Biomonitoring of environmental tobacco smoke (ETS)-related exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

The exposure of non-smokers to the tobacco-specific N-nitrosamine 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a rodent lung carcinogen, was determined in the air of various indoor environments as well as by biomonitoring of non-smokers exposed to environmental tobacco smoke (ETS) under real-life conditions using the urinary NNK metabolites 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and [4-(N-methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc). NNK was not detectable (<0.5 ng m^-3) in 11 rooms in which smoking did not occur. The mean NNK concentration in 19 rooms in which smoking took place was 17.5 (2.4-50.0) ng m^-3. The NNK levels significantly correlated with the nicotine levels (r=0.856; p< 0.0001). Of the 29 non-smokers investigated, 12 exhibited no detectable NNAL and NNAL-Gluc excretion (<3 pmol day) in their urine. The mean urinary excretion of NNAL and NNAL-Gluc of the 17 remaining non-smokers was 20.3 (<3-63.2) and 22.9 (<3-90.0) pmol day^-1, respectively. Total NNAL excretion (NNAL+NNAL-Gluc) in all non-smokers investigated significantly correlated with the amount of nicotine on personal samplers worn during the week prior to urine collection (r=0.88; <0.0001) and with the urinary cotinine levels (r=0.40; p=0.038). No correlation was found between NNAL excretion and the reported extent of ETS exposure. Average total NNAL excretion in the non-smokers with detectable NNAL levels was 74 times less than in 20 smokers who were also investigated. The cotinine/total NNAL ratios in urine of smokers (9900) and non-smokers (9300) were similar. This appears to be at variance with the ratios of the corresponding precursors (nicotine/NNK) in mainstream smoke (16400) and ETS (1000). Possible reasons for this discrepancy are discussed. The possible role of NNK as a lung carcinogen in non-smokers is unclear, especially since NNK exposure in non-smokers is several orders of magnitude lower than the ordinary exposure to exogenous and endogenous N-nitrosamines and the role of NNK as a human lung carcinogen is not fully understood.     

Kidney Stones in Primary Hyperoxaluria: New Lessons Learnt

To investigate potential differences in stone composition with regard to the type of Primary Hyperoxaluria (PH), and in relation to the patient’s medical therapy (treatment naïve patients versus those on preventive medication) we examined twelve kidney stones from ten PH I and six stones from four PH III patients. Unfortunately, no PH II stones were available for analysis. The study on this set of stones indicates a more diverse composition of PH stones than previously reported and a potential dynamic response of morphology and composition of calculi to treatment with crystallization inhibitors (citrate, magnesium) in PH I. Stones formed by PH I patients under treatment are more compact and consist predominantly of calcium-oxalate monohydrate (COM, whewellite), while calcium-oxalate dihydrate (COD, weddellite) is only rarely present. In contrast, the single stone available from a treatment naïve PH I patient as well as stones from PH III patients prior to and under treatment with alkali citrate contained a wide size range of aggregated COD crystals. No significant effects of the treatment were noted in PH III stones. In disagreement with findings from previous studies, stones from patients with primary hyperoxaluria did not exclusively consist of COM. Progressive replacement of COD by small COM crystals could be caused by prolonged stone growth and residence times in the urinary tract, eventually resulting in complete replacement of calcium-oxalate dihydrate by the monohydrate form. The noted difference to the naïve PH I stone may reflect a reduced growth rate in response to treatment. This pilot study highlights the importance of detailed stone diagnostics and could be of therapeutic relevance in calcium-oxalates urolithiasis, provided that the effects of treatment can be reproduced in subsequent larger studies.     

An automated immunoassay for early specificity profiling of antibodies

Antibody-based therapeutics are of great value for the treatment of human diseases. In addition to functional activity, affinity or physico-chemical properties, antibody specificity is considered to be one of the most crucial attributes for safety and efficacy. Consequently, appropriate studies are required before entering clinical trials.

High content protein arrays are widely applied to assess antibody specificity, but this commercial solution can only be applied to final therapeutic antibody candidates because such arrays are expensive and their throughput is limited. A flexible, high-throughput and economical assay that allows specificity testing of IgG or Fab molecules during early discovery is described here. The 384-well microtiter plate assay contains a comprehensive panel of 32 test proteins and uses electrochemiluminescence as readout.

The Protein Panel Profiling (3P) was used to analyze marketed therapeutic antibodies that all showed highly specific binding profiles. Subsequently, 3P was applied to antibody candidates from early discovery and the results compared well with those obtained with a commercially available high content protein chip. Our results suggest that 3P can be applied as an additional filter for lead selection, allowing the identification of favorable antibody candidates in early discovery and thereby increasing the speed and possibility of success in drug development.     

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory,GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.     

The Essential Function of Genes for a Hydratase and an Aldehyde Dehydrogenase for Growth of Pseudomonas sp. Strain Chol1 with the Steroid Compound Cholate Indicates an Aldolytic Reaction Step for Deacetylation of the Side Chain

In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C₅ acyl side chain of the C₂₄ steroid compound cholate involves the C₂₂ intermediate 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C₂₄ steroid (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD⁺ as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage.     

Subviral Dense Bodies of Human Cytomegalovirus Stimulate Maturation and Activation of Monocyte-Derived Immature Dendritic Cells

Dendritic cells play a central role in the immune control of human cytomegalovirus (HCMV) infection. This work aimed at investigating the impact of noninfectious, subviral dense bodies of HCMV on the maturation and activation of dendritic cells (DC). Treatment of immature DC with dense bodies led to the maturation of these cells and significantly increased their capacity for cytokine release and antigen presentation. Dense body-activated DC may thereby contribute to the development of antiviral immunity.