Biomonitoring of workers cleaning up ammunition waste sites

2,4,6-Trinitrotoluene (TNT) is an important occupational and environmental pollutant. In TNT-exposed humans, notable toxic manifestations have included aplastic anaemia, toxic hepatitis, cataracts, hepatomegaly and liver cancer. Therefore, it is important to develop protection measures and to monitor workers involved in the clean-up of ammunition sites. Haemoglobin (Hb) adducts of TNT, 4-amino-2,6-dinitrotoluene (4ADNT) and 2-amino-4,6-dinitrotoluene (2ADNT), and the urine metabolites of TNT, 4ADNT and 2ADNT were found in 22–50% of the exposed workers, but not in the control group. The exposed workers were wearing protective equipment. The levels of erythrocytes, haemoglobin, creatinine, serum glutamic pyruvic transaminase and lymphocyte levels were significantly lower in the exposed workers than in the non-exposed workers. The levels of blood urea and reticulocytes were significantly higher in the exposed workers than in the non-exposed workers. Headache (26%), mucous membrane irritation (16%), sick leave (18%), lassitude (8%), anxiety (6%), shortness of breath (3%), nausea (5%) and allergic reactions (8%) were reported by the exposed workers. In a further analysis the U-4ADNT levels and the Hb-adduct levels were compared to the blood parameter and the health effects. The blood parameters were not significantly different between the U-4ADNT positive and U-4ADNT-negative group. Headache, mucous membrane irritation, sick leave, lassitude, anxiety, shortness of breath and allergic reactions were statistically not different between the two groups. Also in the workers with Hb-4ADNT adducts no significant negative changes were seen in regards to the changes of the blood parameters or the health effects. According to the results of the present study, it appears that the blood parameter changes and the health effects are more influenced by other factors than by the internal exposure to TNT.     

IBC’s 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics International Conferences and the 2012 Annual Meeting of The Antibody Society

The 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences, and the 2012 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, were held December 3–6, 2012 in San Diego, CA. The meeting drew over 800 participants who attended sessions on a wide variety of topics relevant to antibody research and development. As a prelude to the main events, a pre-conference workshop held on December 2, 2012 focused on intellectual property issues that impact antibody engineering. The Antibody Engineering Conference was composed of six sessions held December 3–5, 2012: (1) From Receptor Biology to Therapy; (2) Antibodies in a Complex Environment; (3) Antibody Targeted CNS Therapy: Beyond the Blood Brain Barrier; (4) Deep Sequencing in B Cell Biology and Antibody Libraries; (5) Systems Medicine in the Development of Antibody Therapies/Systematic Validation of Novel Antibody Targets; and (6) Antibody Activity and Animal Models. The Antibody Therapeutics conference comprised four sessions held December 4–5, 2012: (1) Clinical and Preclinical Updates of Antibody-Drug Conjugates; (2) Multifunctional Antibodies and Antibody Combinations: Clinical Focus; (3) Development Status of Immunomodulatory Therapeutic Antibodies; and (4) Modulating the Half-Life of Antibody Therapeutics. The Antibody Society’s special session on applications for recording and sharing data based on GIATE was held on December 5, 2012, and the conferences concluded with two combined sessions on December 5–6, 2012: (1) Development Status of Early Stage Therapeutic Antibodies; and (2) Immunomodulatory Antibodies for Cancer Therapy.     

A new semi-nested PCR protocol to amplify large 18S rRNA gene fragments for PCR-DGGE analysis of soil fungal communities

The analysis of soil fungal communities by molecular fingerprinting and subsequent identification of the underlying populations require the amplification of a phylogenetically informative gene fragment. In this study we tested the reliability and suitability of the previously published fungal primer combination (NS1/FR1-GC) that amplifies almost the entire 18S rRNA gene for the DGGE analysis of fungal communities in soil samples from 36 sites. This direct PCR system failed to amplify the fragment of interest from the total DNA extracted from most of the soils tested. Thus, we developed a new semi-nested PCR system based on the initial amplification of over 1,700 bp of the 18S rRNA gene with a new primer combination, followed by a subsequent amplification with NS1/FR1-GC. By means of the PCR approach developed in this study distinct 18S rRNA gene amplicons could be reproducibly generated for all soil samples. Amplification tests with 101 soil fungal isolates showed that with the new semi-nested system 18S rRNA gene fragments could be obtained from more fungi than with the direct approach. The subsequent DGGE separation of community amplicons resulted in a high resolution and revealed reproducible complex soil fungal communities specific for each site, despite a minor variability between replicates of the same sample. The semi-nested PCR system developed in this study, coupled with DGGE fingerprinting, offers a robust, reliable and sensitive tool for the analysis of soil fungal community structure.     

Generation of Amyloid-β Is Reduced by the Interaction of Calreticulin with Amyloid Precursor Protein,Presenilin and Nicastrin

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer''s disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca²⁺- and N-glycan-independent interaction is mediated by amino acids 330–344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β₄₂ levels in culture supernatants, while transfection with the P-domain increases amyloid-β₄₀ levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330–344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β₄₀ and amyloid-β₄₂ levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer''s disease.     

One step cloning of defined DNA fragments from large genomic clones

Recently, the nucleotide sequences of entire genomes became available. This information combined with older sequencing data discloses the exact chromosomal location of millions of nucleotide markers stored in the databases at NCBI, EMBO or DDBJ. Despite having resolved the intron/exon structures of all described genes within these genomes with a stroke of a pen, the sequencing data opens up other interesting possibilities. For example, the genomic mapping of the end sequences of the human, murine and rat BAC libraries generated at The Institute for Genomic Research (TIGR), reveals now the entire encompassed sequence of the inserts for more than a million of these clones. Since these clones are individually stored, they are now an invaluable source for experiments which depend on genomic DNA. Isolation of smaller fragments from such clones with standard methods is a time consuming process. We describe here a reliable one-step cloning technique to obtain a DNA fragment with a defined size and sequence from larger genomic clones in less than 48 hours using a standard vector with a multiple cloning site, and common restriction enzymes and equipment. The only prerequisites are the sequences of ends of the insert and of the underlying genome.     

Efficacy and Safety of Pafuramidine versus Pentamidine Maleate for Treatment of First Stage Sleeping Sickness in a Randomized,Comparator-Controlled,International Phase 3 Clinical Trial

Background

Sleeping sickness (human African trypanosomiasis [HAT]) is a neglected tropical disease with limited treatment options that currently require parenteral administration. In previous studies, orally administered pafuramidine was well tolerated in healthy patients (for up to 21 days) and stage 1 HAT patients (for up to 10 days), and demonstrated efficacy comparable to pentamidine.

Methods

This was a Phase 3, multi-center, randomized, open-label, parallel-group, active control study where 273 male and female patients with first stage Trypanosoma brucei gambiense HAT were treated at six sites: one trypanosomiasis reference center in Angola, one hospital in South Sudan, and four hospitals in the Democratic Republic of the Congo between August 2005 and September 2009 to support the registration of pafuramidine for treatment of first stage HAT in collaboration with the United States Food and Drug Administration. Patients were treated with either 100 mg of pafuramidine orally twice a day for 10 days or 4 mg/kg pentamidine intramuscularly once daily for 7 days to assess the efficacy and safety of pafuramidine versus pentamidine. Pregnant and lactating women as well as adolescents were included.The primary efficacy endpoint was the combined rate of clinical and parasitological cure at 12 months. The primary safety outcome was the frequency and severity of adverse events. The study was registered on the International Clinical Trials Registry Platform at www.clinicaltrials.gov with the number ISRCTN85534673.

Findings/Conclusions

The overall cure rate at 12 months was 89% in the pafuramidine group and 95% in the pentamidine group; pafuramidine was non-inferior to pentamidine as the upper bound of the 95% confidence interval did not exceed 15%. The safety profile of pafuramidine was superior to pentamidine; however, 3 patients in the pafuramidine group had glomerulonephritis or nephropathy approximately 8 weeks post-treatment. Two of these events were judged as possibly related to pafuramidine. Despite good tolerability observed in preceding studies, the development program for pafuramidine was discontinued due to delayed post-treatment toxicity.     

A molecular biological protocol to distinguish potentially human pathogenic Stenotrophomonas maltophilia from plant-associated Stenotrophomonas rhizophila

In recent years, the importance of the Gram-negative bacterium Stenotrophomonas as an opportunistic pathogen as well as in biotechnology has increased. The aim of the present study was to develop new methods for distinguishing between strains closely related to the potentially human pathogenic Stenotrophomonas maltophilia and those closely related to the plant-associated Stenotrophomonas rhizophila. To accomplish this, 58 strains were characterized by 16S rDNA sequencing and amplified ribosomal DNA restriction analysis (ARDRA), and the occurrence of specific functional genes. Based on 16S rDNA sequences, an ARDRA protocol was developed which allowed differentiation between strains of the S. maltophilia and the S. rhizophila group. As it was known that only salt-treated cells of S. rhizophila were able to synthesize the compatible solute glucosylglycerol (GG), the ggpS gene responsible for GG synthesis was used for differentiation between both species and it was confirmed that it only occurred in S. rhizophila strains. As a further genetic marker the smeD gene, which is part of the genes coding for the multidrug efflux pump SmeDEF from S. maltophilia, was used. Based on the results we propose a combination of fingerprinting techniques using the 16S rDNA and the functional genes ggpS and smeD to distinguish both Stenotrophomonas species.     

Oxidative stress and lipid mediators induced in alveolar macrophages by ultrafine particles

In ambient aerosols, ultrafine particles (UFP) and their agglomerates are considered to be major factors contributing to adverse health effects. Reactivity of agglomerated UFP of elemental carbon (EC), Printex 90, Printex G, and diesel exhaust particles (DEP) was evaluated by the capacity of particles to oxidize methionine in a cell-free in vitro system for determination of their innate oxidative potential and by alveolar macrophages (AMs) to determine production of arachidonic acid (AA), including formation of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), reactive oxygen species (ROS), and oxidative stress marker 8-isoprostane. EC exhibiting high oxidative potential induced generation of AA, PGE2, LTB4, and 8-isoprostane in canine and human AMs. Printex 90, Printex G, and DEP, showing low oxidative capacity, still induced formation of AA and PGE2, but not that of LTB4 or 8-isoprostane. Aging of EC lowered oxidative potential while still inducing production of AA and PGE2 but not that of LTB4 and 8-isoprostane. Cellular ROS production was stimulated by all particles independent of oxidative potential. Particle-induced formation of AA metabolites and ROS was dependent on mitogen-activated protein kinase kinase 1 activation of cytosolic phospholipase A2 (cPLA2) as shown by inhibitor studies. In conclusion, cPLA2, PGE2, and ROS formation was activated by all particle types, whereas LTB4 production and 8-isoprostane were strongly dependent on particles' oxidative potential. Physical and chemical parameters of particle surface correlated with oxidative potential and stimulation of AM PGE2 and 8-isoprostane production.     

Multiscale modeling of the cardiovascular system: application to the study of pulmonary and coronary perfusions in the univentricular circulation

The objective of this study is to compare the coronary and pulmonary blood flow dynamics resulting from two configurations of systemic-to-pulmonary artery shunts currently utilized during the Norwood procedure: the central (CS) and modified Blalock Taussig (MBTS) shunts. A lumped parameter model of the neonatal cardiovascular circulation and detailed 3-D models of the shunt based on the finite volume method were constructed. Shunt sizes of 3, 3.5 and 4 mm were considered. A multiscale approach was adopted to prescribe appropriate and realistic boundary conditions for the 3-D models of the Norwood circulation. Results showed that the average shunt flow rate is higher for the CS option than for the MBTS and that pulmonary flow increases with shunt size for both options. Cardiac output is higher for the CS option for all shunt sizes. Flow distribution between the left and the right pulmonary arteries is not completely balanced, although for the CS option the discrepancy is low (50-51% of the pulmonary flow to the right lung) while for the MBTS it is more pronounced with larger shunt sizes (51-54% to the left lung). The CS option favors perfusion to the right lung while the MBTS favors the left. In the CS option, a smaller percentage of aortic flow is distributed to the coronary circulation, while that percentage rises for the MBTS. These findings may have important implications for coronary blood flow and ventricular function.