A simple scoring system using type IV collagen 7S and aspartate aminotransferase for diagnosing nonalcoholic steatohepatitis and related fibrosis.

BACKGROUND: Recently we reported novel noninvasive scoring systems for diagnosing nonalcoholic steatohepatitis (NASH) and related fibrosis, namely FM-NASH index and FM-fibro index. They are highly accurate, however, they contain some items not widely used in clinical practice and require six or more items to diagnose both NASH and related fibrosis. By focusing on widely used items, we tried to identify convenient markers in common with the both diagnoses. METHODS: To explore the markers for NASH and related fibrosis in nonalcoholic fatty liver disease (NAFLD) patients, we used data of 24 clinical items in our previous report. By logistic regression analysis, we identified items suitable for the both diagnoses. We then evaluated their accuracies by area under the receiver operator characteristic curves (AUROCs) on independent validation data. RESULTS: We identified the combination of type IV collagen 7S and aspartate aminotransferase (AST) as the predictor both for NASH and related fibrosis. We developed a scoring system based on the combination and evaluated the prediction accuracy: the AUROCs for training/validation data sets are 0.857/0.769 for NASH and 0.918/0.842 for NASH-related fibrosis. The former was higher than that of NAFIC score, and the latter was higher than those of existing fibrosis markers: BARD score, FIB-4 index and NAFLD fibrosis score but lower than FM-fibro index. CONCLUSIONS: The scoring system using type IV collagen 7S and AST named CA index can predict both NASH and related fibrosis in NAFLD patients with sufficient accuracy and could be a convenient diagnostic and screening tool for NASH and related fibrosis. The scoring system needs to be validated in independent larger populations from multiple clinical centers.

Novel Drosophila model for psychiatric disorders including autism spectrum disorder by targeting of ATP-binding cassette protein A.

Autism spectrum disorder (ASD) is characterized by persistent deficits in social communication and social interactions, as well as restricted, stereotyped patterns of behavior and interests. In addition, alterations in circadian sleep-wake rhythm are common in young children with ASD. Mutations in ATP binding cassette subfamily A member 13 (ABCA13) have been recently identified in a monkey that displays behavior associated with ASD. ABCA13, a member of the ABCA family of proteins, is predicted to transport lipid molecules and is expressed in the human trachea, testis, bone marrow, hippocampus, cortex, and other tissues. However, its physiological function remains unknown. Drosophila CG1718 shows high homology to human ABCA genes including ABCA13 and is thus designated as Drosophila ABCA (dABCA). To elucidate the physiological role of dABCA, we specifically knocked down dABCA in all neurons of flies and investigated their phenotypes. The pan-neuron-specific knockdown of dABCA resulted in increased social space with the closest neighbor in adult male flies but exerted no effect on their climbing ability, indicating that the increase in social space is not due to a defect in their climbing ability. An activity assay with adult male flies revealed that knockdown of dABCA in all neurons induces early onset of evening activity in adult flies followed by relatively high activity during morning peaks, evening peaks, and midday siesta. These phenotypes are similar to defects observed in human ASD patients, suggesting that the established dABCA knockdown flies are a promising model for ASD. In addition, an increase in satellite boutons in presynaptic terminals of motor neurons was observed in dABCA knockdown third instar larvae, suggesting that dABCA regulates the formation and/or maintenance of presynaptic terminals of motor neurons.

Distributions of Globotriaosylceramide Isoforms, and Globotriaosylsphingosine and Its Analogues in an α-Galactosidase A Knockout Mouse, a Model of Fabry Disease.

Fabry disease is caused by deficient activity of α-galactosidase A (GLA) and characterized by systemic accumulation of glycosphingolipids, substrates of the enzyme. To gain insight into the pathogenesis of Fabry disease based on accumulated substrates, we examined the tissue and plasma distributions of globotriaosylceramide (Gb3) isoforms, and globotriaosylsphingosine (lyso-Gb3) and its analogues in a GLA knockout mouse, a model of Fabry disease, by means of liquid chromatography-mass spectrometry and nano-liquid chromatography-tandem mass spectrometry, respectively. The results revealed that the contents of these substrates in the liver, kidneys, heart, and plasma of GLA knockout mice were apparently higher than in those of wild-type ones, and organ specificity in the accumulation of Gb3 isoforms was found. Especially in the kidneys, accumulation of a large amount of Gb3 isoforms including hydroxylated residues was found. In the GLA knockout mice, the proportion of hydrophobic Gb3 isoforms was apparently higher than that in the wild-type mice. On the other hand, hydrophilic residues were abundant in plasma. Unlike that of Gb3, the concentration of lyso-Gb3 was high in the liver, and the lyso-Gb3/Gb3 ratio in plasma was significantly higher than those in the organs. The concentration of lyso-Gb3 was apparently higher than those of its analogues in the organs and plasma from both the GLA knockout and wild-type mice. This information will be useful for elucidating the basis of Fabry disease.

Identification of a novel set of biomarkers for evaluating phospholipidosis-inducing potential of compounds using rat liver microarray data measured 24-h after single dose administration.

Phospholipid accumulation manifests as an adverse effect of cationic amphiphilic drugs in particular. Detection, however, by histopathology examination is time-consuming and may require repeated administration of compounds for several weeks. To eliminate compounds with potential for inducing phospholipidosis from the discovery pipeline, we have identified and validated a set of biomarkers for predicting the phospholipidosis-inducing potential utilizing a comprehensive rat transcriptome microarray database created by the Japanese Toxicogenomics and Toxicogenomics Informatics Projects (TGP/TGP2) together with in-house data. The set of biomarkers comprising 25 Affymetrix GeneChip probe sets was identified using genetic algorithm optimization on 24-h time-point microarray data from rats treated with single doses of hepatotoxic compounds including amiodarone, clomipramine, haloperidol, hydroxyzine, imipramine, and perhexiline. The set of novel biomarkers represents an early time-point gene-expression pattern characteristic for a condition eventually leading to phospholipidosis. This implies significant advantages in terms of time and resources over currently published biomarkers derived using repeated-dosing late time-point data. The biomarker set was validated by 11 independent compounds. Accuracy, sensitivity, and specificity values were 82%, 67%, and 100%, respectively and the area under the receiver operating characteristic curve was 0.97. These results show that the biomarker set possesses a high classification accuracy for novel compounds. Pathway analysis was carried out for the biomarkers and the detection of pathways related to lipid-metabolism was statistically significant. These pathways most probably reflect lipid metabolism changes associated with phospholipidosis supporting the validity of our novel biomarkers.

A novel approach for Aß1₋40 quantification using immuno-PCR.

Several lines of evidence suggest that aggregation of the amyloid ß-peptide (Aß) in the brain is a trigger of Alzheimer's disease (AD). Thus, quantification of Aß in several different types of samples from brain is fundamental for understanding AD pathogenesis. For analysis of the low levels of Aß present in microdissected neurons, a more sensitive system than the ELISAs used today would be helpful. Here, we report a novel immuno-PCR (IPCR) system in which the lowest quantitative level of Aß1₋40 is 2 attomol/µL. We use the novel IPCR to quantify the intracellular Aß1₋40 levels in pyramidal neurons microdissected from human brain. We show that the level of Aß1₋40 is around 10 attomol/neuron, and thus, only 3 neurons are needed for analysis.

Analysis of microdissected neurons by 18O mass spectrometry reveals altered protein expression in Alzheimer's disease.

It is evident that the symptoms of Alzheimer's disease (AD) are derived from severe neuronal damage, and especially pyramidal neurons in the hippocampus are affected pathologically. Here, we analysed the proteome of hippocampal neurons, isolated from post-mortem brains by laser capture microdissection. By using (18)O labelling and mass spectrometry, the relative expression levels of 150 proteins in AD and controls were estimated. Many of the identified proteins are involved in transcription and nucleotide binding, glycolysis, heat-shock response, microtubule stabilization, axonal transport or inflammation. The proteins showing the most altered expression in AD were selected for immunohistochemical analysis. These analyses confirmed the altered expression levels, and showed in many AD cases a pathological pattern. For comparison, we also analysed hippocampal sections by Western blot. The expression levels found by this method showed poor correlation with the neuron-specific analysis. Hence, we conclude that cell-specific proteome analysis reveals differences in the proteome that cannot be detected by bulk analysis.

The application of mass spectrometry to proteomics and metabolomics in biomarker discovery and drug development.

Drugs are launched to market after the lengthy process of development. Despite careful preclinical assessment, there is still a significant risk that a drug candidate may elicit adverse effects or display a low level of efficacy during clinical trials. If a drug candidate fails in the latter stages of the clinical process, the overall loss, both in terms of time and money, is enormous. A major concern for the pharmaceutical companies is to improve the drug development process to make it faster and more cost-effective by adoption of new technologies. Biomarkers are emerging as a key tool in identifying potential drug failures at an early stage or in helping to make go/no-go decisions, which should significantly accelerate drug development. Omics technologies play an important role in biomarker discovery as well as in other stages of the drug discovery and development (e.g. target discovery, mechanism of action or predicting toxicity). In particular, recent progress in mass spectrometry techniques such as selected reaction monitoring (SRM) and novel high-resolution features have helped facilitate the realization of the inherent power of proteomics and metabolomics in biomarker discovery, validation and qualification. In this manuscript, we review the current state of proteomics and metabolomics in conjunction with recent technical advances in mass spectrometry with some examples of applications in biomarker research. In addition, we discuss the possible impact of biomarker research with these technologies in drug discovery and development.

Effects of DMSO on gene expression in human and rat hepatocytes.

Dimethyl sulfoxide (DMSO) is a very common organic solvent used for dissolving lipophilic substances, for example for in vitro cell-based assays. At the same time, DMSO is known to be cytotoxic at high concentrations. Therefore, it is important to define threshold concentrations of DMSO for cells but relevant data at the molecular level are very limited. We have focused on conducting microarray analyses of human and rat hepatocytes treated with more than 100 chemicals in attempts to identify candidate biomarker genes. In the present study, the effects of DMSO on gene expression and cytotoxicity were assessed in human cryopreserved hepatocytes and rat primary cultured hepatocytes. A cytotoxicity test with lactate dehydrogenase (LDH) activity demonstrated DMSO to be noncytotoxic up to a concentration of 2% (v/v) in both cases and there were only few effects on the gene expression profiles up to 0.5% (v/v). The observed differences from controls were considered to be of little toxicological importance, but still need to be taken into account in interpretation of findings when DMSO is used at high concentration.

Progressive neuropathology and cognitive decline in a single Arctic APP transgenic mouse model.

The Arctic APP mutation (E693G) leads to dementia with clinical features similar to Alzheimer disease (AD), but little is known about the pathogenic mechanism of this mutation. To address this question, we have generated a transgenic mouse model, TgAPParc, with neuron-specific expression of human APP with the Arctic mutation (hAPParc). Heterozygous mice from two separate founder lines with different levels of expression of hAPParc were analyzed with respect to brain morphology and behavior every 3 months until the age of 18 months. Standard histological stainings and immunohistochemistry using a panel of Aß antibodies showed an age- and dose-dependant progression of amyloid deposition in the brain, starting in the subiculum and spreading to the thalamus. Cognitive behavioral testing revealed deficits in hippocampus-dependent spatial learning and memory in the Barnes maze test. This study demonstrates that the Arctic APP mutation is sufficient to cause amyloid deposition and cognitive dysfunction, and thus the TgAPParc mouse model provides a valuable tool to study the effect of the Arctic mutation in vivo without possible confounding effect of other APP mutations.

Generating induced pluripotent stem cells from common marmoset (Callithrix jacchus) fetal liver cells using defined factors, including Lin28.

Although embryonic stem (ES) cell-like induced pluripotent stem (iPS) cells have potential therapeutic applications in humans, they are also useful for creating genetically modified human disease models in nonhuman primates. In this study, we generated common marmoset iPS cells from fetal liver cells via the retrovirus-mediated introduction of six human transcription factors: Oct-3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28. Four to five weeks after introduction, several colonies resembling marmoset ES cells were observed and picked for further expansion in ES cell medium. Eight cell lines were established, and validation analyses of the marmoset iPS cells followed. We detected the expression of ES cell-specific surface markers. Reverse transcription-PCR showed that these iPS cells expressed endogenous Oct-3/4, Sox2, Klf4, c-Myc, Nanog and Lin28 genes, whereas all of the transgenes were silenced. Karyotype analysis showed that two of three iPS cell lines retained a normal karyotype after a 2-month culture. Both embryoid body and teratoma formation showed that marmoset iPS cells had the developmental potential to give rise to differentiated derivatives of all three primary germ layers. In summary, we generated marmoset iPS cells via the transduction of six transcription factors; this provides a powerful preclinical model for studies in regenerative medicine.

Analysis of microdissected human neurons by a sensitive ELISA reveals a correlation between elevated intracellular concentrations of Abeta42 and Alzheimer's disease neuropathology.

In Alzheimer's disease (AD), Purkinje neurons in the cerebellum are spared, while, for instance, pyramidal neurons in the hippocampus are neuropathologically affected. Several lines of evidence suggest that the pathogenesis could be induced by the concentration-dependent polymerization of the amyloid beta-peptide (Abeta) into extracellular oligomers. The role of intracellular Abeta is not fully investigated, but recent data indicate that also this pool could be of importance. Here, we use laser capture microdissection microscopy for isolation of Purkinje neurons from AD cases and controls, and quantify the low levels of intracellular Abeta using a novel and highly sensitive ELISA. Similar to Cornu Ammonis 1 pyramidal neurons, the intracellular levels of the most toxic variant, Abeta42, as well as the Abeta42/Abeta40 ratio, were increased in Purkinje neurons from sporadic AD cases as compared to controls. However, the levels of Abeta42 as well as Abeta40 were clearly lower in Purkinje neurons than in pyramidal neurons. Based on the volume of the captured Purkinje neurons, the intraneuronal concentrations of Abeta42 were calculated to be 200 nM in sporadic AD cases and 90 nM in controls. The corresponding concentrations in pyramidal neurons from hippocampus were 3 muM and 660 nM, respectively. The Abeta40 concentration was not significantly altered in AD cases compared to controls. However, we found ten times higher concentration of Abeta40 in pyramidal neurons (10 muM) compared to Purkinje neurons (1 muM). Finally, we suggest that high concentration of intracellular Abeta42 correlates with vulnerability to AD neuropathology.

Synaptic and endosomal localization of active gamma-secretase in rat brain.

BACKGROUND: A key player in the development of Alzheimer's disease (AD) is the gamma-secretase complex consisting of at least four components: presenilin, nicastrin, Aph-1 and Pen-2. gamma-Secretase is crucial for the generation of the neurotoxic amyloid beta-peptide (Abeta) but also takes part in the processing of many other substrates. In cell lines, active gamma-secretase has been found to localize primarily to the Golgi apparatus, endosomes and plasma membranes. However, no thorough studies have been performed to show the subcellular localization of the active gamma-secretase in the affected organ of AD, namely the brain. PRINCIPAL FINDINGS: We show by subcellular fractionation of rat brain that high gamma-secretase activity, as assessed by production of Abeta40, is present in an endosome- and plasma membrane-enriched fraction of an iodixanol gradient. We also prepared crude synaptic vesicles as well as synaptic membranes and both fractions showed high Abeta40 production and contained high amounts of the gamma-secretase components. Further purification of the synaptic vesicles verified the presence of the gamma-secretase components in these compartments. The localization of an active gamma-secretase in synapses and endosomes was confirmed in rat brain sections and neuronal cultures by using a biotinylated gamma-secretase inhibitor together with confocal microscopy. SIGNIFICANCE: The information about the subcellular localization of gamma-secretase in brain is important for the understanding of the molecular mechanisms of AD. Furthermore, the identified fractions can be used as sources for highly active gamma-secretase.

Affinity pulldown of γ-secretase and associated proteins from human and rat brain.

γ-Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins, including amyloid precursor protein (APP) and Notch. A functional complex is dependent on the assembly of four proteins: presenilin (PS), nicastrin, Aph-1 and Pen-2. Little is known about how the substrates are selected by γ-secretase, but it has been suggested that γ-secretase associated proteins (GSAPs) could be of importance. For instance, it was recently reported from studies in cell lines that TMP21, a transmembrane protein involved in trafficking, binds to γ-secretase and regulates the processing of APP-derived substrates without affecting Notch cleavage. Here, we present an efficient and selective method for purification and analysis of γ-secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a γ-secretase inhibitor coupled to biotin via a long linker and a S-S bridge. After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to LC-MS/MS analysis, and proteins were identified by sequence data from MS/MS spectra. All of the known γ-secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ-secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the γ-secretase complex.

CD147, a gamma-secretase associated protein is upregulated in Alzheimer's disease brain and its cellular trafficking is affected by presenilin-2.

Gamma-secretase activity has been extensively investigated due to its role in Alzheimer's disease. Here, we studied the association of CD147, a transmembrane glycoprotein belonging to the immunoglobulin family, with gamma-secretase and its expression in Alzheimer's disease and control tissues. Subcellular fractionation of postmitochondrial supernatant from rat brain on step iodixanol gradient in combination with co-immunoprecipitation using an anti-nicastrin antibody showed association of limited amount of CD147 to gamma-secretase. By immunoblotting of postnuclear pellets from Alzheimer's disease and control human brain tissues we showed that CD147 with molecular weight 75 kDa is upregulated in frontal cortex and thalamus of the Alzheimer's disease brains. Immunohistochemistry of brain tissues from Alzheimer's disease and control revealed specific upregulation of CD147 in neurons, axons and capillaries of Alzheimer's disease frontal cortex and thalamus. The effect of presenilin-1 and -2, which are the catalytic subunits of gamma-secretase, on CD147 expression and subcellular localization was analyzed by confocal microscopy in combination with flow cytometry and showed that PS2 affected the subcellular localization of CD147 in mouse embryonic fibroblast cells. We suggest that a small fraction of CD147 present in the brain is associated with the gamma-secretase, and can be involved in mechanisms dysregulated in Alzheimer's disease brain.

Amyloid beta-peptide levels in laser capture microdissected cornu ammonis 1 pyramidal neurons of Alzheimer's brain.

Deposition of the amyloid beta-peptide (Abeta) is a pathophysiological event associated with Alzheimer's disease. Although much is known about the molecular composition of extracellular Abeta deposits, the role of the intracellular pool of Abeta is not fully understood. We investigated whether Abeta levels are increased in cornu ammonis 1 pyramidal neurons of Alzheimer's disease hippocampus, using laser capture microdissection to isolate the neurons and enzyme-linked immunosorbent assay for quantification. Our results showed increased Abeta42 levels and an elevated Abeta42/Abeta40 ratio in neurons from sporadic as well as from familial cases of Alzheimer's disease, whereas Abeta40 levels remain unchanged between the cases and controls. We speculate that intracellular accumulation of Abeta42 increase vulnerability of cornu ammonis 1 pyramidal neurons in Alzheimer's disease.

Active gamma-secretase is localized to detergent-resistant membranes in human brain.

Several lines of evidence suggest that polymerization of the amyloid beta-peptide (Abeta) into amyloid plaques is a pathogenic event in Alzheimer's disease (AD). Abeta is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by beta-secretase and gamma-secretase, and it has been suggested that these enzymes could be targets for treatment of AD. gamma-Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines have shown that gamma-secretase is partially localized to lipid rafts, which are detergent-resistant membrane microdomains enriched in cholesterol and sphingolipids. Here, we studied gamma-secretase in detergent-resistant membranes (DRMs) prepared from human brain. DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in gamma-secretase components and activity. The DRM fraction was subjected to size-exclusion chromatography in CHAPSO, and all of the gamma-secretase components and a lipid raft marker were found in the void volume (> 2000 kDa). Co-immunoprecipitation studies further supported the notion that the gamma-secretase components are associated even at high concentrations of CHAPSO. Preparations from rat brain gave similar results and showed a postmortem time-dependent decline in gamma-secretase activity, suggesting that DRMs from fresh rat brain may be useful for gamma-secretase activity studies. Finally, confocal microscopy showed co-localization of gamma-secretase components and a lipid raft marker in thin sections of human brain. We conclude that the active gamma-secretase complex is localized to lipid rafts in human brain.

Rat brain gamma-secretase activity is highly influenced by detergents.

Gamma-secretase is important for the development of Alzheimer's disease, since it is a crucial enzyme for the generation of the pathogenic amyloid beta-peptide (Abeta). Most data on gamma-secretase is derived from studies in cell lines overexpressing gamma-secretase components or amyloid precursor protein (APP), and since gamma-secretase is a transmembrane protein complex, detergents have been frequently used to facilitate the studies. However, no extensive comparison of the influence of different detergents at different concentrations on gamma-secretase activity in preparations from brain has been made. Here, we establish the optimal conditions for gamma-secretase activity in rat brain, using an activity assay detecting endogenous production of the APP intracellular domain, which is generated when gamma-secretase cleaves the APP C-terminal fragments. We performed a subcellular fractionation and noted the highest gamma-secretase activity in the 100000g pellet and that the optimal pH was around 7. We found that gamma-secretase was active for at least 16 h at 37 degrees C and that the endogenous substrate levels were sufficient for activity measurements. The highest activity was obtained in 0.4% CHAPSO, which is slightly below the critical micelle concentration (0.5%) for this detergent, but the complex was not solubilized efficiently at this concentration. On the other hand, 1% CHAPSO solubilized a substantial amount of the gamma-secretase components, but the activity was low. The activity was fully restored by diluting the sample to 0.4% CHAPSO. Therefore, using 1% CHAPSO for solubilization and subsequently diluting the sample to 0.4% is an appropriate procedure for obtaining a soluble, highly active gamma-secretase from rat brain.

RNA interference in immune cells by use of osmotic delivery of siRNA.

Delivery of siRNA to immune cells has been one of the major obstacles to widespread application of RNAi in the immunology field. Here, we report that osmotic delivery of siRNA can be used to silence genes in macrophage RAW264.7 without incurring either cytotoxic or immunomodulatory activity. We also showed usefulness of the osmotic delivery in other types of cells including T cell DO11.10. By repeated osmotic delivery of siRNA, long-term gene silencing was readily achieved. When TLR4 was disrupted in RAW264.7 cells for 48 h and the cells were stimulated with the TLR4 ligand LPS, a significant decrease in TNFalpha production was observed. DNA microarray-based gene expression profile analysis showed that gene silencing by osmotic delivery of siRNA was target-specific and the delivery method itself had little influence on overall gene expression.

Prediction of siRNA functionality using generalized string kernel and support vector machine.

Small interfering RNAs (siRNAs) are becoming widely used for sequence-specific gene silencing in mammalian cells, but designing an effective siRNA is still a challenging task. In this study, we developed an algorithm for predicting siRNA functionality by using generalized string kernel (GSK) combined with support vector machine (SVM). With GSK, siRNA sequences were represented as vectors in a multi-dimensional feature space according to the numbers of subsequences in each siRNA, and subsequently classified with SVM into effective or ineffective siRNAs. We applied this algorithm to published siRNAs, and could classify effective and ineffective siRNAs with 90.6%, 86.2% accuracy, respectively.

Gene-inducing program of human dendritic cells in response to BCG cell-wall skeleton (CWS), which reflects adjuvancy required for tumor immunotherapy.

Adjuvants induce the expression of a number of genes in dendritic cells (DCs), which facilitate effective antigen-presentation and cytokine/chemokine liberation. It has been accepted that the toll-like receptor (TLR) family governs the adjuvant activity in DCs. An adjuvant with a long history is mycobacteria in an oil-in-water emulsion, namely Freund's complete adjuvant. Since the active center for the adjuvancy in mycobacteria is the cell-wall skeleton (CWS), we used the bacillus Calmette-Guerin cell-wall skeleton (BCG-CWS) to test DC maturation by GeneChip analysis. We identified the genes supporting an efficient DC response and output. Approximately 2000 genes were up-regulated by BCG-CWS stimulation. BCG-CWS-, peptidoglycan (PGN)- and lipopolysaccharide (LPS)-stimulation generally up-regulated some gene clusters including genes for inflammatory cytokines (TNF, IL1alpha, IL1beta, IL6, IL12 p40, IL23 p19, etc.), chemokines (CCL20, IL8, etc.), cell adhesion molecules (ICAM-1, etc.), apoptosis-related proteins (GADD45B, BCL2A1, etc.), metabolic enzymes (PTGS2, SOD2, etc.) and miscellaneous proteins (EHD1, TNFAIP6, etc.). LPS-stimulation, but not BCG-CWS- or PGN-stimulation, up-regulated the interferon-inducible antiviral proteins, including IFIT1, IFIT2, IFIT4, CXCL10, ISG15, OASL, IFITM1 and MX1. We also found that the BCG-CWS- or PGN-stimulation up-regulated CXCL5, MMP1, etc. We discussed their properties in association with TLRs and recently discovered TLR adapters.