Clinical Implications and Molecular Characterization of Drebrin-Positive, Tumor-Infiltrating Exhausted T Cells in Lung Cancer.

T cells express an actin-binding protein, drebrin, which is recruited to the contact site between the T cells and antigen-presenting cells during the formation of immunological synapses. However, little is known about the clinical implications of drebrin-expressing, tumor-infiltrating lymphocytes (TILs). To address this issue, we evaluated 34 surgical specimens of pathological stage I-IIIA squamous cell lung cancer. The immune context of primary tumors was investigated using fluorescent multiplex immunohistochemistry. The high-speed scanning of whole-slide images was performed, and the tissue localization of TILs in the tumor cell nest and surrounding stroma was automatically profiled and quantified. Drebrin-expressing T cells were characterized using drebrin+ T cells induced in vitro and publicly available single-cell RNA sequence (scRNA-seq) database. Survival analysis using the propensity scores revealed that a high infiltration of drebrin+ TILs within the tumor cell nest was independently associated with short relapse-free survival and overall survival. Drebrin+ T cells induced in vitro co-expressed multiple exhaustion-associated molecules. The scRNA-seq analyses confirmed that the exhausted tumor-infiltrating CD8+ T cells specifically expressed drebrin. Our study suggests that drebrin-expressing T cells present an exhausted phenotype and that tumor-infiltrating drebrin+ T cells affect clinical outcomes in patients with resectable squamous cell lung cancer.

Determination of optimal 25-hydroxyvitamin D cutoff values for the evaluation of restless legs syndrome among pregnant women.

STUDY OBJECTIVES: Vitamin D deficiency is associated with restless legs syndrome (RLS). However, a cutoff value for serum 25-hydroxyvitamin D (25[OH]D) level associated with RLS has yet to be clearly determined. We evaluated the association between 25(OH)D and RLS in pregnant women. METHODS: Data from 203 pregnant women were evaluated using blood samples taken in the third trimester. Liquid chromatography-tandem mass spectrometry and ligand binding assays were used to measure 25(OH)D. RLS was diagnosed based on International Classification of Sleep Disorders, third edition, criteria. The cutoff value for serum 25(OH)D associated with RLS was explored using receiver operating characteristic (ROC) curve and classification and regression tree (CART) analyses. RESULTS: The results of liquid chromatography-tandem mass spectrometry (x) and ligand binding assays (y) for serum 25(OH)D in the RLS (n = 35, 17.2%) and non-RLS (n = 168) groups showed a relationship of y = -2.65 + 0.08x . The RLS group showed lower serum 25(OH)D and folate levels. ROC curve and CART analyses revealed cutoff values of 10-12.7 ng/mL and 6.6-7.2 ng/mL for 25(OH)D and folate, respectively. Of the 5 women with RLS symptoms persisting at a moderate-to-severe level after delivery, 4 had 25(OH)D levels < 10 ng/mL and all had folate levels < 6 ng/mL. CONCLUSIONS: Vitamin D and folate deficiency were associated with RLS in pregnant women and may be associated with persistent moderate-to-severe postpartum RLS symptomatology; it is essential to examine associations with RLS while accounting for measurement methods and assay systems. CITATION: Miyazaki A, Takahashi M, Shuo T, Eto H, Kondo H. Determination of optimal 25-hydroxyvitamin D cutoff values for the evaluation of restless legs syndrome among pregnant women. J Clin Sleep Med. 2023;19(1):73-83.

Determination of thyroid hormones in pharmaceutical preparations, after derivatization with 9-anthroylnitrile, by high-performance liquid chromatography with fluorescence detection.

HPLC with fluorescence detection was used for the determination of low levels of liothyronine sodium and levothyroxine sodium in pharmaceutical preparations after fluorogenic derivatization. 9-Anthroylnitrile in dimethyl sulfoxide was used as a precolumn fluorogenic reagent. The 9-anthroylnitrile derivatives of liothyronine sodium and levothyroxine sodium were separated on a reversed-phase column with acetonitrile-0.02 M sodium dodecylsulfate (pH 3.5 with phosphoric acid) as the eluent. The calibration graphs were linear over a sample concentration range of 0.25-2.5 microg/ml. The detection limits for liothyronine sodium and levothyroxine sodium were 0.2 ng per injection. The proposed method was applied to the determination of thyroid hormones in pharmaceutical preparations.

Simultaneous determination of eleven dyes and their aluminum lakes in drugs.

A 3-step extraction method was developed for the simultaneous determination of 11 dyes and their aluminum lakes in drugs. The dyes were first extracted with warm water (approximately 60 degrees C) and were cleaned up by solid-phase extraction with a tC18 cartridge. Aluminum lake dyes that remained in the precipitate were extracted with 0.02M NaOH. Aluminum in the dye lakes was reextracted into the organic layer with acetylacetone-butyl acetate (1 + 9, v/v), as an acetylacetone chelate, and was quantified by atomic absorption spectrometry. The dye portions of the aluminum lakes remained in the aqueous layer and were cleaned up in the same way as the dyes. The dyes and the dye portions of the aluminum lakes were quantified by ion-pair liquid chromatography with a photodiode array detector within 20 min. The recoveries of dyes from drug fortified at 10 microg of each dye per pill were 87.0-102.2%, and the recoveries of dyes from drugs fortified at 50 microg of each dye lake per pill were 82.9-101.6%, except for recoveries of indigo carmine. In 40 ethical and over-the-counter drugs, dyes that were not indicated in the package insert information for drugs were detected in 5 samples. The highest amount of dye found in a drug was 1169.5 microg erythrosine, which was detected in a capsule of antibiotic. Aluminum lake dyes were detected in 8 samples of various dosage forms.

[Enantiomeric determination of ephedrine derivatives in unregulated drugs using capillary electrophoresis].

Capillary electrophoresis (CE) was applied to enantiomeric separation of chiral ephedrine derivatives (d/l-ephedrine, d/l-methylephedrine, d/l-pseudoephedrine, and d/l-norephedrine) in unregulated drug products. Unregulated drugs, referred to as dietary supplements in U.S.A., have been used legally as tonic agents, but illegal substances such as ephedrine were often detected. Baseline separation of all enantiomers of ephedrine derivatives was achieved using an electrophoretic solution containing heptakis (2,6-di-O-methyl)-beta-cyclodextrin (DM-CD) as a chiral selector. The optimal conditions were established to be: capillary column of fused silica (50 microm i.d. x 56 cm); running buffer of 20 mM DM-CD with 50 mM potassium dihydrogenphosphate background electrolyte, pH 2.6; capillary temperature of 20 degrees C; applied voltage of 30 kV; on-column detection at 195 nm; and injection pressure of 50 mbar x 3 s. Under these conditions, all four pairs of enantiomers were sufficiently resolved, and eight peaks were observed with resolution factors of greater than 1.5. The calibration curves of all enantiomers showed good linearity over the concentration range of 2.5-10 microg/ml (r =0.999). The present method was used in a survey of marketed products. The resultant chiral contents were reported and the analytical data were also compared with those from HPLC. This method is useful in the simple and rapid analysis of ephedrine derivatives in marketed products.

Creation and application of psychoactive designer drugs data library using liquid chromatography with photodiode array spectrophotometry detector and gas chromatography-mass spectrometry.

In order to quickly confirm a potentially hazardous psychoactive designer drug (a compound in which part of the molecular structure of a stimulant or narcotic has been modified), we created a psychoactive drugs data library by performing analysis using liquid chromatography with photodiode array spectrophotometry (LC/PDA) and gas chromatography-mass spectrometry (GC/MS). The data in this library consist of the LC capacity factor (k') ratios in relation to the internal standard, the ultraviolet (UV) spectra and the MS spectra of 104 compounds. By performing a comparative study of the data in this report with the analytical data for commercial and illegal drug products, it is possible to quickly identify the psychoactive designer drugs in 205 purchased products by using the library. Further, it is possible to analogize the structure of drugs for which there is no matching data in the library using similar data. Furthermore, when structural isomers of controlled substances have detected from the presented library, similarity of their biological effects on human will be predicted, thus leading to regulate their public circulation. Examples of these types of isomers include, for instance, the narcotic 3,4,5-trimethoxyamphetamine (TMA) and its positional isomers 2,4,5-trimethoxyamphetamine (TMA-2) and 2,4,6-trimethoxyamphetamine (TMA-6), or the narcotic 1-(3-chlorophenyl)piperazine (3CPP) and its isomers 1-(o-chlorophenyl)piperazine (2CPP) and 1-(p-chlorophenyl)piperazine (4CPP). Differentiation of these compounds is necessary in regulating them, and we report here the results of a study of a method to confirm these compounds using the present library.

Acute confusional state after designer tryptamine abuse.

A 23-year-old Japanese woman was brought to the emergency department about 6.5 h after taking liquid and later a half tablet purchased on the street. About 4.5 h prior to presentation, she displayed excited and disorganized behavior. On examination, she was not alert or oriented, with a Glasgow Coma Scale score of 13, did not answer any questions from doctors while smirking and looking around restlessly, and sometimes exhibited echolalia, imitating the speech of doctors. She was given intravenous infusion of fluid for 8 h, then discharged. Gas chromatography-mass spectrometry of urine revealed 5-methoxy-diisopropyltryptamine, 5-methoxy-N-methyltryptamine and an unidentified tryptamine. Identifying chemical products based solely on information of users is insufficient, and urinalysis is necessary in cases potentially involving designer drugs.