Exploratory human PET study of the effectiveness of (11)C-ketoprofen methyl ester, a potential biomarker of neuroinflammatory processes in Alzheimer's disease.

INTRODUCTION: Neuroinflammatory processes play an important role in the pathogenesis of Alzheimer's disease (AD). As a biomarker of neuroinflammatory processes, we designed (11)C-labeled ketoprofen methyl ester ([(11)C]KTP-Me) to increase the blood-brain barrier permeability of ketoprofen (KTP), a selective cyclooxygenase-1 (COX-1) inhibitor. Animal studies indicated that [(11)C]KTP-Me enters the brain and accumulates in activated microglia of inflammatory lesions. In a first-in-human study, we reported that [(11)C]KTP-Me is a safe positron emission tomography (PET) tracer and enters the brain; the radioactivity is washed out from normal cerebral tissue. Here we explored the efficacy of [(11)C]KTP-Me as a diagnostic biomarker of neuroinflammatory processes in AD. METHODS: [(11)C]KTP-Me was synthesized by rapid C-[(11)C]methylation of [(11)C]CH3I and the corresponding arylacetate precursor. Nine subjects (four healthy subjects, two Pittsburgh compound-B (PiB)-positive patients with mild cognitive impairment (MCI), and three PiB-positive AD patients) underwent a dynamic brain PET scan for 70min after injection. We evaluated differences in cortical retention and washout rate in the brain between healthy subjects and MCI/AD patients. RESULTS: A brain distribution pattern reflecting blood flow in the early-phase image was seen in both healthy subjects and MCI/AD patients. Cortical activity gradually cleared in all groups. However, we observed no obvious difference in the washout rate between healthy subjects and MCI/AD patients or between MCI and AD patients. CONCLUSIONS: [(11)C]KTP-Me cannot be useful as a potential diagnostic biomarker for MCI/AD. Further improvements in binding affinity and specificity, etc., are needed to be a diagnostic biomarker of neuroinflammation in AD. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: [(11)C]KTP-Me is a new tracer that targets COX-1. [(11)C]KTP-Me is expected to be a diagnostic biomarker of neuroinflammation in AD in the future. The effectiveness was limited in a small number of AD patients. Therefore, further studies are needed to clarify the usefulness of [(11)C]KTP-Me.

Human whole-body biodistribution and dosimetry of a new PET tracer, [(11)C]ketoprofen methyl ester, for imagings of neuroinflammation.

INTRODUCTION: Neuroinflammatory processes play an important role in the pathogenesis of Alzheimer's disease and other brain disorders, and nonsteroidal anti-inflammatory drugs (NSAIDs) are considered therapeutic candidates. As a biomarker of neuroinflammatory processes, (11)C-labeled ketoprofen methyl ester ([(11)C]KTP-Me) was designed to allow cerebral penetration of ketoprofen (KTP), an active form of a selective cyclooxygenase-1 inhibitor that acts as an NSAID. Rat neuroinflammation models indicate that [(11)C]KTP-Me enters the brain and is retained in inflammatory lesions, accumulating in activated microglia. [(11)C]KTP-Me is washed out from normal tissues, leading to the present first-in-human exploratory study. METHODS: [(11)C]KTP-Me was synthesized by rapid C-[(11)C]methylation of [(11)C]CH3I and the corresponding arylacetate precursor, purified with high-performance liquid chromatography, and prepared as an injectable solution including PEG400, providing radiochemical purity of >99% and specific activity of >25GBq/µmol at injection. Six young healthy male humans were injected with [(11)C]KTP-Me and scanned with PET camera to determine the early-phase brain time course followed by three whole-body scans starting 8, 20, and 40 min post-injection, together with sequential blood sampling and labeled metabolite analysis. RESULTS: No adverse effects were observed during PET scanning after [(11)C]KTP-Me injection. [(11)C]KTP-Me was rapidly metabolized to (11)C-labeled ketoprofen ([(11)C]KTP) within 2-3 min and was gradually cleared from blood. The radioactivity entered the brain with an average peak cortical SUV of 1.5 at 2 min. The cortical activity was gradually washed out. Whole-body images indicated that the urinary bladder was the major excretory pathway. The organ with the highest radiation dose was the urinary bladder (average dose of 41µGy/MBq, respectively). The mean effective dose was 4.7µSv/MBq, which was comparable to other (11)C-labeled radiopharmaceuticals. CONCLUSION: [(11)C]KTP-Me demonstrated a favorable dosimetry, biodistribution, and safety profile. [(11)C]KTP-Me entered the human brain, and the radioactivity was washed out from cerebral tissue. These data warrant further exploratory studies on patients with neuroinflammation.

[Importance of three dimensional-fusion image after stent-assisted coil embolization of unruptured aneurysm].

PURPOSE: We made the fusion image of both stent and platinum coil after embolization of an unruptured aneurysm. METHOD: After scanning with cone beam computed tomography, we made three dimensional (3D) images of stent and coil and fused them. CONCLUSION: We can evaluate unruptured aneurysm after embolization by using a fusion image. 3D-fusion image is useful on clinical cases.

Molecular evolution of vertebrate Toll-like receptors: evolutionary rate difference between their leucine-rich repeats and their TIR domains.

Toll-like receptors (TLRs) that initiate an innate immune response contain an extracellular leucine rich repeat (LRR) domain and an intracellular Toll IL-receptor (TIR) domain. There are fifteen different TLRs in vertebrates. The LRR domains, which adopt a solenoid structure, usually have higher rates of evolution than do the TIR globular domains. It is important to understand the molecular evolution and functional roles of TLRs from this standpoint. Both pairwise genetic distances and Ka/Ks's (the ratios between non synonymous and synonymous substitution rates) were compared between the LRR domain and the TIR domain of 366 vertebrate TLRs from 96 species (from fish to primates). In fourteen members (TLRs 1, 2, 3, 4, 5, 6, 7, 8, 9, 11/12, 13, 14, 21, and 22/23) the LRR domains evolved significantly more rapidly than did the corresponding TIR domains. The evolutionary rates of the LRR domains are significantly different among these members; LRR domains from TLR3 and TLR7 from primates to fishes have the lowest rate of evolution. In contrast, the fifteenth member, TLR10, shows no significant differences; its TIR domain is not highly conserved. The present results suggest that TLR10 may have a different function in signaling from those other members and that a higher conservation of TLR3 and TLR7 may reflect a more ancient mechanism and/or structure in the innate immune response system. Gene conversions are suggested to have occurred in platypus TLR6 and TLR10. This study provides new insight about structural and functional diversification of vertebrate TLRs.

Ablation of TSC2 enhances insulin secretion by increasing the number of mitochondria through activation of mTORC1.

AIM: We previously found that chronic tuberous sclerosis protein 2 (TSC2) deletion induces activation of mammalian target of rapamycin Complex 1 (mTORC1) and leads to hypertrophy of pancreatic beta cells from pancreatic beta cell-specific TSC2 knockout (ßTSC2(-/-)) mice. The present study examines the effects of TSC2 ablation on insulin secretion from pancreatic beta cells. METHODS: Isolated islets from ßTSC2(-/-) mice and TSC2 knockdown insulin 1 (INS-1) insulinoma cells treated with small interfering ribonucleic acid were used to investigate insulin secretion, ATP content and the expression of mitochondrial genes. RESULTS: Activation of mTORC1 increased mitochondrial DNA expression, mitochondrial density and ATP production in pancreatic beta cells of ßTSC2(-/-) mice. In TSC2 knockdown INS-1 cells, mitochondrial DNA expression, mitochondrial density and ATP production were increased compared with those in control INS-1 cells, consistent with the phenotype of ßTSC2(-/-) mice. TSC2 knockdown INS-1 cells also exhibited augmented insulin secretory response to glucose. Rapamycin inhibited mitochondrial DNA expression and ATP production as well as insulin secretion in response to glucose. Thus, ßTSC2(-/-) mice exhibit hyperinsulinemia due to an increase in the number of mitochondria as well as enlargement of individual beta cells via activation of mTORC1. CONCLUSION: Activation of mTORC1 by TSC2 ablation increases mitochondrial biogenesis and enhances insulin secretion from pancreatic beta cells.

A nested leucine rich repeat (LRR) domain: the precursor of LRRs is a ten or eleven residue motif.

BACKGROUND: Leucine rich repeats (LRRs) are present in over 60,000 proteins that have been identified in viruses, bacteria, archae, and eukaryotes. All known structures of repeated LRRs adopt an arc shape. Most LRRs are 20-30 residues long. All LRRs contain LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. Seven classes of LRRs have been identified. However, other LRR classes remains to be characterized. The evolution of LRRs is not well understood. RESULTS: Here we describe a novel LRR domain, or nested repeat observed in 134 proteins from 54 bacterial species. This novel LRR domain has 21 residues with the consensus sequence of LxxLxLxxNxLxxLDLxx(N/L/Q/x)xx or LxxLxCxxNxLxxLDLxx(N/L/x)xx. This LRR domain is characterized by a nested periodicity; it consists of alternating 10- and 11- residues units of LxxLxLxxNx(x/-). We call it "IRREKO" LRR, since the Japanese word for "nested" is "IRREKO". The first unit of the "IRREKO" LRR domain is frequently occupied by an "SDS22-like" LRR with the consensus of LxxLxLxxNxLxxLxxLxxLxx or a "Bacterial" LRR with the consensus of LxxLxLxxNxLxxLPxLPxx. In some proteins an "SDS22-like" LRR intervenes between "IRREKO" LRRs. CONCLUSION: Proteins having "IRREKO" LRR domain are almost exclusively found in bacteria. It is suggested that IRREKO@LRR evolved from a common ancestor with "SDS22-like" and "Bacterial" classes and that the ancestor of IRREKO@LRR is 10 or 11 residues of LxxLxLxxNx(x/-). The "IRREKO" LRR is predicted to adopt an arc shape with smaller curvature in which ß-strands are formed on both concave and convex surfaces.

Analyses of non-leucine-rich repeat (non-LRR) regions intervening between LRRs in proteins.

BACKGROUND: Many proteins have LRR (leucine-rich repeat) units interrupted by non-LRRs which we call IR (non-LRR island region). METHODS: We identified proteins containing LRR@IRs (LRRs having IR) by using a new method and then analyzed their natures and distributions. RESULTS: LRR@IR proteins were found in over two hundred proteins from prokaryotes and from eukaryotes. These are divided into twenty-one different protein families. The IRs occur one to four times in LRR regions and range in length from 5 to 11,265 residues. The IR lengths in Fungi adenylate cyclases (acys) range from 5 to 116 residues; there are 22 LRR repeats. The IRs in Leishmania proteophosphoglycans (ppgs) vary from 105 to 11,265 residues. These results indicate that the IRs evolved rapidly. A group of LRR@IR proteins-LRRC17, chondroadherin-like protein, ppgs, and four Pseudomonas proteins-have a super motif consisting of an LRR block and its adjacent LRR@IR region. This indicates that the entire super motif experienced duplication. The sequence analysis of IRs offers functional similarity in some LRR@IR protein families. GENERAL SIGNIFICANCE: This study suggests that various IRs and super motifs provide a great variety of structures and functions for LRRs.

[Biochemical and bacteriological investigation of six cases of purple urine bag syndrome (PUBS) in a geriatric ward for dementia].

AIM: Purple urine bag syndrome is a condition in which the urinary catheter bag turns purple. A tryptophan-indigo hypothesis has been proposed as the mechanism of PUBS, in which bacterial decomposition of tryptophan in gut associated with chronic constipation, bacterial overgrowth in the urinary tract and alkaline urine causes production of indigo and discoloration. We considered that further investigation of cases was needed. METHODS: We investigated 6 cases exhibiting PUBS (3 males and 3 females). RESULTS: All cases had chronic constipation. Oral ingestion was impossible in one case. PUBS disappeared after antibiotic treatment (3 cases) or spontaneously (one case). Alkaline urine and indicanuria were not found in all cases that showed the disappearance of PUBS. In bacterial culture of urine during the exhibition of PUBS, Enterococcus faecalis was isolated together with Morganella morganii (3 cases) and Pseudomonas aeruginosa (one case). Single infections by Klebsiella pneumoniae or Citrobacter species were also found. After disappearance of PUBS, infected bacterial species changed but no cases showed sterile urine. Urine and blood alpha-amino-n-butyric acid levels reduced after the disappearance of PUBS whereas tryptophan levels did not show related changes. In one case, blood protein concentration increased after the spontaneous disappearance of PUBS. Indicanuria and alkalization of urine from urinary catheter bag were more intense than of fresh urine. CONCLUSIONS: The present results generally support the 'Tryptophan-indigo hypothesis'. Furthermore, it was suggested that additional factors associated with the occurrence of PUBS are an environment that facilitates specific bacterial growth in a hospital as well as abnormal metabolism relating to alpha-amino-n-butyric acid and reduced protein synthesis in patients.

Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors.

BACKGROUND: Toll-like receptors (TLRs) play a central role in innate immunity. TLRs are membrane glycoproteins and contain leucine rich repeat (LRR) motif in the ectodomain. TLRs recognize and respond to molecules such as lipopolysaccharide, peptidoglycan, flagellin, and RNA from bacteria or viruses. The LRR domains in TLRs have been inferred to be responsible for molecular recognition. All LRRs include the highly conserved segment, LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. There are seven classes of LRRs including "typical" ("T") and "bacterial" ("S"). All known domain structures adopt an arc or horseshoe shape. Vertebrate TLRs form six major families. The repeat numbers of LRRs and their "phasing" in TLRs differ with isoforms and species; they are aligned differently in various databases. We identified and aligned LRRs in TLRs by a new method described here. RESULTS: The new method utilizes known LRR structures to recognize and align new LRR motifs in TLRs and incorporates multiple sequence alignments and secondary structure predictions. TLRs from thirty-four vertebrate were analyzed. The repeat numbers of the LRRs ranges from 16 to 28. The LRRs found in TLRs frequently consists of LxxLxLxxNxLxxLxxxxF/LxxLxx ("T") and sometimes short motifs including LxxLxLxxNxLxxLPx(x)LPxx ("S"). The TLR7 family (TLR7, TLR8, and TLR9) contain 27 LRRs. The LRRs at the N-terminal part have a super-motif of STT with about 80 residues. The super-repeat is represented by STTSTTSTT or _TTSTTSTT. The LRRs in TLRs form one or two horseshoe domains and are mostly flanked by two cysteine clusters including two or four cysteine residue. CONCLUSION: Each of the six major TLR families is characterized by their constituent LRR motifs, their repeat numbers, and their patterns of cysteine clusters. The central parts of the TLR1 and TLR7 families and of TLR4 have more irregular or longer LRR motifs. These central parts are inferred to play a key role in the structure and/or function of their TLRs. Furthermore, the super-repeat in the TLR7 family suggests strongly that "bacterial" and "typical" LRRs evolved from a common precursor.

Determining vulnerability to schizophrenia in methamphetamine psychosis using exploratory eye movements.

Patients with methamphetamine (MAP) psychosis whose psychotic symptoms continued after MAP withdrawal were observed at Saitama Prefecture Government Psychiatric Hospital. The purpose of the present study was to ascertain whether some of these MAP psychosis subjects have a vulnerability to schizophrenia. Forty-eight patients with MAP psychosis were divided into three groups based on clinical course: transient type, prolonged type and persistent type. Furthermore, the patients with the persistent type were divided into two groups: one group were moderately disturbed in social adaptive functioning and had Global Assessment Functioning scale (GAF) points >50, and the other group consisted of those who were severely disturbed in social adaptive functioning and who had GAF points of < or =50. These MAP patients were tested for exploratory eye movements, which are the vulnerability marker of schizophrenia, and were compared with 30 patients with schizophrenia and 30 healthy control subjects. The responsive search score of the severely disturbed group of patients of the persistent type was lowest, significantly lower than those of the transient type and the healthy controls. It did not differ from that of the schizophrenic subjects. These results suggest that the severely disturbed group of patients with the persistent type of MAP psychosis have a vulnerability to schizophrenia.