Immune checkpoint therapy and response biomarkers in non-small-cell lung cancer: Serum NY-ESO-1 and XAGE1 antibody as predictive and monitoring markers.

Immune checkpoint inhibitors (ICI) are key drugs in systemic therapy for advanced non-small-cell lung cancer (NSCLC) and have recently been incorporated into neoadjuvant and adjuvant settings for surgical resection. Currently, ICI combinations with cytotoxic agents are frequently used in clinical practice, although several ICI clinical trials have failed to produce long-term clinical benefits. Unfortunately, clinical benefit is moderate and limited considering physical and financial burden. Therefore, selecting appropriate patients and regimens for ICI therapy is important, and biomarkers are necessary for their selection. Tumor PD-L1 expression is universally used as a biomarker; however, PD-L1 assays show low analytical validity and reproducibility due to the visual-scoring system by pathologists. Recent tumor immunology studies explore that neoantigens derived from somatic mutations and the collaboration between T and B cells efficiently elicit antitumor responses. This suggests that high tumor mutational burden and T-cell infiltration are predictive biomarkers. However, B cells producing antibody (Ab) remain poorly understood and analyzed as biomarkers. We found that NY-ESO-1 and XAGE1 of cancer-testis antigen frequently elicit spontaneous humoral and cellular immune responses in NSCLC. Serum Ab against these antigens were detected in approximately 25% of NSCLC patients and predicted ICI monotherapy responses. In addition, the Ab levels were decreased with tumor shrinkage after ICI therapy. Thus, NY-ESO-1 and XAGE1 Ab are potentially biomarkers predicting and monitoring response to ICI therapy. For clinical applications, a fully-automated assay system measuring the Ab was developed. Here, we review current ICI therapy, tumor immunology, and biomarkers in NSCLC, and discuss the applicability of the serum biomarkers NY-ESO-1 and XAGE1 Ab.

Highly sensitive and non-disruptive detection of residual undifferentiated cells by measuring miRNAs in culture supernatant.

The clinical usage of induced pluripotent stem cell (iPSC)-derived regenerative medicine products is limited by the possibility of residual undifferentiated cells forming tumours after transplantation. Most of the existing quality control tests involve crushing of cells. As a result, the cells to be transplanted cannot be directly tested, thereby increasing the cost of transplantation. Therefore, we tested a highly sensitive and non-disruptive quality-testing method that involves measuring microRNAs (miRNAs) in culture supernatants released by cells. By measuring miR-302b in the culture supernatant, residual iPSCs were detected with higher sensitivity than by measuring LIN28 (Lin-28 Homolog A) in the cells. To use this method, we also monitored the progression of differentiation. Our novel highly sensitive and non-disruptive method for detecting residual undifferentiated cells will contribute to reducing the manufacturing cost of iPSC-derived products and improving the safety of transplantation.

A novel automated immunoassay for serum NY-ESO-1 and XAGE1 antibodies in combinatory prediction of response to anti-programmed cell death-1 therapy in non-small-cell lung cancer.

BACKGROUND: Anti-programmed cell death-1 (PD-1) antibodies (Abs) are key drugs in non-small-cell lung cancer (NSCLC) treatment; however, clinical benefits with anti-PD-1 monotherapy are limited. We reported that serum Abs against cancer-testis antigens NY-ESO-1 and XAGE1 predicted clinical benefits. We aimed to develop a fully automated immunoassay system measuring NY-ESO-1/XAGE1 Abs. METHODS: Sera from 30 NSCLC patients before anti-PD-1 monotherapy were reacted with recombinant NY-ESO-1 protein- or synthetic XAGE1 peptide-coated magnetic beads. ALP-conjugated Ab and chemiluminescent substrate were added and luminescence measured. These procedures were automated using high sensitivity chemiluminescent enzyme immunoassay (HISCL™). NY-ESO-1/XAGE1 Ab stability was tested under various conditions. Response prediction accuracy was evaluated using area under receiver operating curve (AUROC). RESULTS: HISCL detected specific serum NY-ESO-1/XAGE1 Abs, which levels in ELISA and HISCL were highly correlated. The Ab levels in HISCL were stable at four temperatures, five freeze/thaw cycles, and long-term storage; the levels were not interfered by common blood components. The Ab levels in 15 NSCLC responders to anti-PD-1 monotherapy were significantly higher than those in non-responders and healthy donors. The AUROC was the highest (0.91; 95% CI, 0.78-1.0) in combinatory prediction with NY-ESO-1/XAGE1 Abs. CONCLUSION: Our immunoassay system is useful to predict clinical benefits with NSCLC immune-checkpoint therapy.

The chemical compound bubblin induces stomatal mispatterning in Arabidopsis by disrupting the intrinsic polarity of stomatal lineage cells.

Stem cell polarization is a crucial step in asymmetric cell division, which is a universal system for generating cellular diversity in multicellular organisms. Several conventional genetics studies have attempted to elucidate the mechanisms underlying cell polarization in plants, but it remains largely unknown. In plants, stomata, which are valves for gas exchange, are generated through several rounds of asymmetric divisions. In this study, we identified and characterized a chemical compound that affects stomatal stem cell polarity. High-throughput screening for bioactive molecules identified a pyridine-thiazole derivative, named bubblin, which induced stomatal clustering in Arabidopsis epidermis. Bubblin perturbed stomatal asymmetric division, resulting in the generation of two identical daughter cells. Both cells continued to express the stomatal fate determinant SPEECHLESS, and then differentiated into mispatterned stomata. Bubblin-treated cells had a defect in the polarized localization of BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), which is required for asymmetric cell fate determination. Our results suggest that bubblin induces stomatal lineage cells to divide without BASL-dependent pre-mitotic establishment of polarity. Bubblin is a potentially valuable tool for investigating cell polarity establishment in stomatal asymmetric division.

Specific detection by the polymerase chain reaction of potentially allergenic salmonid fish residues in processed foods.

Salmonid fish is one of the allergenic items that are recommended to be labeled in the Japanese allergen-labeling system. This study develops a salmonid-specific polymerase chain reaction (PCR) method. A new primer pair, SKE-F/SKE-R, was designed to specifically detect the salmonid fish gene encoding mitochondrial DNA cytochrome b. Genomic DNAs extracted from 58 kinds of seafood and 11 kinds of processed food were individually subjected to PCR by using the primer pair, and a salmonid-specific fragment of 212 bp was only amplified in the salmonid samples and salmonid-containing processed foods. The detection limit of the PCR method was as low as 0.02 fg/µL of salmonid fish DNA (corresponding to 10 copies). There is no ELISA method for salmonid fish, making our PCR method the only reliable measure for detecting salmonid fish in processed foods.

Quantification of pork, chicken and beef by using a novel reference molecule.

A standard plasmid was constructed as a novel reference molecule for use in real-time quantitative PCR assays to verify the identity of beef, pork, chicken, mutton, and horseflesh. The plasmid contained a target domain of the cytochrome b (cyt b) gene and an artificial DNA sequence. Primers CO-F and CO-R, and probe CO-P were specifically designed to detect the artificial sequence. The calculated R² values of the standard curves (10³-107 copies per reaction) for the five species ranged between 0.998 and 0.999 in the quantification analysis. The constructed plasmid provides a universal method for measuring the copy number of cyt b DNA in minced meat. This method would be a useful procedure for verifying food labels.

[Is whole body bone mineral density measured by the dual energy X-ray absorptiometry applied to evaluate risk of osteoporosis among Japanese adult females?].

To measure whole body fat accurately, the dual energy X-ray absorptiometry (DXA) is widely utilized. Simultaneously, bone mineral density (BMD) of the whole body can also be measured. BMD is one of important information to diagnose osteoporosis. However, it is not established to use whole body BMD for this diagnosis. It is recommended that lumbar and/or hip BMD should be used for diagnosing osteoporosis by the guideline for prevention and treatment of osteoporosis. Although it is possible to measure whole body BMD and lumbar and/or hip BMD separately at the same visit, it is inevitable to expose patients to more X-ray. Therefore, an aim of this study is to elucidate the relationship between whole body BMD and lumbar BMD to find the cut off point of whole body BMD for screening of osteoporosis. Two hundred and thirty six Japanese adult females were ascertained to this study. Whole body BMD and lumbar BMD of each subject were measured with the use of Delphi W (Hologic, USA). One hundred and sixty five subjects were judged as possible osteoporosis (less than 80% of young adult mean (YAM) of lumbar BMD and/or definite fracture of lumbar vertebras). The cut off point of whole body BMD for screening possible osteoporosis was estimated by receiver operated characteristic (ROS) analysis. The cut off point of whole body BMD was 84% of YAM, equivalent to 80% of YAM of lumbar BMD, with the following sensitivity and specificity (0.84 and 0.79, respectively), indicating that whole body BMD could be used for screening osteoporosis.

Specific detection of banana residue in processed foods using polymerase chain reaction.

Specific polymerase chain reaction (PCR) methods were developed for the detection of banana residue in processed foods. For high banana specificity, the primer set BAN-F/BAN-R was designed on the basis of the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) genes of chloroplasts and used to obtain amplified products specific to banana by both conventional and real-time PCR. To confirm the specificity of these methods, genomic DNA samples from 31 other species were examined; no amplification products were detected. Subsequently, eight kinds of processed foods containing banana were investigated using these methods to confirm the presence of banana DNA. Conventional PCR had a detection limit of 1 ppm (w/w) banana DNA spiked in 50 ng of salmon testis DNA, whereas SYBR Green I real-time semiquantitative PCR had a detection limit as low as 10 ppm banana DNA. Thus, both methods show high sensitivity and may be applicable as specific tools for the detection of trace amounts of banana in commercial food products.

Detection of walnut residues in processed foods by polymerase chain reaction.

A sensitive qualitative detection method for walnut (Juglans regia) using polymerase chain reaction (PCR) was developed. For detection of walnuts with high specificity, the primer pair WAL-F/WAL-R was designed based on walnut matK genes. Trace amounts of walnuts in commercial food products can be qualitatively detected using this method.

Amplification patterns of three genomic regions predict distant recurrence in breast carcinoma.

Currently used clinical and histopathological parameters imprecisely define the risk of distant recurrence in breast cancer, underscoring the need for more informative prognostic markers. In the present fluorescence in situ hybridization study of archived surgical specimens, we derived an algorithm for computing a prognostic index (PI) from DNA copy numbers of three genomic regions (CYP24, PDCD6IP, and BIRC5) for estrogen/progesterone receptor-positive (ER/PR+) cancers and a distinct PI (based on NR1D1, SMARCE1, and BIRC5) for estrogen/progesterone receptor-negative (ER/PR-) cancers. Among independent test cases stratified by PI, recurrence rates were significantly higher among high-risk patients than low-risk patients for both ER/PR+ (odds ratio = 9.52, 95% confidence interval >2.12, P = 0.0024) and ER/PR- (odds ratio = 12.3, 95% confidence interval >1.45, P = 0.0188) cancers. Among the entire population, recurrences were significantly more prevalent for cases with PI above the medians for both ER/PR+ (Fisher's exact, P = 1.19 x 10(-5)) and ER/PR- (P = 0.0025) patients and for the node-negative subsets (ER/PR+ node-negative, P = 0.042 and ER/PR- node-negative, P = 0.039). In conclusion, these markers perform well in comparison with other criteria for recurrence risk assessment and can be used with routinely formalin-fixed, paraffin-embedded surgical specimens.

Saccharomyces cerevisiae mutant displaying beta-glucans on cell surface.

The deletion of MCD4 leads to an increase in beta-1,6-glucan level and a decrease in glycosylphosphatidylinositol-anchored protein and mannan levels in the cell wall of Saccharomyces cerevisiae, suggesting that mcd4 deletion mutant (mcd4Delta) displays beta-glucans on the cell surface without a mannan cover. An observation of the cell surface of mcd4Delta cells and an examination of the effect of contact between mcd4Delta cells and mouse macrophages indicated that macrophages were activated by contact with mcd4Delta cells displaying beta-glucans on the cell surface. We further examined the effect of intraperitoneal ethanol-fixed mcd4Delta cells on the survival period of mice infected with Candida albicans. mcd4Delta cells prolonged the survival period, implying that mcd4Delta cells may enhance the immune function of mice via macrophage activation. Moreover, we examined the structures of beta-glucans (i.e., alkali- and acetic acid-insoluble beta-glucans) extracted from mcd4Delta with (13)C-NMR and the effect of extracted beta-glucans on TNF-alpha secretion from macrophages. The structures of the beta-glucans from mcd4Delta differed from those of wild type (WT); however, there was no difference in tumor necrosis factor-alpha (TNF-alpha) secretion level between beta-glucans from mcd4Delta and those from WT. The yield of purified beta-glucans obtained from dry cells of mcd4Delta was higher than that obtained from dry cells of WT. mcd4Delta may be a superior strain for the preparation of beta-glucans.

Comparison of body composition measurements obtained by two fan-beam DXA instruments.

Although dual-energy X-ray absorptiometry (DXA) has been widely used for measuring body composition, discrepancies have been reported to exist among results obtained from different instruments. In the course of longitudinal studies lasting for many years, old instruments may be required to be replaced with new ones, necessitating comparison and validation between the values obtained by the old and new instruments. We compared the data obtained by the two fan-beam DXA instruments, QDR-2000 (Hologic, Waltham, MA) and Delphi (Hologic). Body composition was first measured by the Hologic QDR-2000 and next by the Delphi W within 30 days in 99 healthy subjects. Whole-body fat mass (FM), percentage of FM, arm FM, and leg FM measured by the Hologic QDR-2000 were significantly larger than those measured by the Delphi W. Lean tissue mass (LTM), bone mineral content, and bone mineral density of the whole body, trunk FM, arm LTM, and leg LTM measured by the QDR-2000 were significantly smaller than those measured by the Delphi W. After converting the QDR-2000 values by equations developed by multiple regression analysis, they were not significantly different from the corresponding Delphi values. Measurements by the QDR-2000 and the Delphi W were not interchangeable and the conversion equations reduced the discrepancy to a level that enabled direct comparison of the data obtained by the two instruments. However, cautious interpretation is necessary when the conversion equations are applied to other instruments even of the same type or when evaluating data of individual subjects.

Patients with dilated cardiomyopathy possess insulin resistance independently of cardiac dysfunction or serum tumor necrosis factor-alpha.

It has recently been reported that insulin resistance is prevalent in patients with dilated cardiomyopathy (DCM); however, it remains unclear whether insulin resistance is directly induced by DCM or if it is caused by congestive heart failure associated with DCM. We evaluated homeostasis model assessment insulin resistance (HOMA-R) in 14 patients with DCM in comparison with 9 patients with valvular heart diseases (VHD). We also measured the level of serum tumor necrosis factor (TNF)-alpha as a possible causative factor for inducing insulin resistance. Even after the adjustment for age, body mass index, and cardiac function, HOMA-R was significantly higher in patients with DCM than in those with VHD (P = 0.012) (mean +/- SEM: 3.51 +/- 0.59, and 0.80 +/- 0.64, respectively). The serum TNF-alpha level tended to be higher in patients with DCM than in those with VHD; however, the difference was not significant. In conclusion, patients with DCM possess insulin resistance independently of the severity of cardiac dysfunction or serum TNF-alpha, suggesting that insulin resistance in patients with DCM may be closely associated with the pathogenic condition of DCM itself.

Deletion of MCD 4 involved in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an increase in beta-1,6-glucan level and a decrease in GPI-anchored protein and mannan levels in the cell wall of Saccharomyces cerevisiae.

Most proteins involved in the synthesis of the GPI core structure of Saccharomyces cerevisiae are essential for growth. To explore the relationship between the GPI anchor structure and beta-1,6-glucan synthesis, we screened deletion mutants in genes involved in GPI synthesis for osmotic remedial growth. Heterozygous diploid strains were dissected on medium with osmotic support and slow growth of the mcd 4 deletion mutant was observed. The mcd 4 mutant showed abnormal morphology and cell aggregation, and was hypersensitive to SDS, hygromycin B and K1 killer toxin. Incorporation of GPI cell wall proteins was examined using a GPI-Flo 1 fusion protein. The result suggested that the mcd 4 deletion causes a decrease in GPI cell wall proteins levels. The mutation also caused a decrease in mannan levels and an increase in alkali-insoluble beta-1,6-glucan and chitin levels in the cell wall.

A novel in vitro system, the integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug toxicity: comparative cytotoxicity of tamoxifen towards normal human cells from five major organs and MCF-7 adenocarcinoma breast cancer cells.

In vitro assays involving primary cells are used routinely to evaluate organ-specific toxic effects, for instance, the use of primary hepatocytes to evaluate hepatotoxicity. A major drawback of an in vitro system is the lack of multiple organ interactions as observed in a whole organism. A novel cell culture system, the integrated discrete multiorgan cell culture system (IdMOC), is described here. The IdMOC is based on the "wells within a well" concept, consisting of a cell culture plate with larger, containing wells, within each of which are multiple smaller wells. Cells from multiple organs can be cultured initially in the small wells (one organ per well, each in its specialized medium). On the day of toxicity testing, a volume of drug-containing medium is added to the containing well to flood all inner wells, thereby interconnecting all the small wells. After testing, the overlying medium is removed and each cell type is evaluated for toxicity using appropriate endpoints. We report here the application of IdMOC in the evaluation of the cytotoxicity of tamoxifen, an anticancer agent with known human toxicity, on primary cells from multiple human organs: liver (hepatocytes), kidney (kidney cortical cells), lung (small airway epithelial cells), central nervous system (astrocytes), blood vessels (aortic endothelial cells) as well as the MCF-7 human breast adenocarcinoma cells. IdMOC produced results that can be used for the quantitative evaluation of its anticancer effects (i.e., cytotoxicity towards MCF-7 cells) versus its toxicity toward normal organs (i.e., liver, kidney, lung, CNS, blood vessels).

Applications of microarrays with toxicologically relevant genes (tox genes) for the evaluation of chemical toxicants in Sprague Dawley rats in vivo and human hepatocytes in vitro.

Microarrays with toxicologically relevant genes (tox genes) have been developed in our laboratory for toxicogenomics studies in rat, dog and man. The genes were chosen using published information as well as a discovery process for genes responsive to toxic treatments using transcription profiling experiments conducted with rats and dogs. In addition to published information human tox genes were derived from rat tox genes based on gene homology. Using the microarray with rat-specific tox genes, a database containing gene expression, histopathology, and clinical chemistry findings has been generated for 89 compounds. Analysis of the database indicates that treatment with toxic compounds induces specific gene expression patterns. Dose- and time-dependent response relationships in gene expression were observed for treatment with toxic compounds. Gene expression at 24h was found to correlate well with organ toxicity observed at 72 h. Mining of the database led to the selection of specific groups of genes (predictive gene sets) whose expression patterns are predictive of organ toxicity with a high degree of accuracy (approximately 90%). The data also provide insight on toxic mechanism and gene regulation pathways. For instance, carbon tetrachloride and chloroform treatments were found to decrease the expression of the cytochrome P450 isoform 3A1 gene while enhancing the expression of the multiple drug resistance gene MDR1 in liver, clearly demonstrating that the CYP3A1 and MDR1 genes were not co-regulated as postulated by some researchers. This approach, the use of gene expression as an endpoint to define organ toxicity, is extended to the definition of human drug toxicity using primary human hepatocytes as a test system. Preliminary results demonstrate that the toxic drug, troglitazone, can be clearly distinguished from the less toxic analogues, rosiglitazone and pioglitazone based on their effects on tox gene expression in human hepatocytes. Our results with both rats in vivo and human hepatocytes in vitro suggest that microarrays with toxicologically relevant genes can be used routinely for the evaluation of chemical toxicity.

Effect of bupropion on CYP2B6 and CYP3A4 catalytic activity, immunoreactive protein and mRNA levels in primary human hepatocytes: comparison with rifampicin.

Animals treated with multiple doses of bupropion have had increased bupropion clearance or increased liver weight, suggesting induction of drug-metabolizing activity. The possibility of cytochrome p450 (CYP) induction by bupropion (10 microM) was evaluated in-vitro by comparing catalytic activity, immunoreactive protein and CYP mRNA levels from human hepatocytes in primary culture versus cells treated with vehicle (0.5% methanol) and with rifampicin (rifampin) as a positive control. mRNA levels were analysed using a branched DNA luminescent assay. CYP2B6 activity, protein and mRNA levels were increased by 2.5, 1.5 and 2.1 fold, respectively, by 20 microM rifampicin. However, 10 microM bupropion minimally altered CYP2B6 (1.4, 1.1, 0.8 fold). Although CYP3A4 activity, protein, and mRNA levels were increased by 4.0, 2.3, and 14.0 fold, respectively, by 20 microM rifampicin, 10 microM bupropion minimally altered CYP3A4 (1.4, 1.0, 0.8 fold). Rifampicin (20 microM) increased CYP2E1 protein by 2.1 fold, while 10 microM bupropion minimally altered CYP2E1 protein (1.2 fold). Overall, results of this study suggest that multiple doses of bupropion are not likely to induce CYP2B6, 3A4 or 2E1 in-vivo in man.

Differential in vitro hepatotoxicity of troglitazone and rosiglitazone among cryopreserved human hepatocytes from 37 donors.

We report here our studies on troglitazone and rosiglitazone cytotoxicity in human hepatocytes isolated from multiple donors to investigate factors responsible for individual differences in sensitivity to the known hepatotoxicity of these antidiabetic drugs. Using cellular adenosine triphosphate (ATP) content as an endpoint, cytotoxicity of both drugs was evaluated in cryopreserved human hepatocytes from 37 donors. We confirmed reports of others that troglitazone was cytotoxic to human hepatocytes using cellular ATP content as an endpoint. In addition, we found that rosiglitazone, although less toxic in the study population, was cytotoxic to hepatocytes in some donors (EC(50)<100 microM). ATP content, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) metabolism, depletion of intracellular glutathione, Alamar Blue metabolism, and neutral red uptake were used as endpoints in a single donor study using freshly isolated human hepatocytes. Troglitazone appeared to be more toxic than rosiglitazone by all endpoints. From the demographic data provided to us for each donor, we were able to establish no direct correlation between cytotoxicity (expressed as EC(50) values) and age, sex, smoking status, or alcohol consumption. We conclude that troglitazone and rosiglitazone are differentially toxic to human hepatocytes, and that toxicity may be independent of age, sex, tobacco use, and alcohol use.

Correlation between troglitazone cytotoxicity and drug metabolic enzyme activities in cryopreserved human hepatocytes.

Troglitazone (TRO) was developed for the treatment of type II diabetes. It was withdrawn from use due to idiosyncratic liver damage and failure. The mechanism of toxicity is still not determined, moreover, it is still not clear whether toxicity is due to the parent compound or its metabolite(s). The cytotoxicity of TRO was evaluated in human hepatocytes using previously cryopreserved hepatocyte suspensions from 27 human donors. Cellular adenosine triphosphate content was used as a viability endpoint. To investigate the role of xenobiotic metabolism in TRO toxicity, the correlation between the drug metabolism activities of the hepatocytes from each donor to EC(50) values TRO cytotoxicity. The activities examined were cytochrome P450 (CYP) isoform activities (CYP2A6, CYP2D6, CYP2C19, CYP1A2, CYP2E1, CYP3A4 and CYP2C9) and phase 2 conjugation enzyme activities (phenol sulfotransferase (PST) and glucuronyl transferase (UGT)). Taken individually, none of the phase 1 or 2 enzyme activities correlated to the EC(50). However, when three enzyme activities ((CYP3A4 x UGT)/PST) were taken into account, a correlation was made (r(2)=0.53). Based on the correlation, we hypothesize that TRO and TRO sulfate are direct acting toxicants, whereas CYP3A4 oxidation and glucuronidation are detoxification pathways.

Expression of AIRE gene in peripheral monocyte/dendritic cell lineage.

The responsible gene for autoimmune polyglandular syndrome type 1, known as autoimmune regulator (AIRE), was identified by positional cloning. The AIRE gene was reported to be expressed in the thymus medulla and lymph nodes. However, an expression of the AIRE gene in peripheral blood cells has not yet been reported. In the present study, we found that the AIRE gene was restrictively expressed in peripheral CD14-positive monocytes but not in CD4-positive T cells nor polymorphonuclear cells, as assessed by RT-PCR. Moreover, immunocytochemical study revealed the expression of the AIRE protein not only in CD14-positive monocytes but also in differentiated dendritic cells, cultured in RPMI1640 medium containing 800 U/ml GM-CSF, 1000 U/ml IL-4 and 100 U/ml TNF-alpha. Thus, it was concluded that the AIRE gene is restrictively expressed in the peripheral monocyte/dendritic cell lineage.