Humoral immune response as an indicator for protection against Covid-19 after anti-SARS-COV2-booster vaccination in hematological and oncological patients.

Cancer patients are at a higher risk to develop severe COVID-19 symptoms after SARS-CoV-2 infection compared to the general population and regularly show an impaired immune response to SARS-CoV-2 vaccination. In our oncological center, 357 patients with hematological and oncological diseases were monitored for neutralizing antibodies from October 2021 over 12 months. All patients had received three anti-SARS-CoV-2 vaccinations with an mRNA-(Comirnaty/BionTech or Spikevax/Moderna) or a vector vaccine (Vakzevria/AstraZeneca or JCOVDEN/Johnson&Johnson). Neutralizing anti-SARS-CoV-2 IgG antibodies in the patients' sera were detected within 3 months before, 3-10 weeks and 5-7 months after the booster vaccination (third vaccination). 112 patients developed a breakthrough SARS-CoV-2 infection during the observation period. High anti-SARS-Cov-2 antibody levels before infection significantly protected against symptomatic Covid-19 disease (p = .003). The median antibody titer in patients with asymptomatic Covid-19 disease was 2080 BAU/ml (binding antibody units per Milliliter) and 765 BAU/ml in symptomatic patients. 98% of the solid tumor patients reached seroconversion after the booster vaccination in comparison to 79% of the hematological patients. High antibody titers of >2080 BAU/ml after the booster vaccination were detected in 61% of the oncological and 34.8% of the hematological patients. 7-10 months after the booster vaccination, the anti-SARS-CoV-2 antibody titer declined to an average of 849 BAU/ml. Considering the heterogenous humoral immune response of cancer patients observed in this study, an individual vaccination strategy based on regular measurement of anti-SARS-CoV-2 antibody levels should be considered in contrast to fixed vaccination intervals.

Integrated genomic surveillance enables tracing of person-to-person SARS-CoV-2 transmission chains during community transmission and reveals extensive onward transmission of travel-imported infections, Germany, June to July 2021.

BackgroundTracking person-to-person SARS-CoV-2 transmission in the population is important to understand the epidemiology of community transmission and may contribute to the containment of SARS-CoV-2. Neither contact tracing nor genomic surveillance alone, however, are typically sufficient to achieve this objective.AimWe demonstrate the successful application of the integrated genomic surveillance (IGS) system of the German city of Düsseldorf for tracing SARS-CoV-2 transmission chains in the population as well as detecting and investigating travel-associated SARS-CoV-2 infection clusters.MethodsGenomic surveillance, phylogenetic analysis, and structured case interviews were integrated to elucidate two genetically defined clusters of SARS-CoV-2 isolates detected by IGS in Düsseldorf in July 2021.ResultsCluster 1 (n = 67 Düsseldorf cases) and Cluster 2 (n = 36) were detected in a surveillance dataset of 518 high-quality SARS-CoV-2 genomes from Düsseldorf (53% of total cases, sampled mid-June to July 2021). Cluster 1 could be traced back to a complex pattern of transmission in nightlife venues following a putative importation by a SARS-CoV-2-infected return traveller (IP) in late June; 28 SARS-CoV-2 cases could be epidemiologically directly linked to IP. Supported by viral genome data from Spain, Cluster 2 was shown to represent multiple independent introduction events of a viral strain circulating in Catalonia and other European countries, followed by diffuse community transmission in Düsseldorf.ConclusionIGS enabled high-resolution tracing of SARS-CoV-2 transmission in an internationally connected city during community transmission and provided infection chain-level evidence of the downstream propagation of travel-imported SARS-CoV-2 cases.

Structure of the GcpE-HMBPP complex from Thermus thermophilius.

Isoprenoid biosynthesis in many bacteria, plant chloroplasts and parasitic protozoa but not in humans proceeds via the mevalonate independent 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. Its penultimate reaction step is catalyzed by (E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase (GcpE/IspG) which transforms 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate (MEcPP) to HMBPP. In this report we present the structure of GcpE of Thermus thermophiles in complex with its product HMBPP at a resolution of 1.65 Å. The GcpE-HMBPP like the GcpE-MEcPP structure is found in a closed, the ligand-free GcpE structure in an open enzyme state. Imposed by the rigid protein scaffold inside the active site funnel, linear HMBPP and circular MEcPP adopt highly similar conformations. The confined space also determines the conformational freedom of transition state intermediates and the design of anti-infective drugs. The apical Fe of the [4Fe-4S] cluster is coordinated to MEcPP in the GcpE-MEcPP complex and to a hydroxyl/water ligand but not to HMBPP in the GcpE-HMBPP complex. The GcpE-HMBPP structure can be attributed to one step in the currently proposed GcpE reaction cycle.

Phosphoantigen/IL2 expansion and differentiation of Vγ2Vδ2 T cells increase resistance to tuberculosis in nonhuman primates.

Dominant Vγ2Vδ2 T-cell subset exist only in primates, and recognize phosphoantigen from selected pathogens including M. tuberculosis(Mtb). In vivo function of Vγ2Vδ2 T cells in tuberculosis remains unknown. We conducted mechanistic studies to determine whether earlier expansion/differentiation of Vγ2Vδ2 T cells during Mtb infection could increase immune resistance to tuberculosis in macaques. Phosphoantigen/IL-2 administration specifically induced major expansion and pulmonary trafficking/accumulation of phosphoantigen-specific Vγ2Vδ2 T cells, significantly reduced Mtb burdens and attenuated tuberculosis lesions in lung tissues compared to saline/BSA or IL-2 controls. Expanded Vγ2Vδ2 T cells differentiated into multifunctional effector subpopulations capable of producing anti-TB cytokines IFNγ, perforin and granulysin, and co-producing perforin/granulysin in lung tissue. Mechanistically, perforin/granulysin-producing Vγ2Vδ2 T cells limited intracellular Mtb growth, and macaque granulysin had Mtb-bactericidal effect, and inhibited intracellular Mtb in presence of perforin. Furthermore, phosphoantigen/IL2-expanded Vγ2Vδ2 T effector cells produced IL-12, and their expansion/differentiation led to enhanced pulmonary responses of peptide-specific CD4+/CD8+ Th1-like cells. These results provide first in vivo evidence implicating that early expansion/differentiation of Vγ2Vδ2 T effector cells during Mtb infection increases resistance to tuberculosis. Thus, data support a rationale for conducting further studies of the γδ T-cell-targeted treatment of established TB, which might ultimately help explore single or adjunctive phosphoantigen expansion of Vγ2Vδ2 T-cell subset as intervention of MDR-tuberculosis or HIV-related tuberculosis.

Primary MHC-class II(+) cells are necessary to promote resting Vδ2 cell expansion in response to (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate and isopentenyl pyrophosphate.

Human Vγ9δ2 (Vδ2) T cells represent a unique effector T cell population in humans and primates detecting nonpeptid phosphoantigens, playing an important role in antimicrobial and antitumor immunity. Currently, it is believed that various leukocyte subsets can promote phosphoantigen-driven Vδ2 cell expansion, but the essential cell type required remains elusive. We have used high purity cell sorting to analyze the cellular requirements for (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP)-driven Vδ2 cell expansion. To our knowledge, we show for the first time that primary human MHC-class II(+) cells are indispensable for HMBPP- and isopentenylpyrophosphate-driven Vδ2 cell expansion. In contrast, MHC-class II(-) cells are unable to promote Vδ2 cell expansion. Moreover, purified primary human TCRαß(+) T cells, CD4(+), or CD8(+) T cells also failed to promote HMBPP-mediated Vδ2 expansion. Depletion of CD4(+)CD25(+) T cells demonstrated that inability of TCRαß(+) cells to expand Vδ2 cells was not related to the presence of regulatory T cells. Separation of MHC-class II(+) cells into dendritic cells, monocytes, and B cells revealed that dendritic cells were the most potent Vδ2 expanders. Pulsing experiments demonstrated that HMBPP transforms MHC-class II(+) but not negative cells into Vδ2 expanders. MHC-class II-blocking experiments with mAbs and secondary MHC-class II induction on CD4(+) T cells after CD3/CD28 costimulation indicated that MHC-class II is necessary, but not sufficient to promote Vδ2 expansion. Our results provide novel insight into the primary cell-specific requirements for human Vδ2 expansion.

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.

Effective high-throughput RT-qPCR screening for SARS-CoV-2 infections in children.

Systematic SARS-CoV-2 testing is a valuable tool for infection control and surveillance. However, broad application of high sensitive RT-qPCR testing in children is often hampered due to unpleasant sample collection, limited RT-qPCR capacities and high costs. Here, we developed a high-throughput approach ('Lolli-Method') for SARS-CoV-2 detection in children, combining non-invasive sample collection with an RT-qPCR-pool testing strategy. SARS-CoV-2 infections were diagnosed with sensitivities of 100% and 93.9% when viral loads were >106 copies/ml and >103 copies/ml in corresponding Naso-/Oropharyngeal-swabs, respectively. For effective application of the Lolli-Method in schools and daycare facilities, SEIR-modeling indicated a preferred frequency of two tests per week. The developed test strategy was implemented in 3,700 schools and 698 daycare facilities in Germany, screening over 800,000 individuals twice per week. In a period of 3 months, 6,364 pool-RT-qPCRs tested positive (0.64%), ranging from 0.05% to 2.61% per week. Notably, infections correlated with local SARS-CoV-2 incidences and with a school social deprivation index. Moreover, in comparison with the alpha variant, statistical modeling revealed a 36.8% increase for multiple (≥2 children) infections per class following infections with the delta variant. We conclude that the Lolli-Method is a powerful tool for SARS-CoV-2 surveillance and can support infection control in schools and daycare facilities.

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.

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.

The nonmevalonate pathway of isoprenoid biosynthesis in Mycobacterium tuberculosis is essential and transcriptionally regulated by Dxs.

Mycobacterium tuberculosis synthesizes isoprenoids via the nonmevalonate or DOXP pathway. Previous work demonstrated that three enzymes in the pathway (Dxr, IspD, and IspF) are all required for growth in vitro. We demonstrate the essentiality of the key genes dxs1 and gcpE, confirming that the pathway is of central importance and that the second homolog of the synthase (dxs2) cannot compensate for the loss of dxs1. We looked at the effect of overexpression of Dxr, Dxs1, Dxs2, and GcpE on viability and on growth in M. tuberculosis. Overexpression of dxs1 or dxs2 was inhibitory to growth, whereas overexpression of dxr or gcpE was not. Toxicity is likely to be, at least partially, due to depletion of pyruvate from the cells. Overexpression of dxs1 or gcpE resulted in increased flux through the pathway, as measured by accumulation of the metabolite 4-hydroxy-3-methyl-but-2-enyl pyrophosphate. We identified the functional translational start site and promoter region for dxr and demonstrated that it is expressed as part of a polycistronic mRNA with gcpE and two other genes. Increased expression of this operon was seen in cells overexpressing Dxs1, indicating that transcriptional control is effected by the first enzyme of the pathway via an unknown regulator.

Paramagnetic intermediates of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE/IspG) under steady-state and pre-steady-state conditions.

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE/IspG) converts 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the penultimate step of the methyl-erythritol phosphate (MEP) pathway for isoprene biosynthesis. MEcPP is a cyclic compound and the reaction involves the opening of the ring and removal of the C3 hydroxyl group consuming a total of two electrons. The enzyme contains a single [4Fe-4S] cluster in its active site. Several paramagnetic species are observed in steady-state and pre-steady-state kinetic studies. The first signal detected is from a transient species that displays a rhombic electron paramagnetic resonance (EPR) signal with g(xyz) = 2.000, 2.019, and 2.087 (FeS(A)). A second set of EPR signals (FeS(B)) accumulated during the reaction. Labeling studies with (57)Fe showed that all species observed are iron-sulfur-based. (31)P-ENDOR measurements on the FeS(A) species showed a weak (31)P coupling which is in line with binding of the substrate to the enzyme in close proximity of the active-site cluster. On the basis of the EPR/ENDOR measurements, we propose a direct binding of the substrate to the [4Fe-4S] cluster during the reaction, and therefore that the iron-sulfur cluster is directly involved in a reductive elimination of a hydroxyl group. The FeS(B) signal also showed (31)P coupling; in this case, however, it could be shown that the signal is due to the binding of the reaction product HMBPP to the active site cluster.

RlmN and Cfr are radical SAM enzymes involved in methylation of ribosomal RNA.

Posttranscriptional modifications of ribosomal RNA (rRNA) nucleotides are a common mechanism of modulating the ribosome's function and conferring bacterial resistance to ribosome-targeting antibiotics. One such modification is methylation of an adenosine nucleotide within the peptidyl transferase center of the ribosome mediated by the endogenous methyltransferase RlmN and its evolutionarily related resistance enzyme Cfr. These methyltransferases catalyze methyl transfer to aromatic carbon atoms of the adenosine within a complex 23S rRNA substrate to form the 2,8-dimethylated product. RlmN and Cfr are members of the Radical SAM superfamily and contain the characteristic cysteine-rich CX(3)CX(2)C motif. We demonstrate that both enzymes are capable of accommodating the requisite [4Fe-4S] cluster. S-Adenosylmethionine (SAM) is both the methyl donor and the source of a 5'-deoxyadenosyl radical, which activates the substrate for methylation. Detailed analyses of the rRNA requirements show that the enzymes can utilize protein-free 23S rRNA as a substrate, but not the fully assembled large ribosomal subunit, suggesting that the methylations take place during the assembly of the ribosome. The key recognition elements in the 23S rRNA are helices 90-92 and the adjacent single stranded RNA that encompasses A2503. To our knowledge, this study represents the first in vitro description of a methyl transfer catalyzed by a member of the Radical SAM superfamily, and it expands the catalytic repertoire of this diverse enzyme class. Furthermore, by providing information on both the timing of methylation and its substrate requirements, our findings have important implications for the functional consequences of Cfr-mediated modification of rRNA in the acquisition of antibiotic resistance.

Analysis of the isoprenoid biosynthesis pathways in Listeria monocytogenes reveals a role for the alternative 2-C-methyl-D-erythritol 4-phosphate pathway in murine infection.

Most bacteria synthesize isoprenoids through one of two essential pathways which provide the basic building block, isopentyl diphosphate (IPP): either the classical mevalonate pathway or the alternative non-mevalonate 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. However, postgenomic analyses of the Listeria monocytogenes genome revealed that this pathogen possesses the genetic capacity to produce the complete set of enzymes involved in both pathways. The nonpathogenic species Listeria innocua naturally lacks the last two genes (gcpE and lytB) of the MEP pathway, and bioinformatic analyses strongly suggest that the genes have been lost through evolution. In the present study we show that heterologous expression of gcpE and lytB in L. innocua can functionally restore the MEP pathway in this organism and confer on it the ability to induce Vgamma9 Vdelta2 T cells. We have previously confirmed that both pathways are functional in L. monocytogenes and can provide sufficient IPP for normal growth in laboratory media (M. Begley, C. G. Gahan, A. K. Kollas, M. Hintz, C. Hill, H. Jomaa, and M. Eberl, FEBS Lett. 561:99-104, 2004). Here we describe a targeted mutagenesis strategy to create a double pathway mutant in L. monocytogenes which cannot grow in the absence of exogenously provided mevalonate, confirming the requirement for at least one intact pathway for growth. In addition, murine studies revealed that mutants lacking the MEP pathway were impaired in virulence relative to the parent strain during intraperitoneal infection, while mutants lacking the classical mevalonate pathway were not impaired in virulence potential. In vivo bioluminescence imaging also confirmed in vivo expression of the gcpE gene (MEP pathway) during murine 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.

Synthesis of beta- and gamma-oxa isosteres of fosmidomycin and FR900098 as antimalarial candidates.

To expand the structure-activity relationships of fosmidomycin and FR900098, two potent antimalarials interfering with the MEP-pathway, we decided to replace a methylene group in beta-position of the phosphonate moiety of these leads by an oxygen atom. beta-oxa-FR900098 (11) proved equally active as the parent compound. When applied to 4-[hydroxyl(methyl)amino]-4-oxobutyl phosphonic acid, featuring a hydroxamate instead of the retrohydroxamate moiety, a beta-oxa modification yielded a derivative (13) with superior activity against the Plasmodium falciparum 3D7 strain than fosmidomycin, while a gamma-oxa modification resulted in less active derivatives. A bis(pivaloyloxymethyl)ester of phosphonate 13 proved twice as active in inhibiting cultured parasites as a similar prodrug of FR900098.

Towards new antimalarial drugs: synthesis of non-hydrolyzable phosphate mimics as feed for a predictive QSAR study on 1-deoxy-D-xylulose-5-phosphate reductoisomerase inhibitors.

The conversion of 1-deoxy-D-xylulose-5-phosphate (DOXP) to 2-C-methyl-D-erythritol-4-phosphate (MEP) is effectively blocked by 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) inhibitors such as the natural antibiotic fosmidomycin. Prediction of binding affinities for closely related Dxr ligands as well as estimation of the affinities of structurally more distinct inhibitors within this class of non-hydrolyzable phosphate mimics relies on the synthesis of fosmidomycin derivatives with a broad range of target affinity. Maintaining the phosphonic acid moiety, linear modifications of the lead structure were carried out in an effort to expand the SAR of this physicochemically challenging class of compounds. Synthetic access to a set of phosphonic acids with inhibitory activity (IC(50)) in the range from 1 to >30 microM vs. E. coli Dxr and 0.4 to 20 microM against P. falciparum Dxr is reported.

Possible direct involvement of the active-site [4Fe-4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP.

The GcpE enzyme converts 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the penultimate step of the DOXP pathway for isoprene biosynthesis. Purification of the enzyme under exclusion of air leads to a preparation that contains solely [4Fe-4S] clusters. Kinetic studies showed that in the presence of the artificial reductant dithionite and MEcPP a new transient iron-sulfur-based signal is detected in electron paramagnetic resonance (EPR) spectroscopy. Similarity of this EPR signal to that detected in ferredoxin:thioredoxin reductase indicates that during the reaction an intermediate is directly bound to the active-site cluster.

Delayed parasite elimination in human infections treated with clindamycin parallels 'delayed death' of Plasmodium falciparum in vitro.

Clindamycin is safe and effective for the treatment of Plasmodium falciparum malaria, but its use as monotherapy is limited by unacceptably slow initial clinical response rates. To investigate whether the protracted action is due to an accumulative, time of exposure-dependent or a delayed effect on parasite growth, we studied the in vivo and in vitro pharmacodynamic profiles of clindamycin against P. falciparum. In vivo, elimination of young, circulating asexual parasite stages during treatment with clindamycin displayed an unusual biphasic kinetic: a plateau phase was followed by a precipitated decline of asexual parasite densities to nearly undetectable levels after 72 and 60 h in adult patients and asymptomatic children, respectively, suggesting an uninhibited capacity to establish a second, but not third, infectious cycle. In vitro, continuous exposure of a laboratory-adapted P. falciparum strain to clindamycin with concentrations of up to 100 microM for two replication cycles (96 h) did not produce inhibitory effects of >50% compared with drug-free controls as measured by the production of P. falciparum histidine-rich protein II (PfHRP2). PfHRP2 production was completely arrested after the second cycle (96-144h) (>10,000-fold decrease of mean half-inhibitory concentrations measured at 96-144h compared to 48-96h). Furthermore, incubation with clindamycin during only the first (0-48h) versus three (0-144h) parasite replication cycles led to comparable inhibition of PfHRP2 production in the third infectious cycle (96-144h) (mean IC(99) of 27 and 22nM, respectively; P=0.2). When parasite cultures were exposed to different concentrations of clindamycin ranging from 50 to 1,000nM for 72h and followed up in an experiment designed to simulate a typical 3-day treatment regimen, parasitaemia was initially suppressed below the microscopic detection threshold. Nonetheless, parasites reappeared in a dose-dependent manner after removal of drug at 72h but not in continuously drug-exposed controls. The delayed, but potent, antimalarial effect of clindamycin appears to be of greatest potential benefit in new combinations of clindamycin with rapidly acting antimalarial combination partners.

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.

Sex-specific phenotypical and functional differences in peripheral human Vgamma9/Vdelta2 T cells.

Vgamma9/Vdelta2 T cells constitute a minor proportion of human peripheral blood T cells that can expand rapidly upon infection with microbial pathogens. Vgamma9/Vdelta2 T cell numbers change characteristically with age, rising from birth to puberty and gradually decreasing again beyond 30 years of age. In adults, female blood donors have significantly higher levels than males, implying that circulating Vgamma9/Vdelta2 T cells in women remain elevated for a longer period in life and drop less strikingly than in men. This loss in men is accompanied by a substantial depletion of CD27- CD45RA- and CD27- CD45RA+ effector T cells and a parallel increase in CD27+ CD45RA- central memory T cells while in women, the distribution of Vgamma9/Vdelta2 T cell subsets remains virtually unchanged. The phenotypical conversion in men older than 30 years is mirrored by an increased proliferative response of Vgamma9/Vdelta2 T cells and a reduced interferon-gamma secretion upon stimulation with isopentenyl pyrophosphate in vitro.