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Biomedical subjects

Keith Thompson

Publications and source records attributed to Keith Thompson.

15 recordsLinked to original sources
PubMed2006

Molecular mechanisms of action of bisphosphonates: current status.

PURPOSE: Bisphosphonates are currently the most important class of antiresorptive agents used in the treatment of metabolic bone diseases, including tumor-associated osteolysis and hypercalcemia. These compounds have high affinity for calcium ions and therefore target bone mineral, where they are internalized by bone-resorbing osteoclasts and inhibit osteoclast function. EXPERIMENTAL DESIGN: This article reviews the pharmacology of bisphosphonates and the relationship between chemical structure and antiresorptive potency. We also describe new insights into their intracellular molecular mechanisms of action, methods for assessing the effects of bisphosphonates on protein prenylation, and their potential as direct antitumor agents. RESULTS: Nitrogen-containing bisphosphonates act intracellularly by inhibiting farnesyl diphosphate synthase, an enzyme of the mevalonate pathway, thereby preventing prenylation of small GTPase signaling proteins required for normal cellular function. Inhibition of farnesyl diphosphate synthase also seems to account for their antitumor effects observed in vitro and for the activation of gamma,delta T cells, a feature of the acute-phase response to bisphosphonate treatment in humans. Bisphosphonates that lack a nitrogen in the chemical structure do not inhibit protein prenylation and have a different mode of action that seems to involve primarily the formation of cytotoxic metabolites in osteoclasts. CONCLUSIONS: Bisphosphonates are highly effective inhibitors of bone resorption that selectively affect osteoclasts in vivo but could also have direct effects on other cell types, such as tumor cells. After >30 years of clinical use, their molecular mechanisms of action on osteoclasts are finally becoming clear but their exact antitumor properties remain to be clarified.

Animals↗
PubMed2006

Observations of Si field evaporation.

Field evaporation studies of crystalline <100> Si were performed in a three-dimensional atom-probe, which utilized a local electrode geometry. Several distinct phenomena were observed. Si field evaporation rates showed: (1) no measurable dependence on temperature below 110K, (2) an exponential dependence on evaporation rate as a function of temperature above 110K, and (3) no dependence on substrate doping (i.e., electrical conductivity) as high as 10 Omega cm in the temperature range of 40-150K. Two distinct evaporation modes were observed. The first was associated with approximately 1at% H+ in the mass spectrum. Negligible amounts of H were detected in the mass spectra of the second mode. When the pulse fraction (pf) was increased from 5% to 30%, the presence of H+ in the mass spectra, i.e. operation in the first mode, was associated with a degradation in mass resolution by as much as 80% for the 10 Omega cm Si samples. Conversely, no loss in mass resolution was detected for the approximately 0.001 Omega cm samples over the pf range studied.

Journal Article↗
PubMed2006

Recent advances in understanding the mechanism of action of bisphosphonates.

Bisphosphonates (BPs) are widely used in the treatment of diseases associated with excessive osteoclast-mediated bone resorption, such as osteoporosis. Although several years ago the molecular target of the potent nitrogen-containing BPs (N-BPs) was identified as farnesyl diphosphate synthase, an enzyme in the mevalonate pathway, recent data have shed new light on the precise mechanism of inhibition and demonstrated that the acute-phase reaction, an adverse effect of N-BPs, is also caused by inhibition of this enzyme. In addition, the identification of BP analogues that inhibit different enzymes in the mevalonate pathway could lead to the development of novel inhibitors of bone resorption with potential applications in the treatment of bone disease.

Acute-Phase Reaction↗
PubMed2006

Cytosolic entry of bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis.

Bisphosphonates such as alendronate and zoledronate are blockbuster drugs used to inhibit osteoclast-mediated bone resorption. Although the molecular mechanisms by which bisphosphonates affect osteoclasts are now evident, the exact route by which they are internalized by cells is not known. To clarify this, we synthesized a novel, fluorescently labeled analog of alendronate (AF-ALN). AF-ALN was rapidly internalized into intracellular vesicles in J774 macrophages and rabbit osteoclasts; uptake of AF-ALN or [14C]zoledronate was stimulated by the presence of Ca2+ and Sr2+ and could be inhibited by addition of EGTA or clodronate, both of which chelate calcium ions. Both EGTA and clodronate also prevented the bisphosphonate-induced inhibition of Rap1A prenylation, an effect that was reversed by addition of Ca2+. In J774 cells and osteoclasts, vesicular AF-ALN colocalized with dextran (but not wheat germ agglutinin or transferrin), and uptake of AF-ALN or [14C]zoledronate was inhibited by dansylcadaverine, indicating that fluid-phase endocytosis is involved in the initial internalization of bisphosphonate into vesicles. Endosomal acidification then seems to be absolutely required for exit of bisphosphonate from vesicles and entry into the cytosol, because monensin and bafilomycin A1, both inhibitors of endosomal acidification, did not inhibit vesicular uptake of AF-ALN or internalization of [14C]zoledronate but prevented the inhibitory effect of alendronate or zoledronate on Rap1A prenylation. Taken together, these results demonstrate that cellular uptake of bisphosphonate drugs requires fluid-phase endocytosis and is enhanced by Ca2+ ions, whereas transfer from endocytic vesicles into the cytosol requires endosomal acidification.

Alendronate↗
PubMed2006

Characterization of a monoclonal antibody with specificity for holo-transcobalamin.

BACKGROUND: Holotranscobalamin, cobalamin-saturated transcobalamin, is the minor fraction of circulating cobalamin (vitamin B12), which is available for cellular uptake and hence is physiologically relevant. Currently, no method allows simple, direct quantification of holotranscobalamin. We now report on the identification and characterization of a monoclonal antibody with a unique specificity for holotranscobalamin. METHODS: The specificity and affinity of the monoclonal antibodies were determined using surface plasmon resonance and recombinant transcobalamin as well as by immobilizing the antibodies on magnetic microspheres and using native transcobalamin in serum. The epitope of the holotranscobalamin specific antibody was identified using phage display and comparison to a de novo generated three-dimensional model of transcobalamin using the program Rosetta. A direct assay for holotrnscobalamin in the ELISA format was developed using the specific antibody and compared to the commercial assay HoloTC RIA. RESULTS: An antibody exhibiting >100-fold specificity for holotranscobalamin over apotranscobalamin was identified. The affinity but not the specificity varied inversely with ionic strength and pH, indicating importance of electrostatic interactions. The epitope was discontinuous and epitope mapping of the antibody by phage display identified two similar motifs with no direct sequence similarity to transcobalamin. A comparison of the motifs with a de novo generated three-dimensional model of transcobalamin identified two structures in the N-terminal part of transcobalamin that resembled the motif. Using this antibody an ELISA based prototype assay was developed and compared to the only available commercial assay for measuring holotranscobalamin, HoloTC RIA. CONCLUSION: The identified antibody possesses a unique specificity for holotranscobalamin and can be used to develop a direct assay for the quantification of holotranscobalamin.

Journal Article↗
PubMed2006

Idiotype-specific immunotherapy in multiple myeloma: suggestions for future directions of research.

Multiple myeloma (MM) remains a difficult-to-cure cancer and less than 20% of patients achieve long-term survival irrespective of the treatment delivered, including high-dose chemotherapy. Thus, new treatment modalities are urgently needed. Myeloma cells produce a monoclonal immunoglobulin (Ig) which is a truly tumor-specific antigen. The tumor-specific antigenic determinants are localized in the variable regions of the monoclonal Ig and are termed idiotopes (Id). Id-vaccination, i.e., vaccination with the autologous monoclonal Ig, has been performed in MM patients in order to elicit tumor-specific immune responses and possibly elimination of myeloma cells. However, clinical trials have not given the promising results obtained in mice. This review focuses on tolerance mechanisms that might hinder Id-specific immune responses in MM patients. New strategies for Id vaccination in MM are discussed.

Antigens, Neoplasm↗
PubMed2005

Alkylamines cause Vgamma9Vdelta2 T-cell activation and proliferation by inhibiting the mevalonate pathway.

Three general classes of small, nonpeptide "antigens" activate Vgamma9Vdelta2 T cells: pyrophosphomonoesters, such as isopentenyl diphosphate (IPP), nitrogen-containing bisphosphonates (N-BPs), and alkylamines. However, we have shown recently that N-BPs indirectly activate Vgamma9Vdelta2 T cells as a consequence of inhibition of farnesyl diphosphate synthase (a key enzyme of the mevalonate pathway) and the intracellular accumulation of IPP. We now show that alkylamines activate Vgamma9Vdelta2 T cells by the same mechanism. Alkylamines were found to be weak inhibitors of farnesyl diphosphate synthase and caused accumulation of unprenylated Rap1A in peripheral blood mononuclear cells and macrophages, indicative of inhibition of the mevalonate pathway. Furthermore, as with N-BPs, the stimulatory effect of the alkylamines on Vgamma9Vdelta2T cells was abrogated by simultaneous treatment with mevastatin. These findings suggest that only pyrophosphomonoesters such as IPP are true Vgamma9Vdelta2 T-cell agonists, whereas alkylamines and N-BPs indirectly activate Vgamma9Vdelta2 T cells through a common mechanism involving the accumulation of IPP.

Animals↗
PubMed2005

Modified electroporation protocol for Lactobacilli isolated from the chicken crop facilitates transformation and the use of a genetic tool.

Isolates of Lactobacillus spp. from a collection of potentially probiotic strains isolated from the crops of broiler chickens were found to be non-electrotransformable using published techniques. One strain of Lactobacillus salivarius was shown to develop electrocompetence when an overnight culture was incubated in fresh medium. The effect was enhanced if glycine was incorporated into the fresh growth medium. When these modifications were applied to a number of other crop isolates of Lactobacillus spp., electrocompetence could be detected in approximately half the strains tested. Two temperature sensitive plasmid vectors that had been used for the genetic modification of other lactic acid bacteria were introduced into a crop strain of Lb. salivarius. Both showed temperature sensitivity at 42 degrees C and above but were relatively stable at 37 degrees C. The genetic tool harbouring an IS element allowed the delivery of the plasmid to multiple independent sites in the host chromosome. Harnessing such genetic tools will facilitate the future genetic analysis of the host bacterium.

Animals↗
PubMed2005

Identification and characterization of an immunogenic 22 kDa exported protein of Mycobacterium avium subspecies paratuberculosis.

An exported 22 kDa putative lipoprotein was identified in an alkaline phosphatase gene fusion library of Mycobacterium avium subsp. paratuberculosis and expressed in Mycobacterium smegmatis. The full nucleic acid sequence of the gene encoding P22 was determined and the ORF was cloned into a mycobacterial expression vector, enabling full-length P22 to be produced as a C-terminal polyhistidine-tagged protein in M. smegmatis. N-terminal sequencing of the recombinant protein confirmed cleavage of a signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. avium subsp. paratuberculosis strain 316F using rabbit antibody raised to recombinant P22. Investigation of the presence of similar genes in other mycobacterial species revealed that the gene was present in Mycobacterium avium subsp. avium and similar genes existed in Mycobacterium intracellulare and Mycobacterium scrofulaceum. Database searches showed that P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in Mycobacterium leprae and in members of the Mycobacterium tuberculosis complex. P22 shared less than 75% identity to these proteins. Recombinant P22 was able to elicit interferon-gamma secretion in blood from eight of a group of nine sheep vaccinated with a live attenuated strain of M. avium subsp. paratuberculosis (strain 316F) compared to none from a group of five unvaccinated sheep. Antibody to P22 was detected by Western blot analysis in 10 out of 11 vaccinated sheep, in two out of two clinically affected cows and in 11 out of 13 subclinically infected cows.

Animals↗
PubMed2005

Mapping the functional domains of human transcobalamin using monoclonal antibodies.

Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.

Amino Acid Sequence↗
PubMed2003

Statins prevent bisphosphonate-induced gamma,delta-T-cell proliferation and activation in vitro.

UNLABELLED: The acute phase response is the major adverse effect of intravenously administered N-BPs. In this study we show that N-BPs cause gamma,delta-T-cell activation and proliferation in vitro by an indirect mechanism through inhibition of FPP synthase, an effect that can be overcome by inhibiting HMG-CoA reductase with a statin. These studies clarify the probable initial cause of the acute phase response to N-BP drugs and suggest a possible way of preventing this phenomenon. INTRODUCTION: The acute phase response is the major adverse effect of intravenously administered nitrogen-containing bisphosphonate drugs (N-BPs), used in the treatment of metabolic bone diseases. This effect has recently been attributed to their action as non-peptide antigens and direct stimulation of gamma,delta-T-cells. However, because N-BPs are potent inhibitors of farnesyl diphosphate (FPP) synthase, they could cause indirect activation of gamma,delta-T-cells owing to the accumulation of intermediates upstream of FPP synthase in the mevalonate pathway, such as isopentenyl diphosphate/dimethylallyl diphosphate, which are known gamma,delta-T-cell agonists. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers and treated with N-BP, statin, or intermediates/inhibitors of the mevalonate pathway for 7 days in the presence of interleukin (IL)-2. Flow cytometric analysis of the T-cell-gated population was used to quantify the proportion of gamma,delta-T-cells in the CD3+ population. RESULTS AND CONCLUSIONS: The ability of N-BPs to stimulate proliferation of CD3+ gamma,delta-T-cells in human PBMC cultures matched the ability to inhibit FPP synthase. Gamma,delta-T-cell proliferation and activation (interferon gamma [IFNgamma] and TNFalpha release) was prevented by mevastatin or lovastatin, which inhibit HMG-CoA reductase upstream of FPP synthase and prevent the synthesis of isopentenyl diphosphate/dimethylallyl diphosphate. Desoxolovastatin, an analog of lovastatin incapable of inhibiting HMG-CoA reductase, did not overcome the stimulatory effect of N-BP. Furthermore, statins did not prevent the activation of gamma,delta-T-cells by a synthetic gamma,delta-T-cell agonist or by anti-CD3 antibody. Together, these observations show that N-BPs indirectly stimulate the proliferation and activation of gamma,delta-T-cells caused by inhibition of FPP synthase and intracellular accumulation of isopentenyl diphosphate/ dimethylallyl diphosphate in PBMCs. Because activation of gamma,delta-T-cells could be the initiating event in the acute phase response to bisphosphonate therapy, co-administration of a statin could be an effective approach to prevent this adverse effect.

Alkyl and Aryl Transferases↗
PubMed2002

Identification of a bisphosphonate that inhibits isopentenyl diphosphate isomerase and farnesyl diphosphate synthase.

We and others have recently shown that the major molecular target of nitrogen-containing bisphosphonate drugs is farnesyl diphosphate synthase, an enzyme in the mevalonate pathway. In an in vitro screen, we discovered a bisphosphonate, NE21650, that potently inhibited farnesyl diphosphate synthase but, unlike other N-BPs investigated, was also a weak inhibitor of isopentenyl diphosphate isomerase. NE21650 was a more potent inhibitor of protein prenylation in osteoclasts and macrophages, and a more potent inhibitor of bone resorption in vitro, than alendronate, despite very similar IC(50) values for inhibition of farnesyl diphosphate synthase. Our observations show that minor changes to the structure of bisphosphonates allow inhibition of more than one enzyme in the mevalonate pathway and suggest that loss of protein prenylation due to inhibition of more than one enzyme in the mevalonate pathway may lead to an increase in antiresorptive potency compared to bisphosphonates that only inhibit farnesyl diphosphate synthase.

Alendronate↗
PubMed2002

Pupil location under mesopic, photopic, and pharmacologically dilated conditions.

PURPOSE: To determine whether there are systematic changes in pupil location with changes in the state of pupil size and with other ocular variables. METHODS: High-resolution images of the pupil of the eyes of 70 subjects were taken using an infrared-sensitive camera. Images were obtained under mesopic, photopic, and pharmacologically dilated conditions. From the images, the center and diameter of the corneal limbus and the pupil were computed. In addition, the location of the first Purkinje image was calculated. RESULTS: The pupil center shifted consistently temporally as the pupil dilated. The total motion was relatively small, with a mean distance of 0.133 mm motion between the mesopic and photopic conditions, with the pupil diameter changing from 6.3 to 4.1 mm. Ninety percent of the subjects had a motion of less than 0.3 mm. One patient showed a motion of almost 0.6 mm. The change in location of the pupil center was not significantly related to refractive error, age, or the change of pupil diameter. CONCLUSIONS: Changes in the location of the pupil center with changes in the dilation of the pupil are typically slight, but can be significant in a few subjects, especially in pharmacologically dilated pupils.

Adaptation, Ocular↗