Plasma cell-free DNA methylation marks for episodic memory impairment: a pilot twin study.

Decline in episodic memory performance usually causes the first clinical symptoms of Alzheimer's disease. At present, Alzheimer's disease can only be diagnosed at a very late stage when neurodegeneration and cognitive impairment is already irreversible. New early disease markers are needed for earlier and more efficient Alzheimer's disease intervention. To identify early disease markers, we implemented a genome-wide bisulphite sequencing method for the analysis of plasma cell-free DNA methylation profiles and compared differences associated with episodic memory performance in Finnish twin pairs. A noticeable amount of cell-free DNA was present in plasma, however, the amounts as well as the genomic coverage of these fragments varied substantially between individuals. We found no significant markers associated with episodic memory performance in the twins' plasma cell-free DNA methylation profiles. Furthermore, our results indicate that due to the low genomic coverage of cell-free DNA fragments and the variety in these fragments between individuals, the implemented genome-wide bisulphite sequencing method is not optimal for comparing cell-free DNA methylation differences between large groups of individuals.

Atopic asthma after rhinovirus-induced wheezing is associated with DNA methylation change in the SMAD3 gene promoter.

Children with rhinovirus-induced severe early wheezing have an increased risk of developing asthma later in life. The exact molecular mechanisms for this association are still mostly unknown. To identify potential changes in the transcriptional and epigenetic regulation in rhinovirus-associated atopic or nonatopic asthma, we analyzed a cohort of 5-year-old children (n = 45) according to the virus etiology of the first severe wheezing episode at the mean age of 13 months and to 5-year asthma outcome. The development of atopic asthma in children with early rhinovirus-induced wheezing was associated with DNA methylation changes at several genomic sites in chromosomal regions previously linked to asthma. The strongest changes in atopic asthma were detected in the promoter region of SMAD3 gene at chr 15q22.33 and introns of DDO/METTL24 genes at 6q21. These changes were validated to be present also at the average age of 8 years.

New Insights and Biomarkers for Type 1 Diabetes: Review for Scandinavian Journal of Immunology.

The increasing incidence of type 1 diabetes observed in the past 60 years has spawned massive efforts in multiple research fields to elucidate the aetiology of this disease. While GWAS studies provide a good genetic basis for the current knowledge, it is clear that environmental triggers and their influence in disease prevalence and origin are highly important. The realization of disease heterogeneity has created a requirement for better biomarkers to complement the known autoantibody markers and to more successfully predict the severity and onset time of the disease. Such biomarkers would be needed both for prevention as well as for monitoring disease activity and response to preventive and therapeutic measures. Systematic holistic approaches concentrating on the triggering molecular mechanisms, pancreatic beta cells, immune response, as well as the influence of diet and environment, are necessary to understand the disease pathogenesis and find a cure. The current genomic knowledge is being broadened with accompanying studies in epigenetics and transcriptomic regulation, metabolomics, proteomics and lipidomics, covering the whole system from beta cells, the profile and cellular balance of the infiltrating lymphocytes, to gut microbiota and viral infections. Here we highlight interesting recent findings in type 1 diabetes research.

Holistic systems biology approaches to molecular mechanisms of human helper T cell differentiation to functionally distinct subsets.

Current knowledge of helper T cell differentiation largely relies on data generated from mouse studies. To develop therapeutical strategies combating human diseases, understanding the molecular mechanisms how human naïve T cells differentiate to functionally distinct T helper (Th) subsets as well as studies on human differentiated Th cell subsets is particularly valuable. Systems biology approaches provide a holistic view of the processes of T helper differentiation, enable discovery of new factors and pathways involved and generation of new hypotheses to be tested to improve our understanding of human Th cell differentiation and immune-mediated diseases. Here, we summarize studies where high-throughput systems biology approaches have been exploited to human primary T cells. These studies reveal new factors and signalling pathways influencing T cell differentiation towards distinct subsets, important for immune regulation. Such information provides new insights into T cell biology and into targeting immune system for therapeutic interventions.

Introduction of complementary foods in infancy and atopic sensitization at the age of 5 years: timing and food diversity in a Finnish birth cohort.

OBJECTIVE: To study the associations between timing and diversity of introduction of complementary foods during infancy and atopic sensitization in 5-year-old children. METHODS: In the Finnish DIPP (type 1 diabetes prediction and prevention) birth cohort (n = 3781), data on the timing of infant feeding were collected up to the age of 2 years and serum IgE antibodies toward four food and four inhalant allergens measured at the age of 5 years. Logistic regression was used for the analyses. RESULTS: Median duration of exclusive and total breastfeeding was 1.4 (interquartile range: 0.2-3.5) and 7.0 (4.0-11.0) months, respectively. When all the foods were studied together and adjusted for confounders, short duration of breastfeeding decreased the risk of sensitization to birch allergen; introduction of oats <5.1 months and barley <5.5 months decreased the risk of sensitization to wheat and egg allergens, and oats additionally associated with milk, timothy grass, and birch allergens. Introduction of rye <7.0 months decreased the risk of sensitization to birch allergen. Introduction of fish <6 months and egg ≤11 months decreased the risk of sensitization to all the specific allergens studied. The introduction of <3 food items at 3 months was associated with sensitization to wheat, timothy grass, and birch allergens; the introduction of 1-2 food items at 4 months and ≤4 food items at 6 months was associated with all endpoints, but house dust mite. These results were particularly evident among high-risk children when the results were stratified by atopic history, indicating the potential for reverse causality. CONCLUSIONS: The introduction of complementary foods was consecutively done, and with respect to the timing of each food, early introduction of complementary foods may protect against atopic sensitization in childhood, particularly among high-risk children. Less food diversity as already at 3 months of age may increase the risk of atopic sensitization.

Enterovirus-induced gene expression profile is critical for human pancreatic islet destruction.

AIMS/HYPOTHESIS: Virally induced inflammatory responses, beta cell destruction and release of beta cell autoantigens may lead to autoimmune reactions culminating in type 1 diabetes. Therefore, viral capability to induce beta cell death and the nature of virus-induced immune responses are among key determinants of diabetogenic viruses. We hypothesised that enterovirus infection induces a specific gene expression pattern that results in islet destruction and that such a host response pattern is not shared among all enterovirus infections but varies between virus strains. METHODS: The changes in global gene expression and secreted cytokine profiles induced by lytic or benign enterovirus infections were studied in primary human pancreatic islet using DNA microarrays and viral strains either isolated at the clinical onset of type 1 diabetes or capable of causing a diabetes-like condition in mice. RESULTS: The expression of pro-inflammatory cytokine genes (IL-1-α, IL-1-ß and TNF-α) that also mediate cytokine-induced beta cell dysfunction correlated with the lytic potential of a virus. Temporally increasing gene expression levels of double-stranded RNA recognition receptors, antiviral molecules, cytokines and chemokines were detected for all studied virus strains. Lytic coxsackievirus B5 (CBV-5)-DS infection also downregulated genes involved in glycolysis and insulin secretion. CONCLUSIONS/INTERPRETATION: The results suggest a distinct, virus-strain-specific, gene expression pattern leading to pancreatic islet destruction and pro-inflammatory effects after enterovirus infection. However, neither viral replication nor cytotoxic cytokine production alone are sufficient to induce necrotic cell death. More likely the combined effect of these and possibly cellular energy depletion lie behind the enterovirus-induced necrosis of islets.

Identification of a c-Jun N-terminal kinase-2-dependent signal amplification cascade that regulates c-Myc levels in ras transformation.

Ras is one of the most frequently activated oncogenes in cancer. Two mitogen-activated protein kinases (MAPKs) are important for ras transformation: extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase 2 (JNK2). Here we present a downstream signal amplification cascade that is critical for ras transformation in murine embryonic fibroblasts. This cascade is coordinated by ERK and JNK2 MAPKs, whose Ras-mediated activation leads to the enhanced levels of three oncogenic transcription factors, namely, c-Myc, activating transcription factor 2 (ATF2) and ATF3, all of which are essential for ras transformation. Previous studies show that ERK-mediated serine 62 phosphorylation protects c-Myc from proteasomal degradation. ERK is, however, not alone sufficient to stabilize c-Myc but requires the cooperation of cancerous inhibitor of protein phosphatase 2A (CIP2A), an oncogene that counteracts protein phosphatase 2A-mediated dephosphorylation of c-Myc. Here we show that JNK2 regulates Cip2a transcription via ATF2. ATF2 and c-Myc cooperate to activate the transcription of ATF3. Remarkably, not only ectopic JNK2, but also ectopic ATF2, CIP2A, c-Myc and ATF3 are sufficient to rescue the defective ras transformation of JNK2-deficient cells. Thus, these data identify the key signal converging point of JNK2 and ERK pathways and underline the central role of CIP2A in ras transformation.

Exosomes - nanovesicles with possible roles in allergic inflammation.

Exosomes are nano-sized membrane vesicles which are released extracellularly after fusion of multivesicular endosomes with the cell membrane. Despite their characteristic composition of proteins compared to the cell membrane, no exosome-specific molecule has so far been characterized. Exosomes are found in bronchoalveolar lavage (BAL), urine, serum and breast milk, and are released from several cells implicated in allergy including mast cells, dendritic cells (DC), T cells and epithelial cells. Antigen-loaded exosomes have been shown to be highly immunogenic and we propose that exosomes could be a modulating factor in allergic responses. Allergen-presenting exosomes could transport allergen and stimulate allergen-specific T cells, and possibly also biasing T cell responses depending on the molecules present on the exosome surface. Furthermore, exosomes from mast cells, highly active in allergic reactions, have been found to induce DC maturation and also to be able to transport functional RNA to recipient cells, suggesting a new pathway for cell communication. Reversely, tolerizing exosomes e.g. tolerosomes, from gut or breast milk, could block an allergic response or prevent allergy development. A better understanding of the role of exosomes in allergies could make us understand how allergy can be prevented or lead to the development of more efficient treatments.

The RhoA transcriptional program in pre-T cells.

The GTPase RhoA is essential for the development of pre-T cells in the thymus. To investigate the mechanisms used by RhoA to control thymocyte development we have used Affymetrix gene profiling to identify RhoA regulated genes in T cell progenitors. The data show that RhoA plays a specific and essential role in pre-T cells because it is required for the expression of transcription factors of the Egr-1 and AP-1 families that have critical functions in thymocyte development. Loss of RhoA function in T cell progenitors causes a developmental block that pheno-copies the consequence of losing pre-TCR expression in Recombinase gene 2 (Rag2) null mice. Transcriptional profiling reveals both common and unique gene targets for RhoA and the pre-TCR indicating that RhoA participates in the pre-TCR induced transcriptional program but also mediates pre-TCR independent gene transcription.

Allergen-induced in vitro expression of IL-18, SLAM and GATA-3 mRNA in PBMC during sublingual immunotherapy.

BACKGROUND: Signalling lymphocytic activation molecule (SLAM) and interleukin (IL)-18 induce interferon (IFN)-gamma production from Th1 cells. The allergen-induced SLAM and IL-18 mRNA expressions are increased during subcutaneous immunotherapy (SCIT), but nothing is known about their role during sublingual immunotherapy (SLIT). Transcription factor GATA-3 is associated with Th2 cells but its role in SCIT and SLIT is yet unexplored. This study was undertaken to analyse the allergen induced in vitro mRNA expression of IL-18, SLAM and GATA-3 in peripheral blood mononuclear cells (PBMC) of children with allergic rhinitis (AR) during SLIT. METHODS: Ten patients with AR undergoing pollen SLIT with a weekly dose of 200,000 SQ-U, 10 with 24,000 SQ-U of mixture of Betula verrucosa, Corylus avellana and Alnus glutinosa and 10 with placebo were included. Peripheral blood mononuclear cell were stimulated with birch extract prior to, after 1 and 2 years of the treatment. The mRNA expression was assessed using kinetic real-time RT-PCR (TaqMan); Applied Biosystems, Foster City, CA, USA). RESULTS: The expression of IL-18 mRNA was increased in the high-dose group in comparison to the placebo group after 1 year of therapy (P = 0.028) and had an inverse correlation with the late phase skin reaction after the second study year (r = -0.41, P = 0.041). SLAM mRNA expression increased in the high-dose group from baseline to 1 year (P = 0.028) and correlated with IL-10 (r = 0.96, P < 0.0001) and transforming growth factor-beta (r = 0.80, P = 0.0037) mRNA expression. No significant changes were seen in GATA-3 mRNA expression. CONCLUSIONS: During SLIT, IL-18 and SLAM are upregulated, suggesting that the Th2 type inflammatory response is downregulated during SLIT by increased Th1 type response.

Intranasal administration of chlamydial outer protein N (CopN) induces protection against pulmonary Chlamydia pneumoniae infection in a mouse model.

Chlamydia pneumoniae is an intracellular pathogen that grows inside a vacuole, referred to as an inclusion. C. pneumoniae possess a type III secretion system (TTSS), which allows them to secrete effector molecules into the inclusion membrane and to the host cell cytosol. Proteins such as chlamydial outer protein N (CopN) that associate with the inclusion membrane are potential targets for the host's MHC-dependent antigen presentation, thereby representing ideal antigen candidates for T cell-based vaccination. The results of this study showed that intranasal immunization of BALB/c mice with heat-aggregated CopN protein and an Escherichia coli heat-labile toxin (LT) induced a strong immune response, detected as antigen-specific antibody production, lymphocyte proliferation and IFN-gamma production. Furthermore, the immunization induced statistically significant protection against intranasal C. pneumoniae challenge, the level of which correlated with the magnitude of CopN-specific lymphocyte proliferation. Both heat-aggregation of the antigen and the presence of LT adjuvant were required for maximal protective effect.

Clearance of Chlamydia pneumoniae infection in H-2 class I human leucocyte antigen-A2.1 monochain transgenic mice.

CD8+ T cells have been suggested to play an important role in protective immunity against pulmonary Chlamydia pneumoniae infection in mice. Moreover, several classical major histocompatibility complex class I - restricted cytotoxic CD8+ T lymphocytes (CTL) specific for C. pneumoniae- derived peptides have been identified. Here, we studied the outcome of C. pneumoniae infection in human leucocyte antigen (HLA)-A2.1 transgenic mice (HHD mice) that are only able to express a classical human class I molecule (HLA-A2.1). C. pneumoniae infection was self-restricted in HHD mice which were able to develop specific immune responses and a protective immunity against a subsequent rechallenge in a manner comparable to wildtype mice. Furthermore, accumulation of functional and C. pneumoniae-specific T cells to the site of infection was detected after challenge. Antigen processing and HLA-A2.1-dependent presentation was studied by immunizing the HHD mice with chlamydial outer protein N (CopN). Isolation of a peptide-specific CTL line from the CopN-immunized mice suggests that the HLA-A2.1 molecule can support the development of CTL response against a chlamydial protein in mice. These findings suggest that the transgenic mouse model can be used for further characterization of the HLA-A2.1-restricted CD8+ T-cell response during C. pneumoniae infection and for identification of CD8 epitopes from chlamydial antigens.

A human ImmunoChip cDNA microarray provides a comprehensive tool to study immune responses.

DNA microarray technology has developed rapidly in recent years and has become an essential tool, providing novel approaches to biomedical research. In this paper, we describe a self-designed ImmunoChip cDNA array for immunological research. With a comprehensive selection of genes of interest, we can focus on key signalling pathways and molecular mechanisms at relatively low cost compared to commercial platforms which are usually targeted at global screening of gene expression. To validate the efficiency of the ImmunoChip, we studied T helper cell polarization to functionally distinct subsets (Th1 and Th2). We also developed a tool for quality control of cDNA microarrays that assesses the technical quality of an ImmunoChip. The information produced with the quality control tool is shown to be valuable for extracting correct information from cDNA microarrays. Gene expression measurements with ImmunoChip are in agreement with the results obtained using oligonucleotide microarrays and with published quantitative RT-PCR data. The ImmunoChip provides reliable measurements and gives new insights into various aspects of human immune responses.

Global profiling of coxsackievirus- and cytokine-induced gene expression in human pancreatic islets.

AIMS/HYPOTHESIS: It is thought that enterovirus infections initiate or facilitate the pathogenetic processes leading to type 1 diabetes. Exposure of cultured human islets to cytolytic enterovirus strains kills beta cells after a protracted period, suggesting a role for secondary virus-induced factors such as cytokines. METHODS: To clarify the molecular mechanisms involved in virus-induced beta cell destruction, we analysed the global pattern of gene expression in human islets. After 48 h, RNA was extracted from three independent human islet preparations infected with coxsackievirus B5 or exposed to interleukin 1beta (50 U/ml) plus interferon gamma (1,000 U/ml), and gene expression profiles were analysed using Affymetrix HG-U133A gene chips, which enable simultaneous analysis of 22,000 probe sets. RESULTS: As many as 13,077 genes were detected in control human islets, and 945 and 1293 single genes were found to be modified by exposure to viral infection and the indicated cytokines, respectively. Four hundred and eighty-four genes were similarly modified by the cytokines and viral infection. CONCLUSIONS/INTERPRETATION: The large number of modified genes observed emphasises the complex responses of human islet cells to agents potentially involved in insulitis. Notably, both cytokines and viral infection significantly (p<0.02) increased the expression of several chemokines, the cytokine IL-15 and the intercellular adhesion molecule ICAM-1, which might contribute to the homing and activation of mononuclear cells in the islets during infection and/or an early autoimmune response. The present results provide novel insights into the molecular mechanisms involved in viral- and cytokine-induced human beta cell dysfunction and death.

DNA microarray-based gene expression profiles of cytomegalovirus infection and acute rejection in liver transplants.

An association between cytomegalovirus (CMV) infection and alloresponse has been suggested. CMV increases inflammation and adhesion molecule expression in graft, and induces cytokines and growth factors, linked with transplant vasculopathy and chronic rejection. We have investigated the gene expression of various inflammatory factors in the CMV-associated immune response and compared this with the immune response of acute rejection in liver transplants by using DNA microarray technology. Gene expression was studied at mRNA level in biopsies from liver transplant patients experiencing CMV infection or acute rejection. RNA extracted from liver grafts after reperfusion was used as control material. Among the strongly upregulated genes in the specimens obtained from liver transplants during CMV infection were IFN-gamma, caspases 1 and 3, granzymes A and B, TGF-beta receptors II and III, IL-10 receptor alpha, VCAM-1, TNF receptor, IL-4, TNF-alpha, IL-10, IL-2 receptor beta, IL-1beta, PDGF-receptor beta, vascular adhesion protein-1, TGF-beta2, and ICAM-1. In biopsies with acute liver allograft rejection, the most significantly upregulated genes were MHC class II, IFN-gamma, caspases 1 and 3, IL-2R beta and gamma, granzymes A and B, VLA-4, L-selectin, E-selectin, VCAM-1, and IL-1beta. Upregulated genes common for CMV and alloresponse were granzyme A and B, E-selection, IFN-gamma, VCAM-1, VLA-4, TNF, caspases 1, 3, and 8, and PDGF. Microarray analysis defined different entities in the immune responses of CMV infection and acute rejection. The differences and similarities of the gene expression profiles related to those in CMV infection and rejection may help to understand the intragraft immunologic events.

Association study of 15 novel single-nucleotide polymorphisms of the T-bet locus among Finnish asthma families.

OBJECTIVE: T-box expressed in T cells (T-bet) is a transcription factor regulating the commitment of T helper (Th) cells by driving the cells into the Th1 direction. Abnormal Th1/Th2 balance may lead to complex disorders like asthma or autoimmune diseases. Recent studies have suggested that T-bet might be a candidate gene for asthma. This led us to screen 23 Finnish individuals for single-nucleotide polymorphisms (SNPs) in the T-bet locus and study the association between the SNPs and high serum IgE level and asthma. METHODS: We screened all six exons, adjacent intronic areas and 2 kb of the 5'-flanking region from 23 individuals utilizing WAVE trade mark technology. To explore whether T-bet is associated in serum IgE regulation or asthma we genotyped the SNPs in a Finnish asthmatic founder population. The association analyses were made using haplotype pattern mining. RESULTS: Fifteen novel SNPs were found in the T-bet gene. Within the Finnish asthmatic founder population, there was no association between T-bet SNPs and high serum IgE level or asthma. CONCLUSIONS: The genetic variability in the T-bet gene does not play a role in the pathogenesis of human asthma. Our results provide a novel panel of SNPs in T-bet and will help determine whether the SNPs have a functional role in other T cell-mediated diseases.

Downregulation of mafB expression in T-helper cells during early differentiation in vitro.

We have studied the expression of a human homologue of mafB (maf-1), a member of the family of large maf transcription factors. In support of the suggested key role that mafB expression plays in differentiating macrophages, we found mafB to be expressed at a very high level in monocytic U937 and THP-1 cell lines. However, we show here that mafB transcription is not restricted to myeloid cells but can also be detected in lymphoid cells, indicating transcriptional plasticity during haematopoiesis. In conclusion, strong proliferative signals mediated by T-cell activation and interleukins (IL-4 and IL-12) downregulate the mafB messenger RNA transcript level when resting naïve CD4+ T-helper cells enter the differentiation pathway in vitro.

Identification and validation of endogenous reference genes for expression profiling of T helper cell differentiation by quantitative real-time RT-PCR.

Real-time RT-PCR method was exploited to identify endogenous reference genes in differentiating human T helper cells. When using this technology in our experimental system, finding a set of genes whose mRNA expression levels would not change appeared to be very challenging. Our initial plan to use the expression level of GAPDH in normalizing the results failed, because the mRNA expression of GAPDH underwent significant changes during the cell culture. Additional studies on the transcription of several other classical housekeeping genes led to similar results. Our second approach was to use results from an extensive survey of gene expression done by oligonucleotide microarrays and to select another panel of genes for testing. This resulted in the identification of three genes whose expression was relatively stable in our experimental system and, therefore, suitable as endogenous reference genes in these cells. The results indicate that the expression level of a constitutively expressed gene may change during the cell culture in vitro, which emphasizes again the importance of carefully validating endogenous control genes for comparative quantification.

Distinct gene expression profiles of human type 1 and type 2 T helper cells.

BACKGROUND: The development and activation of CD4+ helper T cell (Th) subsets with distinct patterns of unbalanced production of cytokines play an important part in infectious, allergic and autoimmune diseases. Human neonatal cord blood CD4+ Th cells can be polarized into type 1 or type 2-like effector cells in vitro by culturing them in the presence of interleukin (IL)-12 or IL-4, respectively. We have exploited this experimental system to identify marker genes that are differentially expressed by polarized Th1 and Th2 cells. An oligonucleotide microarray specifically designed to screen for inflammation-related candidate genes was used and the differential expression was further validated with a quantitative real-time RT-PCR method. RESULTS: In addition to the previously described marker genes of Th cells, we report subtle changes in the expression of several other genes that represent growth factors, receptors and other signaling molecules in polarized Th1 and Th2 cell subsets. Additionally, we describe a novel set of genes as Th1/Th2 differentiation markers for cells activated by anti-CD3 and anti-CD28 antibodies. CONCLUSIONS: This study demonstrates the power of the targeted use of microarrays in combination with quantitative real-time RT-PCR in identifying and validating new marker genes for gene expression studies.

A proteome database of human primary T helper cells.

We have established the first public database of human primary T helper cell proteome using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry. For the database, CD4+ human T cells were activated with anti-CD3+anti-CD28 antibodies and metabolically labeled with [35S]methionine for 24 h. Cells were lysed and proteins were separated by 2-DE. About 1500 protein spots were detected in the resulting 2-DE gels with silver staining, and 2000 spots with autoradiography. We have identified 91 proteins from the 2-DE gels using peptide mass fingerprinting, and annotated them to our database. The identified proteins are also linked to SWISS-PROTand NCBI protein databases. Our database is available via the Internet at http://www3.btk.utu.fi:8080/Genomics/Proteomics/Database.