PET Imaging of Lung Inflammation with [18F]FEDAC, a Radioligand for Translocator Protein (18 kDa)

Purpose

The translocator protein (18 kDa) (TSPO) is highly expressed on the bronchial and bronchiole epithelium, submucosal glands in intrapulmonary bronchi, pneumocytes and alveolar macrophages in human lung. This study aimed to perform positron emission tomography (PET) imaging of lung inflammation with [¹⁸F]FEDAC, a specific TSPO radioligand, and to determine cellular sources enriching TSPO expression in the lung.

Methods

An acute lung injury model was prepared by intratracheal administration of lipopolysaccharide (LPS) to rat. Uptake of radioactivity in the rat lungs was measured with small-animal PET after injection of [¹⁸F]FEDAC. Presence of TSPO was examined in the lung tissue using Western blot and immunohistochemical assays.

Results

The uptake of [¹⁸F]FEDAC increased in the lung with the progress of inflammation by treatment with LPS. Pretreatment with a TSPO-selective ligand PK11195 showed a significant decrease in the lung uptake of [¹⁸F]FEDAC due to competitive binding to TSPO. TSPO expression was elevated in the inflamed lung section and its level responded to the [¹⁸F]FEDAC uptake and severity of inflammation. Increase of TSPO expression was mainly found in the neutrophils and macrophages of inflamed lungs.

Conclusion

From this study we conclude that PET with [¹⁸F]FEDAC may be a useful tool for imaging TSPO expression and evaluating progress of lung inflammation. Study on human lung using [¹⁸F]FEDAC-PET is promising.     

Gene expression and immunolocalisation of a calcium-activated chloride channel during the stratification of cultivated and developing corneal epithelium

The spatial and temporal localisation of a calcium-activated chloride channel (CLCA) and its mRNA was investigated, during the in vivo and in vitro development of stratified epithelia, by fluorescence immunohistochemistry and quantitative polymerase chain reaction in embryonic chicken corneas and the expansion of excised human corneal stem cells on amniotic membrane. Single-layered human epithelial cultures on amniotic membrane and early day embryonic chicken corneas expressed relatively little human CLCA2 or its chicken homologue. However, as the epithelium in both models matured and the number of cell-layers increased, the gene expression level and protein staining intensity increased, primarily within the basal cells of both the cultured and embryonic tissues. These results demonstrate that human CLCA2 protein and mRNA expression are elevated during epithelial stratification, suggesting that this protein plays a role in the growth of multi-layered corneal epithelia during both natural development and tissue cultivation. This work was supported by the Japanese Society for the Promotion of Science (CJC) and The Royal Society (C.J.C.).     

Reduced expression of perchloric acid-soluble protein after partial hepatectomy in rats

We examined the expression of perchloric acid-soluble protein (PSP) during liver regeneration after partial hepatectomy (PH) in rats. Liver regeneration was almost complete at 7-d after PH. Expression of PSP protein and mRNA decreased and then gradually increased during liver regeneration. An immunohistochemical study showed that PSP is distributed in cytosol and nuclei in normal liver, but localization in the nuclei was not be recognized in the regenerated liver.     

Chemical induction of rapid and reversible plastid filamentation in Arabidopsis thaliana roots

Plastids assume various morphologies depending on their developmental status, but the basis for developmentally regulated plastid morphogenesis is poorly understood. Chemical induction of alterations in plastid morphology would be a useful tool for studying this; however, no such chemicals have been identified. Here, we show that antimycin A, an effective respiratory inhibitor, can change plastid morphology rapidly and reversibly in Arabidopsis thaliana. In the root cortex, hypocotyls, cotyledon epidermis and true leaf epidermis, significant differences in mitochondrial morphology were not observed between antimycin‐treated and untreated tissues. In contrast, antimycin caused extreme filamentation of plastids in the mature cortices of main roots. This phenomenon was specifically observed in the mature root cortex. Other mitochondrial respiratory inhibitors (rotenone and carbonyl cyanide m‐chlorophenylhydrazone), hydrogen peroxide, S‐nitroso‐N‐acetylpenicillamine [a nitric oxide (NO) donor] and 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea did not mimic the phenomenon under the present study conditions. Antimycin‐induced plastid filamentation was initiated within 5 min after the onset of chemical treatment and appeared to complete within 1 h. Plastid morphology was restored within 7 h after the washout of antimycin, suggesting that the filamentation was reversible. Co‐applications of antimycin and cytoskeletal inhibitors (demecolcine or latrunculin B) or protein synthesis inhibitors (cycloheximide or chloramphenicol) still caused plastid filamentation. Antimycin A was also effective for plastid filamentation in the chloroplast division mutants atftsZ1‐1 and atminE1. Salicylhydroxamic acid, an alternative oxidase inhibitor, was solely found to suppress the filamentation, implying the possibility that this phenomenon was partly mediated by an antimycin‐activated alternative oxidase in the mitochondria.     

Loop-Sheet Mechanism of Serpin Polymerization Tested by Reactive Center Loop Mutations

The serpin mechanism of protease inhibition involves the rapid and stable incorporation of the reactive center loop (RCL) into central β-sheet A. Serpins therefore require a folding mechanism that bypasses the most stable “loop-inserted” conformation to trap the RCL in an exposed and metastable state. This unusual feature of serpins renders them highly susceptible to point mutations that lead to the accumulation of hyperstable misfolded polymers in the endoplasmic reticulum of secretory cells. The ordered and stable protomer-protomer association in serpin polymers has led to the acceptance of the “loop-sheet” hypothesis of polymerization, where a portion of the RCL of one protomer incorporates in register into sheet A of another. Although this mechanism was proposed 20 years ago, no study has ever been conducted to test its validity. Here, we describe the properties of a variant of α₁-antitrypsin with a critical hydrophobic section of the RCL substituted with aspartic acid (P8–P6). In contrast to the control, the variant was unable to polymerize when incubated with small peptides or when cleaved in the middle of the RCL (accepted models of loop-sheet polymerization). However, when induced by guanidine HCl or heat, the variant polymerized in a manner indistinguishable from the control. Importantly, the Asp mutations did not affect the ability of the Z or Siiyama α₁-antitrypsin variants to polymerize in COS-7 cells. These results argue strongly against the loop-sheet hypothesis and suggest that, in serpin polymers, the P8–P6 region is only a small part of an extensive domain swap.     

Identification of the transgenic integration site in immunodeficient tgε26 human CD3ε transgenic mice

A strain of human CD3ε transgenic mice, tgε26, exhibits severe immunodeficiency associated with early arrest of T cell development. Complete loss of T cells is observed in homozygous tgε26 mice, but not in heterozygotes, suggesting that genomic disruption due to transgenic integration may contribute to the arrest of T cell development. Here we report the identification of the transgenic integration site in tgε26 mice. We found that multiple copies of the human CD3ε transgene are inserted between the Sstr5 and Metrn loci on chromosome 17, and that this is accompanied by duplication of the neighboring genomic region spanning 323 kb. However, none of the genes in this region were abrogated. These results suggest that the severe immunodeficiency seen in tgε26 mice is not due to gene disruption resulting from transgenic integration.     

Development of microsatellite markers for <Emphasis Type="Italic">Bambusa arnhemica</Emphasis> (Poaceae: Bambuseae), a bamboo endemic to northern Australia

Bambusa arnhemica is a bamboo species endemic to northern Australia. We isolated and characterized nine microsatellite loci from this species. The number of alleles ranged from 2 to 16 with an average of 6.8, and expected heterozygosities from 0.40 to 0.84 with an average of 0.69. The markers described here will be useful to investigate clump structure, evolution of the bamboo flowering wave, patterns of gene flow, and the biogeographic history of B. arnhemica in Australia.