Clinical applications of MALDI imaging using sliced sections of formalin-fixed paraffin-embedded tissues and longitudinal sliced hairs

MALDI-imaging MS (IMS) with MSMS analysis is a new powerful tool for the identification of not only disease-related proteins in formalin-fixed paraffin-embedded (FFPE) tissue sections but also protein/peptides/drugs/medicine in fresh-frozen tissues. IMS is used to reveal the mass profiles and spatial distribution of proteins in tissue sections and/or digested peptides derived from deposited protein in pathologic organs and then MSMS analysis identifies the amino acid sequence of the detected proteins in the tissue section. Moreover, on-tissue digestion combined with the MALDI-IM-TOF-IMS approach allows a proteomics "bottom-up" strategy with clinical samples, especially perioperative isolated tissues and FFPE tissues conserved for a long time in a clinical sample bank. The mass barcode-like image (MBI) on a longitudinal sliced hair by IMS is used in the selected reaction monitoring mode for serially chronological monitoring and traceability every few hours after drug and medicine intake. The advances of quantitative MBI for sliced sections of hair allow a new universal standardized assessment of drugs and medicines throughout the drug history.     

Antagonism of NMDA receptors by butanesulfonyl-homospermine guanidine and neuroprotective effects in in vitro and in vivo

The polyamine derivative BsHSPMG (butanesulfonyl-homospermine with guanidine group) was found to inhibit macroscopic currents strongly at heteromeric N-methyl-d-aspartate (NMDA) receptors (NR1/NR2A and NR1/NR2B) and Ca²⁺-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (homomeric glutamate receptor 1) receptors expressed in Xenopus laevis oocytes on voltage-clamp recording. The IC₅₀ values of BsHSPMG for NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D receptors were 0.016, 0.021, 5.4, and 9.0 μM, respectively. BsHSPMG inhibited the activity of NR1/NR2A and NR1/NR2B receptors more strongly and did it for those of NR1/NR2C and NR1/NR2D receptors more weakly than a therapeutic drug of Alzheimer's disease, memantine. The inhibition by BsHSPMG was voltage-dependent, since it was prominent at −100 mV compared to that at −20 mV. Mutations including NR1 N616Q, E621Q, N650A, L655A, T807C, NR2B W559L, M562S, W607L, N616Q, and V620E, among others, reduced the inhibition by BsHSPMG, suggesting that BsHSPMG penetrates the channel pore of NMDA receptors deeply. The toxicity of BsHSPMG in neuroblastoma SH-SY5Y cells was much weaker than that of memantine. The effect of BsHSPMG was measured on the focal cerebral ischemia induced by occlusion (1 h) of the middle cerebral artery in mice. BsHSPMG applied before or after occlusion greatly reduced the volume of infarct in mice. These findings demonstrate that BsHSPMG penetrates the NMDA channel pore and exhibits neuroprotective effects against excitatory toxicity in mice.     

Actions of Midazolam on GABAergic Transmission in Substantia Gelatinosa Neurons of Adult Rat Spinal Cord Slices

Background: Although intrathecal administration of midazolam has been found to produce analgesia, how midazolam exerts this effect is not understood fully at the neuronal level in the spinal cord.

Methods: The effects of midazolam on either electrically evoked or spontaneous inhibitory transmission and on a response to exogenous [gamma]-aminobutyric acid (GABA), a GABAA-receptor agonist, muscimol, or glycine were evaluated in substantia gelatinosa neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique.

Results: Bath-applied midazolam (1 [mu]M) prolonged the decay phase of evoked and miniature inhibitory postsynaptic currents (IPSCs), mediated by GABAA receptors, without a change in amplitudes, while not affecting glycine receptor-mediated miniature inhibitory postsynaptic currents in both the decay phase and the amplitude. Either GABA- or muscimol-induced currents were enhanced in amplitude by midazolam (0.1 [mu]M) in a manner sensitive to a benzodiazepine receptor antagonist, flumazenil (1 [mu]M); glycine currents were, however, unaltered by midazolam.     

Nontuberculous mycobacterial and Aspergillus infections among cadaveric lung transplant recipients in Japan

Background

Lung transplantation is an effective treatment modality for respiratory failure. Chronic lung infections, including infections caused by nontuberculous mycobacteria (NTM) and Aspergillus, are difficult to control, and uncontrolled infections are relative contraindications for lung transplantation. However, few reports have documented the incidence and outcome of these infections in lung transplant recipients.

Unexpected metastasis of intraductal papillary neoplasm of the bile duct without an invasive component to the brain and lungs:A case report

BACKGROUND Despite an expanding number of studies on intraductal papillary neoplasm of the bile duct(IPNB),distant metastasis remains unexplained especially in cases of carcinoma in situ.In the present study,we report a rare and interesting case of IPNB without invasive components that later metastasized to lungs and brain.CASE SUMMARY A 69-year-old male was referred to our hospital due to suspected cholangiocarcinoma.Laboratory tests on admission reported a mild elevation of alkaline phosphatase,γ-glutamyl transpeptidase,and total bilirubin in serum.Endoscopic retrograde cholangiography revealed a filling defect in the common bile duct(CBD)extending to the left hepatic duct.Peroral cholangioscopy delineated a tumor in the CBD that had a papillary pattern.Multidetector computed tomography and magnetic resonance cholangiopancreatography detected partial blockage ot interlude in the CBD leading to cholestasis without evidence of metastasis.Therefore,a diagnosis of IPNB cT1N0M0 was established.Left hepatectomy with bile duct reconstruction was performed.Pathological examination confirmed an intraepithelial neoplasia pattern without an invasive component and an R0 resection achievement.The patient was monitored carefully by regular examinations.However,at 32 mo after the operation,a 26 mm tumor in the lungs and a 12 mm lesion in the brain were detected following a suspicious elevated CA 19-9 level.Video-assisted thoracoscopic surgery of left upper lobectomy and stereotactic radiotherapy are indicated.In addition to histopathological results,a genomic profiling analysis using whole exome sequencing subsequently confirmed lung metastasis originating from bile duct cancer.CONCLUSION This case highlights the important role of genomic profiling analysis using whole exome sequencing in identifying the origin of metastasis in patients with IPNB.     

Close association of Chlamydia pneumoniae IgA seropositivity by ELISA with the presence of coronary artery stenosis in haemodialysis patients.

BACKGROUND: Traditional risk factors of cardiovascular disease do not fully explain the accelerated atherosclerosis present in patients with end-stage renal disease (ESRD). The goal of this study was to identify the association of clinical and laboratory factors including seropositivity for Chlamydia pneumoniae determined by a specific enzyme-linked immunosorbent assay (ELISA) with the presence of coronary artery disease identified by coronary angiography in ESRD patients. METHODS: We prospectively enrolled 161 consecutive ESRD patients undergoing haemodialysis for >6 months (106 men, 55 women; mean age 63.1+/-10.2 years; mean dialysis duration 91.3+/-90.1 months). All patients underwent coronary angiography within 1 week after blood sampling. The associations of coronary artery disease with clinical parameters including C. pneumoniae IgA and IgG seropositivity were analysed using multiple logistic regression models. RESULTS: Coronary stenosis >50% was found in 102 of 161 haemodialysis patients (63.4%). Of the 102 patients, 75.5% were asymptomatic. Seropositivity for C. pneumoniae IgA was found in patients with coronary stenosis (77 out of 102, 75.5%) more frequently (P<0.001) than in patients without coronary stenosis (10 out of 59, 16.9%). Seropositivity for C. pneumoniae IgA but not IgG was strongly associated with the presence of coronary stenosis in multiple logistic regression analysis (odds ratio, 18.440; 95% confidence interval, 7.500-45.337), independently of the Framingham coronary risk factors, factors peculiar to ESRD or serum C-reactive protein levels. CONCLUSIONS: C. pneumoniae IgA seropositivity determined by ELISA is an independent laboratory factor indicating the presence of coronary artery stenosis in ESRD patients undergoing maintenance haemodialysis.     

Effects of slow-releasing colistin microspheres on endotoxin-induced sepsis

Lipopolysaccharide (LPS) is a major contributing factor to endotoxic shock. Colistin specifically binds to LPS. However, it has the disadvantages that adverse reactions are common and it has a short half-life. To overcome these disadvantages, we prepared slow-releasing colistin microspheres and examined the efficacy of these colistin microspheres in a mouse model of endotoxin-induced sepsis. We prepared the colistin microspheres using poly-lactic-co-glycolic acid. For acute toxicity investigations, mice were overdosed with colistin sulfate or colistin microspheres. The group administered with colistin microspheres was associated with less acute toxicity and fewer nephrotoxic changes on histopathological examination compared to the group administered with colistin sulfate alone. For pharmacokinetic analysis, mice were subcutaneously administered with colistin microspheres or colistin sulfate alone. The plasma concentration of colistin was higher in the colistin microspheres group than in the colistin sulfate group at 12 and 24 h after administration. Moreover, mice were intraperitoneally injected with LPS and then immediately subcutaneously administered with blank microspheres, colistin microspheres or colistin sulfate alone. The levels of endotoxin in the sera and cytokine in the spleens were then measured. A significant reduction in the serum endotoxin level in the colistin microspheres group was observed at 24 h. The reduced endotoxin levels in the sera were correlated with the lower cytokine levels in the spleens of mice treated with colistin microspheres. Our results suggest that the use of colistin microspheres may help to maintain a higher colistin concentration in blood, reduce the levels of endotoxin and cytokines in endotoxin-induced sepsis, and lead to decreased toxicity.     

The effect of methylprednisolone on cisplatin-induced nephrotoxicity in rat renal cortical slices

To investigate the protective action of methylprednisolone against cisplatin nephrotoxicity, the effect of in vivo pretreatment with methylprednisolone on the cisplatin-induced reduction inp-aminohippurate (PAH) accumulation and gluconeogenesis was examined using renal cortical slices prepared from Sprague-Dawley rats. The PAH accumulation in the kidney slices prepared from methylprednisolone-pretreated rats was significantly reduced following in vitro incubation with 2 mM cisplatin, to a degree equal to that observed in the slices prepared from untreated rats. However, the inhibitory effect of cisplatin on gluconeogenesis in the renal cortical slices obtained from methylpredimisolone-pretreated rats was significantly smaller than that seen in the slices from untreated rats. Our present studies suggest that in vivo pretreatment with methylprednisolone may contribute to its protective effect against cisplatin nephrotoxicity through the process of gluconeogenesis in renal epithelial cells.