Fluorescently Activated Cell Sorting Followed by Microarray Profiling of Helper T Cell Subtypes from Human Peripheral Blood

Background

Peripheral blood samples have been subjected to comprehensive gene expression profiling to identify biomarkers for a wide range of diseases. However, blood samples include red blood cells, white blood cells, and platelets. White blood cells comprise polymorphonuclear leukocytes, monocytes, and various types of lymphocytes. Blood is not distinguishable, irrespective of whether the expression profiles reflect alterations in (a) gene expression patterns in each cell type or (b) the proportion of cell types in blood. CD4⁺ Th cells are classified into two functionally distinct subclasses, namely Th1 and Th2 cells, on the basis of the unique characteristics of their secreted cytokines and their roles in the immune system. Th1 and Th2 cells play an important role not only in the pathogenesis of human inflammatory, allergic, and autoimmune diseases, but also in diseases that are not considered to be immune or inflammatory disorders. However, analyses of minor cellular components such as CD4⁺ cell subpopulations have not been performed, partly because of the limited number of these cells in collected samples.

Methodology/Principal Findings

We describe fluorescently activated cell sorting followed by microarray (FACS–array) technology as a useful experimental strategy for characterizing the expression profiles of specific immune cells in the circulation. We performed reproducible gene expression profiling of Th1 and Th2, respectively. Our data suggest that this procedure provides reliable information on the gene expression profiles of certain small immune cell populations. Moreover, our data suggest that GZMK, GZMH, EOMES, IGFBP3, and STOM may be novel markers for distinguishing Th1 cells from Th2 cells, whereas IL17RB and CNTNAP1 can be Th2-specific markers.

Conclusions/Significance

Our approach may help in identifying aberrations and novel therapeutic or diagnostic targets for diseases that affect Th1 or Th2 responses and elucidating the involvement of a subpopulation of immune cells in some diseases.     

Structural Biology of Presenilins and Signal Peptide Peptidases

Presenilin and signal peptide peptidase are multispanning intramembrane-cleaving proteases with a conserved catalytic GxGD motif. Presenilin comprises the catalytic subunit of γ-secretase, a protease responsible for the generation of amyloid-β peptides causative of Alzheimer disease. Signal peptide peptidase proteins are implicated in the regulation of the immune system. Both protease family proteins have been recognized as druggable targets for several human diseases, but their detailed structure still remains unknown. Recently, the x-ray structures of some archaeal GxGD proteases have been determined. We review the recent progress in biochemical and biophysical probing of the structure of these atypical proteases.     

Biosynthesis of leukotoxin, 9,10-epoxy-12 octadecenoate, by leukocytes in lung lavages of rat after exposure to hyperoxia

In lung lavages of rat after pure oxygen breathing, a toxic linoleate peroxide, 9,10-epoxy-12-octadecenoate, and its isomer, 12,13-epoxy-9-octadecenoate were detected by HPLC analyses. The epoxide(s) was demonstrated to be biosynthesized by incubating linoleate with leukocytes collected from lung lavages, thus nominated to be leukotoxin. The chemical structures of leukotoxin and its isomer were determined by gas-chromatography/mass spectrometry and nuclear magnetic resonance measurements. Leukotoxin showed a potent uncoupling activity to rat liver mitochondrial respiration and a dose-dependent relaxation of rat stomach smooth muscle. These findings were discussed with 'oxygen toxicity' on the lung.     

Two types of growth inhibitor in rat platelets for primary cultured rat hepatocytes

Rat platelets contain two types of growth inhibitor of adult rat hepatocytes in primary culture. One, named platelet derived growth inhibitor (PDGI)-alpha, is a heat- and acid-labile protein with a molecular weight of over 200 KD that is not released on thrombin treatment. The other, named PDGI-beta, is a heat- and acid-stable factor with a molecular weight of 24 KD that is released by thrombin. Both PDGI-alpha and -beta were inactivated by treatment with dithiothreitol. They both caused dose-dependent inhibition of DNA synthesis stimulated by insulin plus epidermal growth factor. These inhibitions were closely correlated with marked decrease in the labeling index. Neither PDGI-alpha nor -beta had a cytotoxic effect as judged by phase-contrast microscopic examination of the cells nor inhibition of protein synthesis. The properties of PDGI-beta suggest that it may be identical with transforming growth factor-beta. These results indicate that rat platelets contain not only a growth factor (HGF), but also growth inhibitors that affect adult rat hepatocytes.     

Molecular Markers of Tubulointerstitial Fibrosis and Tubular Cell Damage in Patients with Chronic Kidney Disease

In chronic kidney disease (CKD), progressive nephron loss causes glomerular sclerosis, as well as tubulointerstitial fibrosis and progressive tubular injury. In this study, we aimed to identify molecular changes that reflected the histopathological progression of renal tubulointerstitial fibrosis and tubular cell damage. A discovery set of renal biopsies were obtained from 48 patients with histopathologically confirmed CKD, and gene expression profiles were determined by microarray analysis. The results indicated that hepatitis A virus cellular receptor 1 (also known as Kidney Injury Molecule-1, KIM-1), lipocalin 2 (also known as neutrophil gelatinase-associated lipocalin, NGAL), SRY-box 9, WAP four-disulfide core domain 2, and NK6 homeobox 2 were differentially expressed in CKD. Their expression levels correlated with the extent of tubulointerstitial fibrosis and tubular cell injury, determined by histopathological examination. The expression of these 5 genes was also increased as kidney damage progressed in a rodent unilateral ureteral obstruction model of CKD. We calculated a molecular score using the microarray gene expression profiles of the biopsy specimens. The composite area under the receiver operating characteristics curve plotted using this molecular score showed a high accuracy for diagnosing tubulointerstitial fibrosis and tubular cell damage. The robust sensitivity of this score was confirmed in a validation set of 5 individuals with CKD. These findings identified novel molecular markers with the potential to contribute to the detection of tubular cell damage and tubulointerstitial fibrosis in the kidney.     

Characterization of Two Members among the Five ADP-Forming Acyl Coenzyme A (Acyl-CoA) Synthetases Reveals the Presence of a 2-(Imidazol-4-yl)Acetyl-CoA Synthetase in Thermococcus kodakarensis

The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α₂β₂ structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS III_Tk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICS_Tk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICS_Tk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales.     

Both Notch1 and Notch2 contribute to the regulation of melanocyte homeostasis

Notch signaling affects a variety of mammalian stem cells, but there has been limited evidence that a specific Notch molecule regulates adult stem cells. Recently, it was reported that the reduced Notch signaling initiated at the embryonic stage results in a gradual hair graying phenotype after birth. Here we demonstrate that the oral administration of a gamma-secretase inhibitor (GSI) to wild-type adult C57/Bl6 mice led to a gradual increase in gray spots, which remained unchanged for at least 20 weeks after discontinuing the GSI. In GSI-treated mice, there was a severe decrease in unpigmented melanocytes in the bulge/subbulge region where melanocyte stem cells are located. While we confirmed that Notch1+/-Notch2+/- double heterozygous mice with a C57/Bl6 background were born with a normal hair color phenotype and gradually turned gray after the second hair cycle, in the c-kit mutant Wv background, Notch1+/- and Notch2+/- mice had larger white spots on the first appearance of hair than did the Wv/+ mice, which did not change throughout life. Notch1+/-Notch2+/-Wv/+ mice had white hair virtually all over the body at the first appearance of hair and the depigmentation continued to progress thereafter. Using a neural crest organ culture system, GSI blocked the generation of pigmented melanocytes when added to the culture during the period of melanoblast proliferation, but not during the period of differentiation. These observations imply roles of Notch signaling in both development of melanocyte during embryogenesis and maintenance of melanocyte stem cells in adulthood, while the degree of requirement is distinct in these settings: the latter is more sensitive than the former to the reduced Notch signaling. Furthermore, Notch1 and Notch2 cooperates with c-kit signaling during embryogenesis, and they cooperate with each other to regulate melanocyte homeostasis after birth.