Pooled genome-wide analysis to identify novel risk loci for pediatric allergic asthma

Background

Genome-wide association studies of pooled DNA samples were shown to be a valuable tool to identify candidate SNPs associated to a phenotype. No such study was up to now applied to childhood allergic asthma, even if the very high complexity of asthma genetics is an appropriate field to explore the potential of pooled GWAS approach.

Methodology/Principal Findings

We performed a pooled GWAS and individual genotyping in 269 children with allergic respiratory diseases comparing allergic children with and without asthma. We used a modular approach to identify the most significant loci associated with asthma by combining silhouette statistics and physical distance method with cluster-adapted thresholding. We found 97% concordance between pooled GWAS and individual genotyping, with 36 out of 37 top-scoring SNPs significant at individual genotyping level. The most significant SNP is located inside the coding sequence of C5, an already identified asthma susceptibility gene, while the other loci regulate functions that are relevant to bronchial physiopathology, as immune- or inflammation-mediated mechanisms and airway smooth muscle contraction. Integration with gene expression data showed that almost half of the putative susceptibility genes are differentially expressed in experimental asthma mouse models.

Conclusion/Significance

Combined silhouette statistics and cluster-adapted physical distance threshold analysis of pooled GWAS data is an efficient method to identify candidate SNP associated to asthma development in an allergic pediatric population.     

A stapled chromogranin A-derived peptide homes in on tumors that express αvβ6 or αvβ8 integrins

Rationale: The αvβ6- and αvβ8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFβ complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called “5a”), which selectively recognizes the LAP/TGFβ complex-binding site of αvβ6 and αvβ8.Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with ¹⁸F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvβ6/αvβ8 integrins and various αvβ6/αvβ8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvβ8-positive prostate tumors.Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFβ activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvβ6/αvβ8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvβ6/αvβ8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvβ8-positive prostate tumors.Conclusions: The results indicate that 5a can home to αvβ6- and/or αvβ8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvβ6/αvβ8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFβ activators.     

Metabolic effect of telmisartan and losartan in hypertensive patients with metabolic syndrome

Background

Metabolic syndrome is a cluster of common cardiovascular risk factors that includes hypertension and insulin resistance. Hypertension and diabetes mellitus are frequent comorbidities and, like metabolic syndrome, increase the risk of cardiovascular events. Telmisartan, an antihypertensive agent with evidence of partial peroxisome proliferator-activated receptor activity-gamma (PPARγ) activity, may improve insulin sensitivity and lipid profile in patients with metabolic syndrome.

Methods

In a double-blind, parallel-group, randomized study, patients with World Health Organization criteria for metabolic syndrome received once-daily doses of telmisartan (80 mg, n = 20) or losartan (50 mg, n = 20) for 3 months. At baseline and end of treatment, fasting and postprandial plasma glucose, insulin sensitivity, glycosylated haemoglobin (HBA_1c) and 24-hour mean systolic and diastolic blood pressures were determined.

Results

Telmisartan, but not losartan, significantly (p < 0.05) reduced free plasma glucose, free plasma insulin, homeostasis model assessment of insulin resistance and HbA_ic. Following treatment, plasma glucose and insulin were reduced during the oral glucose tolerance test by telmisartan, but not by losartan. Telmisartan also significantly reduced 24-hour mean systolic blood pressure (p < 0.05) and diastolic blood pressure (p < 0.05) compared with losartan.

Conclusion

As well as providing superior 24-hour blood pressure control, telmisartan, unlike losartan, displayed insulin-sensitizing activity, which may be explained by its partial PPARγ activity.     

The BTB-kelch protein KLHL6 is involved in B-lymphocyte antigen receptor signaling and germinal center formation

BTB-kelch proteins can elicit diverse biological functions but very little is known about the physiological role of these proteins in vivo. Kelch-like protein 6 (KLHL6) is a BTB-kelch protein with a lymphoid tissue-restricted expression pattern. In the B-lymphocyte lineage, KLHL6 is expressed throughout ontogeny, and KLHL6 expression is strongly upregulated in germinal center (GC) B cells. To analyze the role of KLHL6 in vivo, we have generated mouse mutants of KLHL6. Development of pro- and pre-B cells was normal but numbers of subsequent stages, transitional 1 and 2, and mature B cells were reduced in KLHL6-deficient mice. The antigen-dependent GC reaction was blunted (smaller GCs, reduced B-cell expansion, and reduced memory antibody response) in the absence of KLHL6. Comparison of mutants with global loss of KLHL6 to mutants lacking KLHL6 specifically in B cells demonstrated a B-cell-intrinsic requirement for KLHL6 expression. Finally, B-cell antigen receptor (BCR) cross-linking was less sensitive in KLHL6-deficient B cells compared to wild-type B cells as measured by proliferation, Ca2+ response, and activation of phospholipase Cgamma2. Our results strongly point to a role for KLHL6 in BCR signal transduction and formation of the full germinal center response.     

Immunoprevention and immunotherapy of cancer in ageing

Over the last few years there has been a growing interest in geriatric oncology, mainly because of the evidence that advanced age is the greatest risk factor for the development of cancer and that, since the elderly population is rapidly expanding, so too will the number of cancer patients. This forecast necessitates the development of new and more specific strategies for the prevention and cure of cancer in the elderly and as a result an ever-increasing need for oncologists, geriatricians and researchers to work closely together. The increased incidence of cancer in elderly people has been related to the age-associated changes occurring in the immune system, the so-called immunosenescence. This phenomenon is best characterised by a remodelling of the immune system, which appears early on and progresses throughout a persons life and mainly involves a decrease in cellular functions. This review aims to provide a rationale for the development of specific immunotherapeutic and immunopreventive regimens for the elderly. We also include a discussion on the influence that immunosenescence has on the growth of tumours and the effectiveness of immunogene therapy and cancer vaccination following a brief analysis of the age-related alterations of the cell populations involved in antitumour immunity.     

Evaluation of genotoxic effects induced by exposure to antineoplastic drugs in lymphocytes and exfoliated buccal cells of oncology nurses and pharmacy employees

The continuous introduction of new antineoplastic drugs and their use as complex mixture emphasize the need to carry out correct health risk assessment. The aim of this study was to evaluate genotoxic effects of antineoplastic drugs in nurses (n=25) and pharmacy technicians (n=5) employed in an oncology hospital. The nurses administered antineoplastic drugs in the day-care hospital (n=12) and in the wards (n=13), and pharmacy technicians prepared the drugs in the central pharmacy. We performed the micronucleus (MN) test with lymphocytes and exfoliated buccal cells and conducted traditional analysis of chromosomal aberrations (CA). Thirty healthy subjects were selected as controls. Monitoring of surface contamination with cyclophosphamide, 5-fluorouracil, ifosfamide, cytarabine, and gemcitabine showed the presence of detectable levels only for cyclophosphamide, 5-fluorouracil and ifosfamide. In addition, we measured the 5-fluorouracil metabolite alpha-F-betaalanine in the urine of all subjects and found significant concentrations only in 3 out of 25 nurses. The micronucleus assay with lymphocytes did not show significant differences between exposed and control groups, while the same test with exfoliated buccal cells found higher values in nurses administering antineoplastic drugs than in pharmacy employees. In the CA analysis, we detected in exposed groups a significant increase (about 2.5-fold) of structural CA, particularly breaks (up to 5.0-fold). Our results confirm the genotoxic effect of antineoplastic drugs in circulating blood lymphocytes. Moreover, in exfoliated buccal cells the data show more consistent genetic damage induced during administration of the antineoplastic drugs than during their preparation. The data also stress the use of this non-invasive sampling, to assess occupational exposure to mixture of chemicals at low doses.     

Aerobic cometabolism of chloroform by butane-grown microorganisms: long-term monitoring of depletion rates and isolation of a high-performing strain

The focus of this microcosm study was to monitor the performances of 17 butane-utilizing microcosms during a long-term (100–250 days) aerobic cometabolic depletion of chloroform (CF). The depletion of the contaminant began after a lag-time variable between 0 and 23 days. All microcosms quickly reached a pseudo steady-state condition, in terms of biomass concentration (with an average of 9.3 × 10⁶ CFU ml^–1), chloroform depletion rate (5 mol l^–1 d^–1) and butane utilization rate (730 mol l^–1 d^–1). After about 100 days of CF depletion, a sudden 5- to 7-fold increase of the chloroform rate was observed in two microcosms, where the highest amount of contaminant had been depleted. In one of these high-performing microcosms, an experiment of chloroform depletion in the absence of butane resulted in the depletion of a surprisingly high amount of contaminant (765 mol_CF kg_{dry soil}^–1 in 2 months) and in a marked selection of a single bacterial strain. Bioaugmentation assays conducted with the biomass selected in this microcosm and with a pure culture of the selected strain immediately resulted in very high chloroform depletion rates. Preliminary results of a study conducted with resting cells of the selected strain indicated that it can degrade chloroform concentrations up to 119 M (14.2 mg l^–1) without any sign of substrate toxicity, and that it is able to transform vinyl chloride and 1,1,2-trichloroethane.     

4-Alkyliden-beta-lactams conjugated to polyphenols: synthesis and inhibitory activity

A series of compounds combining the beta-lactam and polyphenol scaffold have been prepared and evaluated for inhibition of human leukocyte elastase and matrix metallo-proteases MMP-2 and MMP-9. The design of these compounds has been based on the 'overlapping-type' strategy where two pharmacophores are linked in a single molecule. The most powerful compound against elastase was an N-galloyl-4-alkyliden beta-lactam, [3-[1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-4-oxo-1-(3,4,5-tris-benzyloxy-benzoyl)-azetidin-2-ylidene]-acetic acid ethylester, with an IC50 of 0.5 microM; while the most powerful against MMP-2 was a 4-alkyliden beta-lactam arylated on the C-3 hydroxy side chain (3,5-bis-benzyloxy-4-hydroxy-benzoic acid 1-(2-benzyloxycarbonylmethylene-4-oxo-azetidin-3-yl)-ethyl ester) with an IC50 of 4 microM. Of the total 35 compounds tested, high levels of inhibition of elastase and of MMPs were separately exerted by distinct molecules.     

Highlights from the 5th Annual Meeting of the Italian Society of Virology

The 5th National Congress of the Italian Society of Virology (SIV) was attended by junior- and senior-level virologists to promote interactions and scientific collaborations among the different areas of Virology and allied sciences. The invited and selected lecturers covered the following topics: General Virology and Viral Genetics; Virus-host Interaction and Pathogenesis; Viral Oncogenesis; Viral Immunology and Vaccines; Anti-viral Therapy; Innovative Diagnostics; Viral Biotechnologies and Cell and Gene Therapy. As in the previous editions (Salata and Palù, 2004; Salata et al., 2005), a specific topic was thoroughly covered in a roundtable. This year the elected subject was "HIV: determinants of pathogenicity and clinical implications." The final program and the abstract book can be found at the web site http://www.siv-virologia.it. This report summarizes the lessons learned from the plenary lectures and the selected oral presentations of the 2005 meeting.