diabetes among children exposed to traumatic father or sibling deaths (aIRR: 2.03, 1.22–3.38); the association was mainly seen for girls (aIRR: 2.91, 1.61–5.26).

Conclusions

We found evidence to suggest that female fetuses exposed to severe prenatal stress are at increased risk for developing type-1 diabetes.     

Rapid Implementation of an Integrated Large-Scale HIV Counseling and Testing,Malaria, and Diarrhea Prevention Campaign in Rural Kenya

Background

Integrated disease prevention in low resource settings can increase coverage, equity and efficiency in controlling high burden infectious diseases. A public-private partnership with the Ministry of Health, CDC, Vestergaard Frandsen and CHF International implemented a one-week integrated multi-disease prevention campaign.

Method

Residents of Lurambi, Western Kenya were eligible for participation. The aim was to offer services to at least 80% of those aged 15–49. 31 temporary sites in strategically dispersed locations offered: HIV counseling and testing, 60 male condoms, an insecticide-treated bednet, a household water filter for women or an individual filter for men, and for those testing positive, a 3-month supply of cotrimoxazole and referral for follow-up care and treatment.

Findings

Over 7 days, 47,311 people attended the campaign with a 96% uptake of the multi-disease preventive package. Of these, 99.7% were tested for HIV (87% in the target 15–49 age group); 80% had previously never tested. 4% of those tested were positive, 61% were women (5% of women and 3% of men), 6% had median CD4 counts of 541 cell/µL (IQR; 356, 754). 386 certified counselors attended to an average 17 participants per day, consistent with recommended national figures for mass campaigns. Among women, HIV infection varied by age, and was more likely with an ended marriage (e.g. widowed vs. never married, OR.3.91; 95% CI. 2.87–5.34), and lack of occupation. In men, quantitatively stronger relationships were found (e.g. widowed vs. never married, OR.7.0; 95% CI. 3.5–13.9). Always using condoms with a non-steady partner was more common among HIV-infected women participants who knew their status compared to those who did not (OR.5.4 95% CI. 2.3–12.8).

Conclusion

Through integrated campaigns it is feasible to efficiently cover large proportions of eligible adults in rural underserved communities with multiple disease preventive services simultaneously achieving various national and international health development goals.     

GUB06‐046, a novel secretin/glucagon‐like peptide 1 co‐agonist,decreases food intake,improves glycemic control,and preserves beta cell mass in diabetic mice

Bariatric surgery is currently the most effective treatment of obesity, which has spurred an interest in developing pharmaceutical mimetics. It is thought that the marked body weight‐lowering effects of bariatric surgery involve stimulated secretion of appetite‐regulating gut hormones, including glucagon‐like peptide 1. We here report that intestinal expression of secretin is markedly upregulated in a rat model of Roux‐en‐Y gastric bypass, suggesting an additional role of secretin in the beneficial metabolic effects of Roux‐en‐Y gastric bypass. We therefore developed novel secretin‐based peptide co‐agonists and identified a lead compound, GUB06‐046, that exhibited potent agonism of both the secretin receptor and glucagon‐like peptide 1 receptor. Semi‐acute administration of GUB06‐046 to lean mice significantly decreased cumulative food intake and improved glucose tolerance. Chronic administration of GUB06‐046 to diabetic db/db mice for 8 weeks improved glycemic control, as indicated by a 39% decrease in fasting blood glucose and 1.6% reduction of plasma HbA1c levels. Stereological analysis of db/db mice pancreata revealed a 78% increase in beta‐cell mass after GUB06‐046 treatment, with no impact on exocrine pancreas mass or pancreatic duct epithelial mass. The data demonstrate beneficial effects of GUB06‐046 on appetite regulation, glucose homeostasis, and beta‐cell mass in db/db mice, without proliferative effects on the exocrine pancreas and the pancreatic duct epithelium. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

The effect of oxycholesterols on thermo-induced membrane dynamics

The effect of temperature change(s) on the dynamics of giant unilamellar vesicles containing oxidized and non-oxidized cholesterol was investigated and characterized. We have demonstrated that (i) major cholesterol auto-oxidation products, 7β-hydroxycholesterol (7β) and 7-ketocholesterol (7keto), rendered vesicles more responsive to temperature changes; (ii) 7keto imparted greater thermo-induced membrane dynamics than 7β; (iii) 7β and 7keto vesicles synergistically were more thermo-responsive than the individual oxysterols; (iv) the thermo-responsiveness of 7keto-containing vesicles was equivalent to that of 25 hydroxycholesterol (25OH)-containing vesicles; and (v) we have characterized the observed membrane dynamics. The results provide a new plausible mechanism: oxidative-stressed membranes in conjunction with temperature change induce membrane dynamics. These findings improve the mechanisms reported previously that attributed the induced dynamics solely to membrane oxidation.