Quantification of lipopolysaccharides in outer membrane vesicle vaccines against meningococcal disease. High-performance liquid chromatographic determination of the constituent 3-hydroxy-lauric acid.

A high-performance liquid chromatographic (HPLC) assay for quantification of lipopolysaccharides (LPSs, endotoxins) in outer membrane vesicle vaccines against meningococcal disease has been developed. The LPS constituent, 3-hydroxy-lauric acid, served as marker substance for the quantification. LPS from the vaccine was precipitated by ethanol and the fatty acid constituents, including 3-hydroxy-lauric acid, were released by acidic hydrolysis, collected and purified by solid phase extraction on C18 disc-cartridges and converted into phenacyl esters for UV detection at 240 nm. Quantification of the derivatized 3-hydroxy-lauric acid was achieved by HPLC using a Brownlee RP-18 reversed phase column with acetonitrile/water (68:32, v/v) as mobile phase. The method was found to be linear over the range 3-49 microg LPS/ml with a sensitivity of 1.6 (microg/ml)(-1). The repeatability (within-day precision) of the method at three levels (3-49 microg LPS/ml) was 6-14% relative standard deviation and the intermediate (between-day) precision was 7% relative standard deviation (at level 15 microg LPS/ml). The method has been successfully used in the quality control of a meningococcal B outer membrane vesicle vaccine, containing 4-8% LPS relative to protein (w/w), in our laboratory for three years.     

integRATE: a desirability-based data integration framework for the prioritization of candidate genes across heterogeneous omics and its application to preterm birth

Background

The integration of high-quality, genome-wide analyses offers a robust approach to elucidating genetic factors involved in complex human diseases. Even though several methods exist to integrate heterogeneous omics data, most biologists still manually select candidate genes by examining the intersection of lists of candidates stemming from analyses of different types of omics data that have been generated by imposing hard (strict) thresholds on quantitative variables, such as P-values and fold changes, increasing the chance of missing potentially important candidates.

Methods

To better facilitate the unbiased integration of heterogeneous omics data collected from diverse platforms and samples, we propose a desirability function framework for identifying candidate genes with strong evidence across data types as targets for follow-up functional analysis. Our approach is targeted towards disease systems with sparse, heterogeneous omics data, so we tested it on one such pathology: spontaneous preterm birth (sPTB).

Results

We developed the software integRATE, which uses desirability functions to rank genes both within and across studies, identifying well-supported candidate genes according to the cumulative weight of biological evidence rather than based on imposition of hard thresholds of key variables. Integrating 10 sPTB omics studies identified both genes in pathways previously suspected to be involved in sPTB as well as novel genes never before linked to this syndrome. integRATE is available as an R package on GitHub (https://github.com/haleyeidem/integRATE).

Conclusions

Desirability-based data integration is a solution most applicable in biological research areas where omics data is especially heterogeneous and sparse, allowing for the prioritization of candidate genes that can be used to inform more targeted downstream functional analyses.

     

Ischemic Heart Disease in Workers at Mayak PA: Latency of Incidence Risk after Radiation Exposure

We present an updated analysis of incidence and mortality from atherosclerotic induced ischemic heart diseases in the cohort of workers at the Mayak Production Association (PA). This cohort constitutes one of the most important sources for the assessment of radiation risk. It is exceptional because it comprises information on several other risk factors. While most of the workers have been exposed to external gamma radiation, a large proportion has additionally been exposed to internal radiation from inhaled plutonium. Compared to a previous study by Azizova et al. 2012, the updated dosimetry system MWDS-2008 has been applied and methods of analysis have been revised. We extend the analysis of the significant incidence risk and observe that main detrimental effects of external radiation exposure occur after more than about 30 years. For mortality, significant risk was found in males with an excess relative risk per dose of 0.09 (95% CI: 0.02; 0.16) while risk was insignificant for females. With respect to internal radiation exposure no association to risk could be established.     

Cerebrovascular Diseases in Workers at Mayak PA: The Difference in Radiation Risk between Incidence and Mortality

A detailed analysis of cerebrovascular diseases (CeVD) for the cohort of workers at Mayak Production Association (PA) is presented. This cohort is especially suitable for the analysis of radiation induced circulatory diseases, due to the detailed medical surveillance and information on several risk factors. The risk after external, typically protracted, gamma exposure is analysed, accounting for potential additional internal alpha exposure. Three different endpoints have been investigated: incidence and mortality from all cerebrovascular diseases and incidence of stroke. Particular emphasis was given to the form of the dose-response relationship and the time dependence of the radiation induced risk. Young attained age was observed to be an important, aggravating modifier of radiation risk for incidence of CeVD and stroke. For incidence of CeVD, our analysis supports a dose response sub-linear for low doses. Finally, the excess relative risk per dose was confirmed to be significantly higher for incidence of CeVD compared to CeVD mortality and incidence of stroke. Arguments are presented for this difference to be based on a true biological effect.     

Lung Cancer Mortality (1950-1999) among Eldorado Uranium Workers: A Comparison of Models of Carcinogenesis and Empirical Excess Risk Models

Lung cancer mortality after exposure to radon decay products (RDP) among 16,236 male Eldorado uranium workers was analyzed. Male workers from the Beaverlodge and Port Radium uranium mines and the Port Hope radium and uranium refinery and processing facility who were first employed between 1932 and 1980 were followed up from 1950 to 1999. A total of 618 lung cancer deaths were observed. The analysis compared the results of the biologically-based two-stage clonal expansion (TSCE) model to the empirical excess risk model. The spontaneous clonal expansion rate of pre-malignant cells was reduced at older ages under the assumptions of the TSCE model. Exposure to RDP was associated with increase in the clonal expansion rate during exposure but not afterwards. The increase was stronger for lower exposure rates. A radiation-induced bystander effect could be a possible explanation for such an exposure response. Results on excess risks were compared to a linear dose-response parametric excess risk model with attained age, time since exposure and dose rate as effect modifiers. In all models the excess relative risk decreased with increasing attained age, increasing time since exposure and increasing exposure rate. Large model uncertainties were found in particular for small exposure rates.     

'Troy-bodies': antibodies as vector proteins for T cell epitopes

A major objective in vaccine development is the design of reagents that give a strong, specific T cell response. Targeting of antigens to antigen presenting cells (APC) results in enhanced antigen presentation and T cell activation. In this paper, we describe a novel targeting reagent denoted 'Troy-bodies', namely recombinant antibodies with APC-specificity and with T cell epitopes integrated in their C regions. We have made such antibodies with V regions specific for either IgD or MHC class II, and five different T cell epitopes have been tested. All epitopes could be introduced into loops of C domains without disrupting immunoglobulin (Ig) folding. Four have been tested in T cell activation studies, and all could be released and presented by APC. Furthermore, whether IgD- or MHC-specific, the molecules tested enhanced T cell stimulation compared to non-specific control antibodies in vitro as well as in vivo. Using this technology, specific reagents can be designed that target selected antigenic peptides to an APC of choice. Troy-bodies may therefore be useful for manipulation of immune responses, and in particular for vaccination purposes.