Error analysis in newborn screening: can quotients support the absolute values?

Newborn screening is performed using modern tandem mass spectrometry, which can simultaneously detect a variety of analytes, including several amino acids and fatty acids. Tandem mass spectrometry measures the diagnostic parameters as absolute concentrations and produces fragments which are used as markers of specific substances. Several prominent quotients can also be derived, which are quotients of two absolute measured concentrations. In this study, we determined the precision of both the absolute concentrations and the derived quotients. First, the measurement error of the absolute concentrations and the measurement error of the ratios were practically determined. Then, the Gaussian theory of error calculation was used. Finally, these errors were compared with one another. The practical analytical accuracies of the quotients were significantly higher (e.g., coefficient of variation (CV) = 5.1% for the phenylalanine to tyrosine (Phe/Tyr) quotient and CV = 5.6% for the Fisher quotient) than the accuracies of the absolute measured concentrations (mean CVs = 12%). According to our results, the ratios are analytically correct and, from an analytical point of view, can support the absolute values in finding the correct diagnosis.

FR-900098, an antimalarial development candidate that inhibits the non-mevalonate isoprenoid biosynthesis pathway, shows no evidence of acute toxicity and genotoxicity.

FR-900098 is an inhibitor of 1-deoxy-d-xylulose-5-phosphate (DXP) reductoisomerase, the second enzyme in the non-mevalonate isoprenoid biosynthesis pathway. In previous studies, FR-900098 was shown to possess potent antimalarial activity in vitro and in a murine malaria model. In order to provide a basis for further preclinical and clinical development, we studied the acute toxicity and genotoxicity of FR-900098. We observed no acute toxicity in rats, i.e. there were no clinical signs of toxicity and no substance-related deaths after the administration of a single dose of 3000 mg/kg body weight orally or 400 mg/kg body weight intravenously. No mutagenic potential was detected in the Salmonella typhimurium reverse mutation assay (Ames test) or an in vitro mammalian cell gene mutation test using mouse lymphoma L5178Y/TK(+/-) cells (clone 3.7.2C), both with and without metabolic activation. In addition, FR-900098 demonstrated no clastogenic or aneugenic capability or significant adverse effects on blood formation in an in vivo micronucleus test with bone marrow erythrocytes from NMRI mice. We conclude that FR-900098 lacks acute toxicity and genotoxicity, supporting its further development as an antimalarial drug.

Fosmidomycin uptake into Plasmodium and Babesia-infected erythrocytes is facilitated by parasite-induced new permeability pathways.

BACKGROUND: Highly charged compounds typically suffer from low membrane permeability and thus are generally regarded as sub-optimal drug candidates. Nonetheless, the highly charged drug fosmidomycin and its more active methyl-derivative FR900098 have proven parasiticidal activity against erythrocytic stages of the malaria parasite Plasmodium falciparum. Both compounds target the isoprenoid biosynthesis pathway present in bacteria and plastid-bearing organisms, like apicomplexan parasites. Surprisingly, the compounds are inactive against a range of apicomplexans replicating in nucleated cells, including Toxoplasma gondii. METHODOLOGY/PRINCIPAL FINDINGS: Since non-infected erythrocytes are impermeable for FR90098, we hypothesized that these drugs are taken up only by erythrocytes infected with Plasmodium. We provide evidence that radiolabeled FR900098 accumulates in theses cells as a consequence of parasite-induced new properties of the host cell, which coincide with an increased permeability of the erythrocyte membrane. Babesia divergens, a related parasite that also infects human erythrocytes and is also known to induce an increase in membrane permeability, displays a similar susceptibility and uptake behavior with regard to the drug. In contrast, Toxoplasma gondii-infected cells do apparently not take up the compounds, and the drugs are inactive against the liver stages of Plasmodium berghei, a mouse malaria parasite. CONCLUSIONS/SIGNIFICANCE: Our findings provide an explanation for the observed differences in activity of fosmidomycin and FR900098 against different Apicomplexa. These results have important implications for future screens aimed at finding new and safe molecular entities active against P. falciparum and related parasites. Our data provide further evidence that parasite-induced new permeability pathways may be exploited as routes for drug delivery.

NMR studies of DOXP reductoisomerase and its inhibitor complex.

1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase (EC1.1.1.267) catalyses the second step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. The enzyme is used by most bacteria, apicomplexan parasites and the plastids of plants, but not by humans, and therefore represents an attractive target for antibacterial, antiparasitic and herbicidal compounds. Fosmidomycin, an inhibitor of DXR, has been found to be active against bacterial infections and malaria in early clinical studies. Here, we report sample optimisation, partial backbone assignment and secondary-structure prediction of E. coli DXR by heteronuclear NMR analysis for further NMR-aided drug discovery. Perdeuterated (15)N,(13)C-labelled samples were prepared under oxygen exclusion in the presence of Mg(2+), NADPH and the inhibitor FR-900098, a close derivative of fosmidomycin. (1)H and (15)N backbone assignment was achieved for 44 % of the primary structure, and (13)C backbone assignment was achieved for 50 % of the primary structure. Comparison with previously solved crystal structures revealed that the assigned fragments were located mainly in helical regions on the solvent-exposed surface of the enzyme. Torsion angle likelihood obtained from shift and sequence similarity (TALOS) was used for secondary structure prediction, resulting in agreement with eight available crystal structures; deviations could be observed for the catalytic loop region.

Characterization of the DMAE-modified juvenile excretory-secretory protein Juv-p120 of Litomosoides sigmodontis.

Juv-p120 is an excretory-secretory 160 kDa glycoprotein of juvenile female Litomosoides sigmodontis and exhibits features typical for mucins. 50% of its molecular mass is attributed to posttranslational modifications with the unusual substituent dimethylaminoethanol (DMAE). By that Juv-p120 corresponds to the surface proteins of the microfilarial sheath, Shp3 and Shp3a. The secreted protein consists of 697 amino acids, organized in two different domains of repeat elements separated by a stretch of polar residues. The N-terminal domain shows fourteen P/S/T/F-rich repeat elements highly modified with phospho-DMAE substituted O-glycans confering a negative charge to the protein. The C-terminal domain is extremely rich in glutamine (35%) and leucine (25%) in less organized repeats and may play a role in oligomerization of Juv-p120 monomers. A protein family with a similar Q/L-rich region and conserved core promoter region was identified in Brugia malayi by homology screening and in Wuchereria bancrofti and Loa loa by database similarity search. One of the Q/L-rich proteins in each genus has an extended S/T-rich region and due to this feature is supposed to be a putative Juv-p120 ortholog. The corresponding modification of Juv-p120 and the microfilarial sheath surface antigens Shp3/3a explains the appearance of anti-sheath antibodies before the release of microfilariae. The function of Juv-p120 is unknown.

Gammadelta T cell immune manipulation during chronic phase of simian-human immunodeficiency virus infection [corrected] confers immunological benefits.

Vgamma2Vdelta2 T cells, a major human gammadelta T cell subset, recognize the phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) produced by mycobacteria and some opportunistic pathogens, and they contribute to innate/adaptive/homeostatic and anticancer immunity. As initial efforts to explore Vgamma2Vdelta2 T cell-based therapeutics against HIV/AIDS-associated bacterial/protozoal infections and neoplasms, we investigated whether a well-defined HMBPP/IL-2 therapeutic regimen could overcome HIV-mediated immune suppression to massively expand polyfunctional Vgamma2Vdelta2 T cells, and whether such activation/expansion could impact AIDS pathogenesis in simian HIV (SHIV)-infected Chinese rhesus macaques. While HMBPP/IL-2 coadministration during acute or chronic phase of SHIV infection induced massive activation/expansion of Vgamma2Vdelta2 T cells, the consequences of such activation/expansions were different between these two treatment settings. HMBPP/IL-2 cotreatment during acute SHIV infection did not prevent the increases in peak and set-point viral loads or the accelerated disease progression seen with IL-2 treatment alone. In contrast, HMBPP/IL-2 cotreatment during chronic infection did not exacerbate disease, and more importantly it could confer immunological benefits. Surprisingly, although viral antigenic loads were not increased upon HMBPP/IL-2 cotreatment during chronic SHIV infection, HMBPP activation of Vgamma2Vdelta2 T cells boosted HIV Env-specific Ab titers. Such increases in Abs were sustained for >170 days and were immediately preceded by increased production of IFN-gamma, TNF-alpha, IL-4, and IL-10 during peak expansion of Vgamma2Vdelta2 T cells displaying memory phenotypes, as well as the short-term increased effector function of Vgamma2Vdelta2 T cells and CD4(+) and CD8(+) alphabeta T cells producing antimicrobial cytokines. Thus, HMBPP/Vgamma2Vdelta2 T cell-based intervention may potentially be useful for combating neoplasms and HMBPP-producing opportunistic pathogens in chronically HIV-infected individuals.

Antigen-specific Vgamma2Vdelta2 T effector cells confer homeostatic protection against pneumonic plaque lesions.

The possibility that Vgamma2Vdelta2 T effector cells can confer protection against pulmonary infectious diseases has not been tested. We have recently demonstrated that single-dose (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) plus IL-2 treatment can induce prolonged accumulation of Vgamma2Vdelta2 T effector cells in lungs. Here, we show that a delayed HMBPP/IL-2 administration after inhalational Yersinia pestis infection induced marked expansion of Vgamma2Vdelta2 T cells but failed to control extracellular plague bacterial replication/infection. Surprisingly, despite the absence of infection control, expansion of Vgamma2Vdelta2 T cells after HMBPP/IL-2 treatment led to the attenuation of inhalation plague lesions in lungs. Consistently, HMBPP-activated Vgamma2Vdelta2 T cells accumulated and localized in pulmonary interstitials surrounding small blood vessels and airway mucosa in the lung tissues with no or mild plague lesions. These infiltrating Vgamma2Vdelta2 T cells produced FGF-7, a homeostatic mediator against tissue damages. In contrast, control macaques treated with glucose plus IL-2 or glucose alone exhibited severe hemorrhages and necrosis in most lung lobes, with no or very few Vgamma2Vdelta2 T cells detectable in lung tissues. The findings are consist with the paradigm that circulating Vgamma2Vdelta2 T cells can traffic to lungs for homeostatic protection against tissue damages in infection.

Structure of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, the terminal enzyme of the non-mevalonate pathway.

Molecular evolution has evolved two metabolic routes for isoprenoid biosynthesis: the mevalonate and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. The MEP pathway is used by most pathogenic bacteria and some parasitic protozoa (including the malaria parasite, Plasmodium falciparum) as well as by plants, but is not present in animals. The terminal reaction of the MEP pathway is catalyzed by (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase (LytB), an enzyme that converts HMBPP into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Here, we present the structure of Aquifex aeolicus LytB, at 1.65 A resolution. The protein adopts a cloverleaf or trefoil-like structure with each monomer in the dimer containing three alpha/beta domains surrounding a central [Fe3S4] cluster ligated to Cys13, Cys96, and Cys193. Two highly conserved His (His 42 and His 124) and a totally conserved Glu (Glu126) are located in the same central site and are proposed to be involved in ligand binding and catalysis. Substrate access is proposed to occur from the front-side face of the protein, with the HMBPP diphosphate binding to the two His and the 4OH of HMBPP binding to the fourth iron thought to be present in activated clusters, while Glu126 provides the protons required for IPP/DMAPP formation.

Prolonged (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vgamma2Vdelta2 T cells in macaques.

Although phosphoantigen-specific Vgamma2Vdelta2 T cells appear to play a role in antimicrobial and anticancer immunity, mucosal immune responses and effector functions of these gammadelta T cells during infection or phospholigand treatment remain poorly characterized. In this study, we demonstrate that the microbial phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) plus IL-2 treatment of macaques induced a prolonged major expansion of circulating Vgamma2Vdelta2 T cells that expressed CD8 and produced cytotoxic perforin during their peak expansion. Interestingly, HMBPP-activated Vgamma2Vdelta2 T cells underwent an extraordinary pulmonary accumulation, which lasted for 3-4 mo, although circulating Vgamma2Vdelta2 T cells had returned to baseline levels weeks prior. The Vgamma2Vdelta2 T cells that accumulated in the lung following HMBPP/IL-2 cotreatment displayed an effector memory phenotype, as follows: CCR5+CCR7-CD45RA-CD27+ and were able to re-recognize phosphoantigen and produce copious amounts of IFN-gamma up to 15 wk after treatment. Furthermore, the capacity of massively expanded Vgamma2Vdelta2 T cells to produce cytokines in vivo coincided with an increase in numbers of CD4+ and CD8+ alphabeta T cells after HMBPP/IL-2 cotreatment as well as substantial perforin expression by CD3+Vgamma2- T cells. Thus, the prolonged HMBPP-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vgamma2Vdelta2 T cells may confer immunotherapeutics against infectious diseases and cancers.

Synthesis of the antimalarial drug FR900098 utilizing the nitroso-ene reaction.

The antimalarial drug FR900098 was prepared from diethyl allylphosphonate involving the nitroso-ene reaction with nitrosocarbonyl methane as the key step followed by hydrogenation and dealkylation. The utilization of dibenzyl allylphosphonate as the starting compound allows one-step hydrogenation with dealkylation, which simplifies the preparative scheme further.

Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum.

In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid-like organelle (apicoplast) by the mevalonate independent 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe-4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin-NADP(+) reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin-NADP(+) reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.

Mycoplasma penetrans is capable of activating V gamma 9/V delta 2 T cells while other human pathogenic mycoplasmas fail to do so.

While most mycoplasma species appear to have evolutionarily lost the ability to synthesize isoprenoid precursors, Mycoplasma penetrans has retained the nonmevalonate pathway that proceeds via the immunogenic intermediate (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). Consequently, this pathogen is capable of stimulating human V gamma 9/V delta 2 T cells.

Determination of fosmidomycin in human serum and urine by capillary electrophoresis.

A capillary electrophoresis method with direct UV detection was developed for the determination of fosmidomycin, a promising new anti-malarial drug, in human serum and urine. Optimization of the separation parameters resulted in a buffer system adjusted to pH 10.8 containing a cationic reagent and an organic modifier. Under these conditions, the migration time of fosmidomycin was 5.2 min with serum and 7.4 min with urine samples. Validation of the method revealed good recoveries, precision and accuracy. The limit of quantification was 0.5 microg/ml in serum and 10 microg/ml in urine. The determination of fosmidomycin in serum was linear over a range of 0.1-150 microg/ml. Short and long-term stability tests resulted in no significant loss of fosmidomycin. The described technique will provide a fast and accurate analytical method for future pharmacokinetic studies.

The interplay between classical and alternative isoprenoid biosynthesis controls gammadelta T cell bioactivity of Listeria monocytogenes.

Isoprenoids are synthesised either through the classical, mevalonate pathway, or the alternative, non-mevalonate, 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The latter is found in many microbial pathogens and proceeds via (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), a potent activator of human Vgamma9/Vdelta2 T cells. Listeria monocytogenes is the only pathogenic bacterium known to contain both pathways concurrently. Strategic gene knockouts demonstrate that either pathway is functional but dispensable for viability. Yet, disrupting the mevalonate pathway results in a complementary upregulation of the MEP pathway. Vgamma9/Vdelta2 T cell bioactivity is increased in DeltalytB mutants where HMB-PP accumulation is expected, and lost in DeltagcpE mutants which fail to produce HMB-PP.

Microbial isoprenoid biosynthesis and human gammadelta T cell activation.

Human Vgamma9/Vdelta2 T cells play a crucial role in the immune response to microbial pathogens, yet their unconventional reactivity towards non-peptide antigens has been enigmatic until recently. The break-through in identification of the specific activator was only possible due to recent success in a seemingly remote field: the elucidation of the reaction steps of the newly discovered 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway of isoprenoid biosynthesis that is utilised by many pathogenic bacteria. Unexpectedly, the intermediate of the MEP pathway, (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate) (HMB-PP), turned out to be by far the most potent Vgamma9/Vdelta2 T cell activator known, with an EC(50) of 0.1 nM.

Replacing the pyrophosphate group of HMB-PP by a diphosphonate function abrogates Its potential to activate human gammadelta T cells but does not lead to competitive antagonism.

The immunological characterization of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), and its methylenediphosphonate analogue, HMB-PCP, is described. With an EC(50) of 0.1-0.2 nM, HMB-PP is significantly more potent in stimulating human Vgamma9/Vdelta2 T cells than any other compound described so far. However, replacing the pyrophosphate by a P-CH(2)-P function abrogates the bioactivity drastically, with HMB-PCP having a EC(50) of only 5.3 microM.

Fosmidomycin, a novel chemotherapeutic agent for malaria.

In previous studies, fosmidomycin has been shown to possess activity against Plasmodium falciparum in vitro and in the mouse model. It has a novel mode of action through inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase, an enzyme of the nonmevalonate pathway of isoprenoid biosynthesis, which is absent in humans. In this open-label, uncontrolled trial, the efficacy and safety of fosmidomycin, in an oral dose of 1,200 mg every 8 h for 7 days, were evaluated in the treatment of acute uncomplicated Plasmodium falciparum malaria in 20 adult subjects in Gabon and Thailand. Clinical assessments were performed and thick blood smears were evaluated every 8 h until parasite clearance and resolution of symptoms were achieved; assessments continued at weekly intervals thereafter for the duration of the 28-day followup period. All subjects were clinically and parasitologically cured on day 7 (primary end point). Parasite and fever clearance were rapid, with means of 44 and 41 h, respectively. On day 28, seven out of nine subjects (78%) were cured in Gabon and two out of nine subjects (22%) were cured in Thailand. The drug was well tolerated, although mild gastrointestinal side effects were recorded for five subjects. Analysis of hematological and biochemical parameters showed no clinically significant changes throughout the study. Fosmidomycin is an effective and safe antimalarial drug, although its use as a single agent is restricted by the occurrence of recrudescent infections. However, its role in combination therapy should be explored.

Functional characterization of GcpE, an essential enzyme of the non-mevalonate pathway of isoprenoid biosynthesis.

The gcpE gene product controls one of the terminal steps of isoprenoid biosynthesis via the mevalonate independent 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. This pathway is utilized by a variety of eubacteria, the plastids of algae and higher plants, and the plastid-like organelle of malaria parasites. Recombinant GcpE protein from the hyperthermophilic bacterium Thermus thermophilus was produced in Escherichia coli and purified under dioxygen-free conditions. The protein was enzymatically active in converting 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the presence of dithionite as reductant. The maximal specific activity was 0.6 micromol x min(-1) x mg(-1) at pH 7.5 and 55 degrees C. The kcat value was 0.4 s(-1) and the K(m) value for HMBPP 0.42 mM.

LytB protein catalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis.

Recombinant LytB protein from the thermophilic eubacterium Aquifex aeolicus produced in Escherichia coli was purified to apparent homogeneity. The purified LytB protein catalyzed the reduction of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in a defined in vitro system. The reaction products were identified as isopentenyl diphosphate and dimethylallyl diphosphate. A spectrophotometric assay was established to determine the steady-state kinetic parameters of LytB protein. The maximal specific activity of 6.6+/-0.3 micromol x min(-1) x mg(-1) protein was determined at pH 7.5 and 60 degrees C. The k(cat) value of the LytB protein was 3.7+/-0.2 s(-1) and the K(m) value for HMBPP was 590+/-60 microM.