Isopentenyl Pyrophosphate Research Articles

Antigen-presenting effects of effector memory Vγ9Vδ2 T cells in rheumatoid arthritis

Rheumatoid arthritis is an autoimmune disease that primarily affects the limbs, but the pathogenic mechanism remains unclear. γδ T cells, a T-cell subpopulation, are characterized by multiple biological functions and associated with a variety of diseases. This study investigated the antigen-presenting effects of γδ T cells and their relationship with rheumatoid arthritis development. We found that Vγ9Vδ2 T cells (the predominant subtype of γδ T cells in peripheral blood) were activated by isopentenyl pyrophosphate to continuously proliferate and differentiate into effector memory cells. The effector memory Vγ9Vδ2 T cells exhibited phenotypic characteristics of specific antigen-presenting cells, including high HLA-DR and CD80/86 expression. These Vγ9Vδ2 T cells could present soluble antigens and synthetic peptides to CD4(+) T cells. Vγ9Vδ2 T cells with different phenotypes showed different cytokine secretion patterns. Effector memory Vγ9Vδ2 T cells simultaneously secreted not only interferon (IFN)-γ but also IL-17. The peripheral blood and joint synovial fluid from RA patients contained numerous heterogeneous γδ T cells that were predominantly effector memory Vγ9Vδ2 T cells with the ability to secrete inflammatory factors. We also found that γδ T cells had a similar antigen-presenting capability to B cells. These results suggest that during the development of rheumatoid arthritis, γδ T cells can aggravate immune dysfunction and produce abnormal immune damage by secreting cytokines and inducing inflammatory cells to participate in synergistic inflammatory responses. Furthermore, γδ T cells can behave similarly to B cells to present viral peptides and autoantigen peptides to CD4(+) T cells, thus sustaining CD4(+) T-cell activation.

Biosynthesis of artemisinin – revisited

Artemisinin is a well-known antimalarial drug isolated from the Artemisia annua plant. The biosynthesis of this well-known molecule has been reinvestigated by using [1-13C]acetate, [2-13C]acetate, and [1,6-13C2]glucose. The 13C peak enrichment in artemisinin was observed in six and nine carbon atoms from [1-13C]acetate and [2-13C]acetate, respectively. The 13C NMR spectra of 13C-enriched artemisinin suggested that the mevalonic acid (MVA) pathway is the predominant route to biosynthesis of this sesquiterpene. On the other hand, the peak enrichment of five carbons of 13C-artemisinin including carbon atoms originating from methyls of dimethylallyl group of geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) was observed from [1,6-13C2]glucose. This suggested that GPP which is supposed to be biosynthesized in plastids travels from plastids to cytosol through the plastidial wall and combines with isopentenyl pyrophosphate (IPP) to form the (E,E)-FPP which finally cyclizes and oxidizes to artemisinin. In this way the DXP pathway also contributes to the biosynthesis of this sesquiterpene.

Indirect Stimulation of Human Vγ2Vδ2 T Cells through Alterations in Isoprenoid Metabolism

Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C-methyl-d-erythritol-4-phosphate pathway used by microbes, and isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Aminobisphosphonates and alkylamines indirectly stimulate Vγ2Vδ2 cells by inhibiting farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, thereby increasing IPP/triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester that directly stimulate. In this study, we further characterize stimulation by these compounds and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by aminobisphosphonates and alkylamines was much more sensitive to statin inhibition than stimulation by prenyl pyrophosphates; however, the continuous presence of aminobisphosphonates was toxic for T cells and blocked their proliferation. Aminobisphosphonate stimulation was rapid and prolonged, independent of known Ag-presenting molecules, and resistant to fixation. New classes of stimulatory compounds-mevalonate, the alcohol of HMBPP, and alkenyl phosphonates-likely stimulate differently. Mevalonate, a rate-limiting metabolite, appears to enter cells to increase IPP levels, whereas the alcohol of HMBPP and alkenyl phosphonates are directly recognized. The critical chemical feature of bisphosphonates is the amino moiety, because its loss switched aminobisphosphonates to direct Ags. Transfection of APCs with small interfering RNA downregulating FDPS rendered them stimulatory for Vγ2Vδ2 cells and increased cellular IPP. Small interfering RNAs for isopentenyl diphosphate isomerase functioned similarly. Our results show that a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells and that pulsing aminobisphosphonates would be more effective for the ex vivo expansion of Vγ2Vδ2 T cells for adoptive cancer immunotherapy.

Expansion of Human Peripheral Blood γδ T Cells using Zoledronate

Human γδ T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity. The majority of γδ T cells in peripheral blood have the Vγ9Vδ2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions. Therefore, γδ T cells are attractive candidate effector cells for cancer immunotherapy. Vγ9Vδ2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis; and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway. In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of γδ T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and γδ T cells activation. Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to γδ T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransformed cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating γδ T cells with aminobisphosphonates. Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. Monocytes that accumulate IPP become antigen-presenting cells and stimulate Vγ9Vδ2 T cells in the peripheral blood. Based on these mechanisms, we developed a technique for large-scale expansion of γδ T cell cultures using zoledronate and interleukin-2 (IL-2). Other methods for expansion of γδ T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP) or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP). All of these methods allow ex vivo expansion, resulting in large numbers of γδ T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded γδ T cells display CD27(-)CD45RA(-) effector memory phenotype and thier function can be evaluated by IFN-γ production assay.

Research of mechanism about enhancing the activity of γδδT cells treated by resveratrol on colonic cancer cell lines SW-1116

Objective To explore the effect of γδT cells on colonic cancer cell lines SW-1116 treated by resveratrol. Methods Amplification γδT cells of human peripheral blood in vitro by using isopentenylpyrophosphate. Different density resveratrol induced γδT cells and colonic cancer cell SW-1 116, methyl thiazolyl tetrazolium(MTT) detectde the effect of growth and proliferation when expression veratrol action to γδT cells and colonic cancer cell lines SW-1116. Flow cytometer( FCM )detectde the expression of porforin, granzymeB and CD107a on γδT cells before it treated by resveratrol and after that. γδT cells against colonic cancer cell lines SW-1116 were detectde by lactate dehydrogenase(LDH) releasing assay before and after treated by resveratrol.Western blot analyzed the liveness of extracellular signal-regulated kinase1/2 of γδT cells before it treated by resveratrol and after that. Results γδTcells would be proliferation when the density of resveratrol in 0.39-3.125 μmol/L, while the effect on colonic cancer cell lines SW-1116 was not significance. There was significant difference of the expression of porforin, ranzymeB and CD107a on γδT cells before it treated by resveratrol and after that ( P＜0.05 ). γδT cells against colonic cancer cell lines SW-1116 treated by resveratrol was enhanced compared with control group( P＜0.05 ) . The p-ERK 1/2expression of γδT cells enhanced when the density of resveratrol treated to γδT cells in 0.1-10 μmol/L( P＜0.05 ). Conclusion Resveratrol could promote γδT cells, proliferation and strengthen the cytotoxicity of γδT cells against colonic cancer cell SW-1116, the mechanism might concerned with activating p-ERK1/2 and enhancing the expression of porforin, granzymeB and CD107a on γδT cells. Key words: Resveratrol; Colon cancer cells; γδT cells

Chemical Rescue of Malaria Parasites Lacking an Apicoplast Defines Organelle Function in Blood-Stage Plasmodium falciparum

Author SummaryMalaria caused by Plasmodium spp parasites is a profound human health problem that has shaped our evolutionary past and continues to influence modern day with a disease burden that disproportionately affects the world's poorest and youngest. New anti-malarials are desperately needed in the face of existing or emerging drug resistance to available therapies, while an effective vaccine remains elusive. A plastid organelle, the apicoplast, has been hailed as Plasmodium's “Achilles' heel” because it contains bacteria-derived pathways that have no counterpart in the human host and therefore may be ideal drug targets. However, more than a decade after its discovery, the essential functions of the apicoplast remain a mystery, and without a specific pathway or function to target, development of drugs against the apicoplast has been stymied. In this study, we use a simple chemical method to generate parasites that have lost their apicoplast, normally a deadly event, but which survive—“rescued” by the addition of an essential metabolite to the culture. This chemical rescue demonstrates that the apicoplast serves only a single essential function, namely isoprenoid precursor biosynthesis during blood-stage growth, validating this metabolic function as a viable drug target. Moreover, the apicoplast-minus Plasmodium strains generated in this study will be a powerful tool for identifying apicoplast-targeted drugs and as a potential vaccine strain with significant advantages over current vaccine technologies.

What Do Human Parasites Do with a Chloroplast Anyway?

Apicomplexans are an important group of pathogens that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. These single-celled eukaryotic parasites evolved from photosynthetic algae. A remnant chloroplast, called the apicoplast, is a telltale hold-over from this more benign past in the ocean. The apicoplast is essential for parasite growth and development and therefore a potential target for drug therapy. The fact that humans and animals lack chloroplasts suggests that using approaches to target the apicoplast may provide parasite specificity. What are the critical functions of the apicoplast that should be targeted? In addition to the obvious medical relevance this question has broader biological implications. Why do organisms maintain an ancient symbiotic relationship when the initial rationale for this relationship has fallen by the evolutionary wayside? A new study by Yeh and DeRisi provides important clues. Their work demonstrates that antibiotic-induced loss of the apicoplast in cultured malaria parasites can be chemically rescued by providing isopentenyl-pyrophosphate (IPP) in the medium. IPP is generated by the apicoplast resident isoprenoid biosynthesis pathway and is apparently the one apicoplast metabolite that the parasite cannot live without in the red blood cell. This finding could be of great importance for the development of drugs and vaccines. The ability to produce and maintain parasite lines that lack the apicoplast also offers exciting experimental possibilities for the future.

Immune Modulation by Zoledronic Acid in Human Myeloma: An Advantageous Cross-Talk between Vγ9Vδ2 T Cells, αβ CD8+ T Cells, Regulatory T Cells, and Dendritic Cells

Vγ9Vδ2 T cells play a major role as effector cells of innate immune responses against microbes, stressed cells, and tumor cells. They constitute <5% of PBLs but can be expanded by zoledronic acid (ZA)-treated monocytes or dendritic cells (DC). Much less is known about their ability to act as cellular adjuvants bridging innate and adaptive immunity, especially in patients with cancer. We have addressed this issue in multiple myeloma (MM), a prototypic disease with several immune dysfunctions that also affect γδ T cells and DC. ZA-treated MM DC were highly effective in activating autologous γδ T cells, even in patients refractory to stimulation with ZA-treated monocytes. ZA inhibited the mevalonate pathway of MM DC and induced the intracellular accumulation and release into the supernatant of isopentenyl pyrophosphate, a selective γδ T cell activator, in sufficient amounts to induce the proliferation of γδ T cells. Immune responses against the tumor-associated Ag survivin (SRV) by MHC-restricted, SRV-specific CD8(+) αβ T cells were amplified by the concurrent activation of γδ T cells driven by autologous DC copulsed with ZA and SRV-derived peptides. Ancillary to the isopentenyl pyrophosphate-induced γδ T cell proliferation was the mevalonate-independent ZA ability to directly antagonize regulatory T cells and downregulate PD-L2 expression on the DC cell surface. In conclusion, ZA has multiple immune modulatory activities that allow MM DC to effectively handle the concurrent activation of γδ T cells and MHC-restricted CD8(+) αβ antitumor effector T cells.

Molecular cloning of mevalonate pathway genes from Taraxacum brevicorniculatum and functional characterisation of the key enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Taraxacum brevicorniculatum is known to produce high quality rubber. The biosynthesis of rubber is dependent on isopentenyl pyrophosphate (IPP) precursors derived from the mevalonate (MVA) pathway. The cDNA sequences of seven MVA pathway genes from latex of T. brevicorniculatum were isolated, including three cDNA sequences encoding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases (TbHMGR1-3). Expression analyses indicate an important role of TbHMGR1 as well as for the HMG-CoA synthase (TbHMGS), the diphosphomevalonate decarboxylase and the mevalonate kinase in the provision of precursors for rubber biosynthesis. The amino acid sequences of the TbHMGRs show the typical motifs described for plant HMGRs such as two transmembrane domains and a catalytic domain containing two HMG-CoA and two NADP(H) binding sites. The functionality of the HMGRs was demonstrated by complementation assay using an IPP auxotroph mutant of Escherichia coli. Furthermore, the transient expression of the catalytic domains of TbHMGR1 and TbHMGR2 in Nicotiana benthamiana resulted in a strong accumulation of sterol precursors, one of the major groups of pathway end-products.

The enzymatic incorporation of isopentenyl pyrophosphate into polyisoprene in rubber particles from Parthenium argentatum Gray

Studies have indicated the presence of both a de novo biosynthesis of new polyisoprene chains and the elongation of pre-existing rubber-PP chains in rubber particles from Ficus elastica Roxb. and small rubber particles from Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. In this paper, we have isolated rubber particles from the cortical parenchyma of Parthenium argentatum Gray with an adsorbed isopentenyl pyrophosphate isomerase that was competent to form polyisoprene to determine the presence of de novo biosynthesis of polyisoprene and the elongation of pre-existing rubber-PP. The gel filtrated rubber particles were supplied saturating concentrations of [ 14C]-isopentenyl pyrophosphate for 1 h and the resulting concentrations of [ 14C]-polyisoprene were isolated and analyzed with a Waters 150C Gel Permeation Chromatograph. The mol wt distribution of the [ 14C]-polyisoprene ranged from 10 3 to 10 6 Da with no formation of high mol wt chains equal to the polyisoprene in the mol wt distribution of natural rubber from P. argentatum. The mol wt distribution of [ 14C]-polyisoprene following 1 h of incubation of the reaction mixture was similar to the mol wt distribution formed by the bound rubber transferase in washed rubber particles of H. brasiliensis and the time-dependent formation of polyisoprene by washed rubber particles from P. argentatum. The data support the conclusion that the washed rubber particles from P. argentatum contain a bound rubber transferase that initiates the formation of new chains of polyisoprene from the dimethylallyl pyrophosphate initiator generated from the added isopentenyl pyrophosphate isomerase followed by the extensive polymerization of isopentenyl pyrophosphate to form a monomodal rubber polymer. There was no evidence in these washed rubber particles of a chain elongation of pre-existing rubber-PP.

Type 1 Responses of Human Vγ9Vδ2 T Cells to Influenza A Viruses

γδ T cells are essential constituents of antimicrobial and antitumor defenses. We have recently reported that phosphoantigen isopentenyl pyrophosphate (IPP)-expanded human Vγ9Vδ2 T cells participated in anti-influenza virus immunity by efficiently killing both human and avian influenza virus-infected monocyte-derived macrophages (MDMs) in vitro. However, little is known about the noncytolytic responses and trafficking program of γδ T cells to influenza virus. In this study, we found that Vγ9Vδ2 T cells expressed both type 1 cytokines and chemokine receptors during influenza virus infection, and IPP-expanded cells had a higher capacity to produce gamma interferon (IFN-γ). Besides their potent cytolytic activity against pandemic H1N1 virus-infected cells, IPP-activated γδ T cells also had noncytolytic inhibitory effects on seasonal and pandemic H1N1 viruses via IFN-γ but had no such effects on avian H5N1 or H9N2 virus. Avian H5N1 and H9N2 viruses induced significantly higher CCL3, CCL4, and CCL5 production in Vγ9Vδ2 T cells than human seasonal H1N1 virus. CCR5 mediated the migration of Vγ9Vδ2 T cells toward influenza virus-infected cells. Our findings suggest a novel therapeutic strategy of using phosphoantigens to boost the antiviral activities of human Vγ9Vδ2 T cells against influenza virus infection.

Screening for Mevalonate Biosynthetic Pathway Inhibitors Using Sensitized Bacterial Strains

A simple, optical density-based assay for inhibitors of the mevalonate-dependent pathway for isoprenoid biosynthesis was developed. The assay uses pathway-sensitized Staphylococcus aureus strains and is fully compatible with high-density screening in a 1536-well format. S. aureus strains were constructed in which genes required for mevalonate-dependent isopentenyl pyrophosphate (IPP) synthesis were regulated by an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible promoter. Inhibitors of the target enzymes displayed greater antibacterial potency in media containing low concentrations of IPTG, and therefore less induction of mevalonate pathway genes, than in media with high IPTG conditions. This differential growth phenotype was exploited to bias the cell-based screening hits toward specific inhibitors of mevalonate-dependent IPP biosynthesis. Screens were run against strains engineered for regulation of the enzymes HMG-CoA synthase (MvaS) and mevalonate kinase (mvaK1), mevalonate diphosphate decarboxylase (mvaD), and phosphomevalonate kinase (mvaK2). The latter three enzymes are regulated as an operon. These assays resulted in the discovery of potent antibacterial hits that were progressed to an active hit-to-lead program. The example presented here demonstrates that a cell sensitization strategy can be successfully applied to a 1.3-million compound high-throughput screen in a high-density 1536-well format.

The volatile metabolome of grapevine roots: first insights into the metabolic response upon phylloxera attack.

Many plant species respond to herbivore attack by an increased formation of volatile organic compounds. In this preliminary study we analysed the volatile metabolome of grapevine roots [Teleki 5C (Vitis berlandieri Planch. × Vitis riparia Michx.)] with the aim to gain insight into the interaction between phylloxera (Daktulosphaira vitifoliae Fitch; Hemiptera: Phylloxeridae) and grapevine roots. In the first part of the study, headspace solid phase microextraction (HS-SPME) coupled to gas chromatography – mass spectrometry (GC–MS) was used to detect and identify volatile metabolites in uninfested and phylloxera-infested root tips of the grapevine rootstock Teleki 5C. Based on the comparison of deconvoluted mass spectra with spectra databases as well as experimentally derived retention indices with literature values, 38 metabolites were identified, which belong to the major classes of plant volatiles including C6-compounds, terpenes (including modified terpenes), aromatic compounds, alcohols and n-alkanes. Based on these identified metabolites, changes in root volatiles were investigated and resulted in metabolite profiles caused by phylloxera infestation. Our preliminary data indicate that defence related pathways such as the mevalonate and/or alternative isopentenyl pyrophosphate-, the lipoxygenase- (LOX) as well as the phenylpropanoid pathway are affected in root galls as a response to phylloxera attack.

UNRAVELING THE MYSTERY OF NATURAL RUBBER BIOSYNTHESIS. PART I: INVESTIGATION OF THE COMPOSITION AND GROWTH OF IN VITRO NATURAL RUBBER USING HIGH RESOLUTION SIZE EXCLUSION CHROMATOGRAPHY

Abstract Monitoring the growth of in vitro natural rubber was accomplished by high resolution size exclusion chromatography, SEC. Washed rubber particles isolated from H. brasiliensis latex, containing the rubber transferase enzyme, were used to catalyze the polymerization of synthetic isopentenyl pyrophosphate monomer in the presence of farnesyl pyrophosphate initiator. The high-resolution SEC was able to detect the formation of new rubber. Changes in the low molecular weight fraction were also detected. Gravimetric analysis revealed ∼30% mass gain after the in vitro synthesis. The overall gel content was found to be reduced, which further supported the formation of new rubber. This is the first report that utilizes high-resolution SEC to monitor the in vitro NR growth without the use of radiolabeling.

Bisphosphonates: The first 40years

The first full publications on the biological effects of the diphosphonates, later renamed bisphosphonates, appeared in 1969, so it is timely after 40years to review the history of their development and their impact on clinical medicine. This special issue of BONE contains a series of review articles covering the basic science and clinical aspects of these drugs, written by some of many scientists who have participated in the advances made in this field. The discovery and development of the bisphosphonates (BPs) as a major class of drugs for the treatment of bone diseases has been a fascinating story, and is a paradigm of a successful journey from 'bench to bedside'. Bisphosphonates are chemically stable analogues of inorganic pyrophosphate (PPi), and it was studies on the role of PPi as the body's natural 'water softener' in the control of soft tissue and skeletal mineralisation that led to the need to find inhibitors of calcification that would resist hydrolysis by alkaline phosphatase. The observation that PPi and BPs could not only retard the growth but also the dissolution of hydroxyapatite crystals prompted studies on their ability to inhibit bone resorption. Although PPi was unable to do this, BPs turned out to be remarkably effective inhibitors of bone resorption, both in vitro and in vivo experimental systems, and eventually in humans. As ever more potent BPs were synthesised and studied, it became apparent that physico-chemical effects were insufficient to explain their biological effects, and that cellular actions must be involved. Despite many attempts, it was not until the 1990s that their biochemical actions were elucidated. It is now clear that bisphosphonates inhibit bone resorption by being selectively taken up and adsorbed to mineral surfaces in bone, where they interfere with the action of the bone-resorbing osteoclasts. Bisphosphonates are internalised by osteoclasts and interfere with specific biochemical processes. Bisphosphonates can be classified into at least two groups with different molecular modes of action. The simpler non-nitrogen containing bisphosphonates (such as etidronate and clodronate) can be metabolically incorporated into non-hydrolysable analogues of ATP, which interfere with ATP-dependent intracellular pathways. The more potent, nitrogen-containing bisphosphonates (including pamidronate, alendronate, risedronate, ibandronate and zoledronate) are not metabolised in this way but inhibit key enzymes of the mevalonate/cholesterol biosynthetic pathway. The major enzyme target for bisphosphonates is farnesyl pyrophosphate synthase (FPPS), and the crystal structure elucidated for this enzyme reveals how BPs bind to and inhibit at the active site via their critical N atoms. Inhibition of FPPS prevents the biosynthesis of isoprenoid compounds (notably farnesol and geranylgeraniol) that are required for the post-translational prenylation of small GTP-binding proteins (which are also GTPases) such as rab, rho and rac, which are essential for intracellular signalling events within osteoclasts. The accumulation of the upstream metabolite, isopentenyl pyrophosphate (IPP), as a result of inhibition of FPPS may be responsible for immunomodulatory effects on gamma delta (γδ) T cells, and can also lead to production of another ATP metabolite called ApppI, which has intracellular actions. Effects on other cellular targets, such as osteocytes, may also be important. Over the years many hundreds of BPs have been made, and more than a dozen have been studied in man. As reviewed elsewhere in this issue, bisphosphonates are established as the treatments of choice for various diseases of excessive bone resorption, including Paget's disease of bone, the skeletal complications of malignancy, and osteoporosis. Several of the leading BPs have achieved 'block-buster' status with annual sales in excess of a billion dollars. As a class, BPs share properties in common. However, as with other classes of drugs, there are obvious chemical, biochemical, and pharmacological differences among the various BPs. Each BP has a unique profile in terms of mineral binding and cellular effects that may help to explain potential clinical differences among the BPs. Even though many of the well-established BPs have come or are coming to the end of their patent life, their use as cheaper generic drugs is likely to continue for many years to come. Furthermore in many areas, e.g. in cancer therapy, the way they are used is not yet optimised. New 'designer' BPs continue to be made, and there are several interesting potential applications in other areas of medicine, with unmet medical needs still to be fulfilled. The adventure that began in Davos more than 40 years ago is not yet over.

Indirect stimulation of human Vγ2Vδ2 T cells through alterations in isoprenoid metabolism: implications for cancer immunotherapy (48.14)

Abstract Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the foreign MEP pathway used by protozoa and bacteria, and isopentenyl pyrophosphate (IPP), an intermediate in the self mevalonate pathway. Synthetic bisphosphonates (BPH), used for cancer immunotherapy, and alkylamines indirectly stimulate Vγ2Vδ2 T cells by inhibiting self farnesyl diphosphate synthase (FDPS), thereby increasing IPP which is directly stimulatory. Here, we further characterize stimulation by these compounds, and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by BPH and alkylamines was much more sensitive to upstream statin inhibition than stimulation by phosphoantigens or mitogens. However, the continuous presence of BPH or statins was toxic, blocking Vγ2Vδ2 T cell proliferation but not cytokine release. SiRNA downregulation of FDPS in APC increased IPP and rendered them stimulatory for Vγ2Vδ2 cells. SiRNAs downregulation of isopentenyl diphosphate isomerase functioned similarly, albeit less potently. New stimulatory compounds-mevalonate and the alcohol of HMBPP-may also stimulate indirectly, but required high statin doses for inhibition. The critical chemical feature of BPH is the amino moiety, since BPH lacking amino moieties acted as direct antigens. Thus, a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells.

Expression of three isoprenoid biosynthesis genes and their effects on the carotenoid production of the zygomycete Mucor circinelloides

The zygomycete Mucor circinelloides accumulates β-carotene as the main carotenoid compound. In this study, the applicability of some early genes of the general isoprenoid pathway to improve the carotenoid production in this fungus was examined. The isopentenyl pyrophosphate isomerase gene ( ipi) was cloned and used together with the genes encoding farnesyl pyrophosphate synthase ( isoA) and geranylgeranyl pyrophosphate synthase ( carG) in overexpression studies. Transformation experiments showed that the first bottleneck in the pathway, from the aspect of carotenoid production, is the step controlled by the carG gene, but overexpression of the ipi and isoA genes also contributes to the availability of the precursors. Transformations with these isoprenoid genes in combination with a bacterial β-carotene ketolase gene yielded Mucor strains producing canthaxanthin and echinenone.

Correlation between time-dependent inhibition of human farnesyl pyrophosphate synthase and blockade of mevalonate pathway by nitrogen-containing bisphosphonates in cultured cells

A class of drugs successfully used for treatment of metabolic bone diseases is the nitrogen-containing bisphosphonates (N-BPs), which act by inhibiting the vital enzyme, farnesyl pyrophosphate synthase (FPPS), of the mevalonate pathway. Inhibition of FPPS by N-BPs results in the intracellular accumulation of isopentenyl pyrophosphate (IPP) and consequently induces the biosynthesis of a cytotoxic ATP analog (ApppI). Previous cell-free data has reported that N-BPs inhibit FPPS by time-dependent manner as a result of the conformational change. This associated conformational change can be measured as an isomerization constant (Kisom) and reflects the binding differences of the N-BPs to FPPS. In the present study, we tested the biological relevance of the calculated Kisom values of zoledronic acid, risedronate and five experimental N-BP analogs in the cell culture model. We used IPP/ApppI formation as a surrogate marker for blocking of FPPS in the mevalonate pathway.As a result, a correlation between the time-dependent inhibition of FPPS and IPP/ApppI formation by N-BPs was observed. This outcome indicates that the time-dependent inhibition of FPPS enzyme is a biologically significant mechanism and further supports the use of the Kisom calculations for evaluation of the overall potency of the novel FPPS inhibitors. Additionally, data illustrates that IPP/ApppI analysis is a useful method to monitor the intracellular action of drugs and drug candidates based on FPPS inhibition.

γδT cells in Juvenile Idiopathic Arthritis: Higher Percentages of Synovial Vδ1+ and Vγ9+ T Cell Subsets Are Associated with Milder Disease

To analyze γδT cell subsets in peripheral blood (PB) and synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA), and to correlate γδT cell subsets with clinical characteristics. γδT cell subsets as percentages of CD3+ T cells in samples of PB (n = 25) and SF (n = 93) were analyzed by flow cytometry in 93 JIA patients. The percentage of Vγ9+ γδT cells after 10 days of in vitro expansion with either interleukin 2 (IL-2) or isopentenyl pyrophosphate (IPP) plus IL-2 was determined. Both Vδ1+ and Vγ9+ γδT cell subsets were detected in SF of all patients, but only the percentage of Vδ1+ cells was higher in SF compared to PB (p < 0.01). The distribution of γδT cell subsets was similar in different JIA subgroups, whereas antinuclear antibody (ANA)-positive patients had a higher percentage of SF Vδ1+ T cells than ANA-negative patients (p < 0.01). The percentage of SF Vδ1+ T cells was inversely associated with age at onset, recurrence of synovitis, and erythrocyte sedimentation rate; and that of SF Vγ9+ T cells was inversely correlated with age at onset and was higher in patients who recovered from disease (n = 15). IPP-induced expansion of SF Vγ9+ T cells correlated with disease remission, whereas the expansion of SF Vγ9+ T cells in media with IL-2 alone was significantly greater in patients with uveitis. The percentage of Vδ1+ and Vγ9+ γδT cells among the SF T cells and their ability to respond to IPP or IL-2 correlated with specific outcomes of JIA, suggesting their role in the immunopathogenesis of this disease.

Isopentenyl pyrophosphate is a novel antinociceptive substance that inhibits TRPV3 and TRPA1 ion channels

Transient receptor potential ion channels (TRPs) expressed in the periphery sense and electrically transduce noxious stimuli to transmit the signals to the brain. Many natural and synthetic ligands for the sensory TRPs have been found, but little is known about endogenous inhibitors of these TRP channels. Recently, we reported that farnesyl pyrophosphate, an endogenous substance produced in the mevalonate pathway, is a specific activator for TRPV3. Here, we show that isopentenyl pyrophosphate (IPP), an upstream metabolite in the same pathway, is a dual inhibitor for TRPA1 and TRPV3. By using Ca 2+ imaging and voltage clamp experiments with human embryo kidney cell heterologous expression system, cultured sensory neurons, and epidermal keratinocytes, we demonstrate that micromolar IPP suppressed responses to specific agonists of TRPA1 and TRPV3. Consistently, peripheral IPP administration attenuated TRPA1 and TRPV3 agonist-specific acute pain behaviors. Furthermore, local IPP pretreatment significantly reversed mechanical and thermal hypersensitivity of inflamed animals. Taken together, the present study suggests that IPP is a novel endogenous TRPA1 and TRPV3 inhibitor that causes local antinociception. Our results may provide useful chemical information to elucidate TRP physiology in peripheral pain sensation. Isopentenyl pyrophosphate, a substance generated in the mevalonate metabolism, inhibits TRPA1 and TRPV3, and it results in peripheral antinociception in a receptor-dependent manner.