Child with serogroup W135 primary meningococcal septic arthritis.

Over the last decade, there has been a concerning increase in the number of invasive meningococcal serotype W infections in Europe. Although sepsis and meningitis are the most feared complications, focal complications of systemic disease such as pneumonia, pericarditis and arthritis can also occur. We present a rare case of isolated meningococcal W135 arthritis of the hip without invasive meningococcal disease in a 6-year-old patient.

[Partial lipodystrophy: a spot diagnosis]. / Partiële lipodystrofie: een diagnose à vue.

BACKGROUND: Partial lipodystrophy is a rare acquired disorder characterised by gradual loss of subcutaneous adipose tissue in the upper half of the body. CASE DESCRIPTION: We saw a 9-year-old girl who had been referred on account of recurrent urinary tract infections. On physical examination, she was noticed to be very thin in the face. Her upper extremities were also skinny. Strikingly, the lower half of her body was normally proportioned, which immediately suggested a diagnosis of partial lipodystrophy. Additional examinations showed a low level of complement factor C3 and the presence of C3 nephritic factor. CONCLUSION: Partial lipodystrophy is rare but it is important to include it in the differential diagnosis of unwanted disproportional subcutaneous fat loss because of the somatic and psychological consequences.

Microdosing of a Carbon-14 Labeled Protein in Healthy Volunteers Accurately Predicts Its Pharmacokinetics at Therapeutic Dosages.

Preclinical development of new biological entities (NBEs), such as human protein therapeutics, requires considerable expenditure of time and costs. Poor prediction of pharmacokinetics in humans further reduces net efficiency. In this study, we show for the first time that pharmacokinetic data of NBEs in humans can be successfully obtained early in the drug development process by the use of microdosing in a small group of healthy subjects combined with ultrasensitive accelerator mass spectrometry (AMS). After only minimal preclinical testing, we performed a first-in-human phase 0/phase 1 trial with a human recombinant therapeutic protein (RESCuing Alkaline Phosphatase, human recombinant placental alkaline phosphatase [hRESCAP]) to assess its safety and kinetics. Pharmacokinetic analysis showed dose linearity from microdose (53 µg) [(14) C]-hRESCAP to therapeutic doses (up to 5.3 mg) of the protein in healthy volunteers. This study demonstrates the value of a microdosing approach in a very small cohort for accelerating the clinical development of NBEs.

Activity-Based Protein Profiling Reveals Broad Reactivity of the Nerve Agent Sarin.

Elucidation of noncholinesterase protein targets of organophosphates, and nerve agents in particular, may reveal additional mechanisms for their high toxicity as well as clues for novel therapeutic approaches toward intoxications with these agents. Within this framework, we here describe the synthesis of the activity-based probe 3, which contains a phosphonofluoridate moiety, a P-Me moiety, and a biotinylated O-alkyl group, and its use in activity-based protein profiling with two relevant biological samples, that is, rhesus monkey liver and cultured human A549 lung cells. In this way, we have unearthed eight serine hydrolases (fatty acid synthase, acylpeptide hydrolase, dipeptidyl peptidase 9, prolyl oligopeptidase, carboxylesterase, long-chain acyl coenzyme A thioesterase, PAF acetylhydrolase 1b, and esterase D/S-formyl glutathione hydrolase) as targets that are modified by the nerve agent sarin. It is also shown that the newly developed probe 3 might find its way into the development of alternative, less laborious purification protocols for human butyrylcholinesterase, a potent bioscavenger currently under clinical investigation as a prophylactic/therapeutic for nerve agent intoxications.

Protein adduct formation by glucuronide metabolites of permethrin.

Biomonitoring of exposure to the insecticide permethrin is usually performed by analysis of its urinary metabolites 3-phenoxybenzoic acid (3-PBA) or cis/ trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Cl 2 CA). We are engaged in the development of a methodology to assess the cumulative internal dose of exposure to permethrin, which is based on the assumption that (reactive) glucuronide conjugates of the major permethrin metabolites 3-PBA and Cl 2 CA will form persistent (weeks to months) adducts to proteins, in analogy with the glucuronide conjugates of structurally related drugs. The 3-PBA and Cl 2 CA beta-glucuronide metabolites of permethrin have been successfully chemically and enzymatically synthesized. Their identities have been assessed by means of (1)H NMR spectroscopy and liquid chromatography-tandem mass spectrometry. The reactivity of these metabolites with various amino acids, peptides, and albumin in human plasma has been studied. Several distinct adducts could be identified by liquid chromatography-tandem mass spectrometry. After pronase digestion of albumin isolated from exposed human plasma, various lysine derivatives resulted with favorable mass spectrometric and chromatographic properties. Covalent binding was quantified by using [(14)C]-3-PBA glucuronide; >1.5% of total radioactivity was bound to proteins. It is envisaged that the obtained results can form a firm basis for the development of a protein adduct-based methodology for biomonitoring exposure to permethrin. In view of the widespread use of permethrin, the toxicological relevance of protein binding by its metabolites will be addressed in more detail in future work.

Retrospective detection of sulfur mustard exposure by mass spectrometric analysis of adducts to albumin and hemoglobin: an in vivo study.

The persistence in rats of sulfur mustard adducts to albumin and hemoglobin was studied in vivo after exposure (intravenously; 0.3 mg/kg; approximately 0.1 LD(50)) of rats to sulfur mustard. The albumin adduct (S-HETE)Cys-Pro-Tyr was detectable up to 7 days after the exposure, while the adduct to the N-terminal valine in hemoglobin was still detected after 28 days. The decrease in adduct levels corresponded well with the half-life time of albumin in rats and with the lifetime of the rat erythrocyte. Remarkably, the N-terminal valine adduct to hemoglobin increased during the first three days, which implies that there is still free sulfur mustard present during that time. In contrast, the corresponding albumin adduct levels did not increase during this time period. The free sulfur mustard might have accumulated in the erythrocyte cell membrane.

Verification of exposure to cholinesterase inhibitors: generic detection of OPCW Schedule 1 nerve agent adducts to human butyrylcholinesterase.

Phosphylated butyrylcholinesterase is one of the most important biomarkers to verify an exposure to nerve agents, and it can be analyzed with liquid chromatography-tandem mass spectrometry (LC-MS-MS) by detection of a phosphylated nonapeptide that results after digestion of butyrylcholinesterase (BuChE) with pepsin. For a sensitive analysis (low degree of BuChE inhibition), the identity of the cholinesterase inhibitor has to be known in order to use the LC-MS-MS instrument in the most sensitive selected reaction monitoring mode. In practice, the identity of the cholinesterase inhibitor will not be known beforehand, and the number of possible organophosphates is greater than 1000. However, the number of possible molecular masses of organophosphates is approximately 170. A method for which only 34 transitions in the multiple reaction monitoring mode have to be acquired in order to screen for an exposure to all Organization for the Prohibition of Chemical Weapons Schedule 1 nerve agents was developed.

Verification of exposure to organophosphates: Generic mass spectrometric method for detection of human butyrylcholinesterase adducts.

We present a generic mass spectrometric method to verify exposure to organophosphates, based on the chemical conversion of the phosphylated peptides obtained after pepsin digestion of human butyrylcholinesterase (HuBuChE) to a common precursor peptide. After exposure of plasma to various organophosphates (nerve agents, pesticides), HuBuChE was isolated from plasma by procainamide affinity-based solid-phase extraction. Upon subsequent pepsin digestion, the respective phosphylated nonapeptides could be identified in the digests. After treatment of the pepsin digests with Ba(OH)2 in the presence of a nucleophilic tag (a thiol or amine), the phosphylated nonapeptides were transformed into a common tagged nonapeptide that could be analyzed sensitively by means of LC tandem MS. So far, best results were obtained with 2-(3-aminopropylamino)ethanol as nucleophilic tag. By applying the presented method, HuBuChE inhibition can now be monitored accurately by mass spectrometry, without advance knowledge of the structure of the inhibitor.

Retrospective detection of exposure to sulfur mustard: improvements on an assay for liquid chromatography-tandem mass spectrometry analysis of albumin-sulfur mustard adducts.

We here report on the further development of the method comprising the pronase digestion of albumin alkylated by sulfur mustard and the subsequent mass spectrometric analysis of an adducted tripeptide. This includes significant improvements in both the albumin isolation procedure and the automation of the microliquid chromatography-electrospray-tandem mass spectrometric analysis. We also report on the results of a small reference range study, in which we have established that there are no detectable interferences in sera from unexposed individuals.

Procedure for monitoring exposure to sulfur mustard based on modified edman degradation of globin.

A procedure for the modified Edman degradation of globin for determination of sulfur mustard adducts to the N-terminal valine residue in human hemoglobin has been developed for use under field laboratory conditions. The minimum detectable exposure level of human blood (in vitro) to sulfur mustard using this procedure is 100 nM. The interindividual and intraindividual variabilities of the procedure were acceptable (standard deviation < 10% and < 20%, respectively). The procedure could be properly set up and carried out in another laboratory within one working day, demonstrating its robustness.