Biomedical subjects

Michael S Rolph

Publications and source records attributed to Michael S Rolph.

15 recordsLinked to original sources

PubMed2006

Regulation of dendritic cell function and T cell priming by the fatty acid-binding protein AP2.

The fatty acid-binding protein (FABP) family consists of a number of conserved cytoplasmic proteins with roles in intracellular lipid transport, storage, and metabolism. Examination of a comprehensive leukocyte gene expression database revealed strong expression of the adipocyte FABP aP2 in human monocyte-derived dendritic cells (DCs). We isolated bone marrow-derived DC from aP2-deficient mice, and showed that expression of DC cytokines including IL-12 and TNF was significantly impaired in these cells. Degradation of IkappaBalpha was also impaired in aP2-deficient DCs, indicative of reduced signaling through the IkappaB kinase-NF-kappaB pathway. The cytokine defect was selective because there was no effect on Ag uptake or expression of MHC class II, CD40, CD80, or CD86. In an MLR, aP2-deficient DCs stimulated markedly lower T cell proliferation and cytokine production than did wild-type DCs. Moreover, aP2-deficient mice immunized with keyhole limpet hemocyanin/CFA showed reduced production of IFN-gamma by restimulated draining lymph node cells, suggesting a similar defect in DC function in vivo. Similarly, infection of aP2-deficient mice with the natural mouse pathogen ectromelia virus resulted in substantially lower production of IFN-gamma by CD8+ T cells. Thus, FABP aP2 plays an important role in DC function and T cell priming, and provides an additional link between metabolic processes and the regulation of immune responses.

Animals↗

PubMed2006

Generalization of DNA microarray dispersion properties: microarray equivalent of t-distribution.

BACKGROUND: DNA microarrays are a powerful technology that can provide a wealth of gene expression data for disease studies, drug development, and a wide scope of other investigations. Because of the large volume and inherent variability of DNA microarray data, many new statistical methods have been developed for evaluating the significance of the observed differences in gene expression. However, until now little attention has been given to the characterization of dispersion of DNA microarray data. RESULTS: Here we examine the expression data obtained from 682 Affymetrix GeneChips with 22 different types and we demonstrate that the Gaussian (normal) frequency distribution is characteristic for the variability of gene expression values. However, typically 5 to 15% of the samples deviate from normality. Furthermore, it is shown that the frequency distributions of the difference of expression in subsets of ordered, consecutive pairs of genes (consecutive samples) in pair-wise comparisons of replicate experiments are also normal. We describe a consecutive sampling method, which is employed to calculate the characteristic function approximating standard deviation and show that the standard deviation derived from the consecutive samples is equivalent to the standard deviation obtained from individual genes. Finally, we determine the boundaries of probability intervals and demonstrate that the coefficients defining the intervals are independent of sample characteristics, variability of data, laboratory conditions and type of chips. These coefficients are very closely correlated with Student's t-distribution. CONCLUSION: In this study we ascertained that the non-systematic variations possess Gaussian distribution, determined the probability intervals and demonstrated that the K(alpha) coefficients defining these intervals are invariant; these coefficients offer a convenient universal measure of dispersion of data. The fact that the K(alpha) distributions are so close to t-distribution and independent of conditions and type of arrays suggests that the quantitative data provided by Affymetrix technology give "true" representation of physical processes, involved in measurement of RNA abundance. REVIEWERS: This article was reviewed by Yoav Gilad (nominated by Doron Lancet), Sach Mukherjee (nominated by Sandrine Dudoit) and Amir Niknejad and Shmuel Friedland (nominated by Neil Smalheiser).

Journal Article↗

PubMed2006

Immune cell transcriptome datasets reveal novel leukocyte subset-specific genes and genes associated with allergic processes.

BACKGROUND: The precise function of various resting and activated leukocyte subsets remains unclear. For instance, mast cells, basophils, and eosinophils play important roles in allergic inflammation but also participate in other immunologic responses. One strategy to understand leukocyte subset function is to define the expression and function of subset-restricted molecules. OBJECTIVE: To use a microarray dataset and bioinformatics strategies to identify novel leukocyte markers as well as genes associated with allergic or innate responses. METHODS: By using Affymetrix microarrays, we generated an immune transcriptome dataset composed of gene profiles from all of the major leukocyte subsets, including rare enigmatic subsets such as mast cells, basophils, and plasma cells. We also assessed whether analysis of genes expressed commonly by certain groups of leukocytes, such as allergic leukocytes, might identify genes associated with particular responses. RESULTS: Transcripts highly restricted to a single leukocyte subset were readily identified (>2000 subset-specific transcripts), many of which have not been associated previously with leukocyte functions. Transcripts expressed exclusively by allergy-related leukocytes revealed well known as well as novel molecules, many of which presumably contribute to allergic responses. Likewise, Nearest Neighbor Analysis of genes coexpressed with Toll-like receptors identified genes of potential relevance for innate immunity. CONCLUSION: Gene profiles from all of the major human leukocyte subsets provide a powerful means to identify genes associated with single leukocyte subsets, or different types of immune response. CLINICAL IMPLICATIONS: A comprehensive dataset of gene expression profiles of human leukocytes should provide new targets or biomarkers for human inflammatory diseases.

Gene Expression Profiling↗

PubMed2006

Positive regulation of immune cell function and inflammatory responses by phosphatase PAC-1.

Mitogen-activated protein kinases facilitate many cellular processes and are essential for immune cell function. Their activity is controlled by kinases and dual-specificity phosphatases. A comprehensive microarray analysis of human leukocytes identified DUSP2 (encoding the phosphatase PAC-1) as one of the most highly induced transcripts in activated immune cells. We generated Dusp2(-/-) mice and found considerably reduced inflammatory responses in the 'K/BxN' model of rheumatoid arthritis. PAC-1 deficiency led to increased activity of Jun kinase (Jnk) but unexpected impairment of the activity of extracellular signal-regulated kinase (Erk) and the kinase p38, reduced activity of the transcription factor Elk1 and a complex of mobilized transcription factor NFAT and the AP-1 transcription factor and decreased effector immune cell function. Thus, PAC-1 is a key positive regulator of inflammatory cell signaling and effector functions, mediated through Jnk and Erk mitogen-activated protein kinase crosstalk.

Animals↗

PubMed2006

Cyclophosphamide augments inflammation by reducing immunosuppression in a mouse model of allergic airway disease.

BACKGROUND: Allergic asthma is a TH2 cell-driven immunological disease, characterized by eosinophilic inflammation. The cytotoxic agent cyclophosphamide paradoxically augments several immune responses. OBJECTIVE: We studied the proposal that cyclophosphamide may aggravate airway inflammation in allergic mice, and these features might result from the loss of regulatory T cells. METHODS: BALB/c mice were immunized with ovalbumin on days 0 and 14 and challenged with aerosolized ovalbumin from days 21 to 27. Some mice also received cyclophosphamide on days -2 and 12. RESULTS: In the lungs of cyclophosphamide-treated animals, pronounced worsening of inflammatory features was noted, including increased eosinophil infiltration, epithelial thickness, mucus occlusion, and eosinophil numbers in bronchoalveolar lavage fluid. There was also increased total and ovalbumin-specific serum IgE, increased IL-4 and IL-5 secretion by peritracheal lymph node cells, and reduced lung mRNA expression of IL-10 and TGF-beta in animals treated with cyclophosphamide. The expression of FoxP3, a marker of regulatory T cells, was significantly reduced in lymphoid organs after the second injection of cyclophosphamide, and in the lung tissue after allergen challenge in cyclophosphamide-treated mice. Lung IL-10+CD4+ T cells and cytotoxic T lymphocyte-associated antigen 4+CD4+ T cells were reduced after allergen challenge in cyclophosphamide-treated mice. CONCLUSION: Cyclophosphamide worsened features of allergic pulmonary inflammation in this model, in association with increased production of IgE and TH2 cytokines. The reduced expression of FoxP3 and immunosuppressive cytokines by cyclophosphamide is consistent with the possibility that toxicity to regulatory T cells may contribute to the increased inflammation.

Adjuvants, Immunologic↗

PubMed2006

The adipocyte fatty acid-binding protein aP2 is required in allergic airway inflammation.

The adipocyte fatty acid-binding protein aP2 regulates systemic glucose and lipid metabolism. We report that aP2, in addition to being abundantly expressed by adipocytes, is also expressed by human airway epithelial cells and shows a striking upregulation following stimulation of epithelial cells with the Th2 cytokines IL-4 and IL-13. Regulation of aP2 mRNA expression by Th2 cytokines was highly dependent on STAT6, a transcription factor with a major regulatory role in allergic inflammation. We examined aP2-deficient mice in a model of allergic airway inflammation and found that infiltration of leukocytes, especially eosinophils, into the airways was highly dependent on aP2 function. T cell priming was unaffected by aP2 deficiency, suggesting that aP2 was acting locally within the lung, and analysis of bone marrow chimeras implicated non-hematopoietic cells, most likely bronchial epithelial cells, as the site of action of aP2 in allergic airway inflammation. Thus, aP2 regulates allergic airway inflammation and may provide a link between fatty acid metabolism and asthma.

3T3 Cells↗

PubMed2005

Identification of T cell-restricted genes, and signatures for different T cell responses, using a comprehensive collection of microarray datasets.

We used a comprehensive collection of Affymetrix microarray datasets to ascertain which genes or molecules distinguish the known major subsets of human T cells. Our strategy allowed us to identify the genes expressed in most T cell subsets: TCR alphabeta+ and gammadelta+, three effector subsets (Th1, Th2, and T follicular helper cells), T central memory, T effector memory, activated T cells, and others. Our genechip dataset also allowed for identification of genes preferentially or exclusively expressed by T cells, compared with numerous non-T cell leukocyte subsets profiled. Cross-comparisons between microarray datasets revealed important features of certain subsets. For instance, blood gammadelta T cells expressed no unique gene transcripts, but did differ from alphabeta T cells in numerous genes that were down-regulated. Hierarchical clustering of all the genes differentially expressed between T cell subsets enabled the identification of precise signatures. Moreover, the different T cell subsets could be distinguished at the level of gene expression by a smaller subset of predictor genes, most of which have not previously been associated directly with any of the individual subsets. T cell activation had the greatest influence on gene regulation, whereas central and effector memory T cells displayed surprisingly similar gene expression profiles. Knowledge of the patterns of gene expression that underlie fundamental T cell activities, such as activation, various effector functions, and immunological memory, provide the basis for a better understanding of T cells and their role in immune defense.

Cluster Analysis↗

PubMed2005

Clues to asthma pathogenesis from microarray expression studies.

Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness (AHR), tissue remodeling, and airflow obstruction. The pathogenesis of asthma is only partly understood, and there is an urgent need for improved therapeutic strategies for this disease. Microarray technology has considerable promise as a tool for discovery of novel asthma therapeutic targets, although the field is still in its infancy. A number of studies have described expression profiles derived from human asthmatic lung tissue, mouse airway tissue, or from key cell types associated with asthma, but to date relatively few studies have exploited these findings to discover new pathways involved in the pathogenesis of asthma. Among the genes to have been identified by array studies and validated by further studies are monokine induced by interferon (IFN)-gamma, fatty acid binding proteins (FABP), and complement factor 5 (C5). Here we provide examples of microarray approaches to the discovery of new molecules associated with asthma. We anticipate that these types of analyses will provide considerable insight into asthma pathogenesis and will provide a wealth of new molecules for downstream analyses such as gene deficient mouse studies, or monoclonal antibody production.

Animals↗

PubMed2005

BAFF augments certain Th1-associated inflammatory responses.

B cell-activating factor belonging to the TNF family (BAFF; BLyS) is a critical regulator of B cell maturation and survival, and its overexpression in BAFF transgenic (Tg) mice results in the development of autoimmune disorders. BAFF also affects T cell function through binding to one of the BAFF receptors, BAFF-R. Using BAFF Tg mice, we examined a typical Th1-mediated response, the cutaneous delayed-type hypersensitivity reaction, and found a much greater degree of paw swelling and inflammation than in control mice. Importantly, delayed-type hypersensitivity scores correlated directly with BAFF levels in serum. Conversely, in a Th2-mediated model of allergic airway inflammation, BAFF Tg mice were largely protected and showed markedly reduced Ag-specific T cell proliferation and eosinophil infiltration associated with the airways. Thus, local and/or systemically distributed BAFF affects Th1 and Th2 responses and impacts on the course of some T cell-mediated inflammatory reactions. Our results are consistent with the idea that BAFF augments T cell as well as B cell responses, particularly Th1-type responses. Results in BAFF Tg mice may reflect the situation in certain autoimmune patients or virally infected individuals, because BAFF levels in blood are comparable.

Adjuvants, Immunologic↗

PubMed2004

Nonspecific antiviral immunity by formalin-fixed Coxiella burnetii is enhanced in the absence of nitric oxide.

Mice treated with a single injection of formalin-fixed Coxiella burnetii showed a significant increase in resistance to vaccinia virus (VV) infection compared to untreated mice. C. burnetii stimulated dramatically high levels of nitric oxide (NO) in the serum of treated mice, suggesting that NO might play a role in resistance to virus infection. To test this hypothesis, the effect of C. burnetii treatment on VV replication was examined in NOS2-/- and wild-type mice. C. burnetii treatment inhibited VV replication in both the knockout and wild-type mice but the effect was significantly greater in the NOS2-/- mice. Experiments in IFNgamma receptor knockout mice indicated that the nonspecific antiviral immunity induced by C. burnetii was dependent on IFNgamma and not NO. In the absence of NO, indoleamine 2,3-dioxygenase (IDO) was increased in C. burnetii-treated mice and this may contribute to the accelerated virus clearance in NOS2-/- mice.

Animals↗

PubMed2004

T follicular helper cells express a distinctive transcriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells.

Effector T cell responses have long been viewed in the context of the Th1/Th2 paradigm. Recently, a third major subset of nonpolarized effector T cells that provides help to B cells has been identified. These T cells, termed T follicular helper (T(FH)) cells, home to the B cell areas of secondary lymphoid tissue, through interactions mediated via the chemokine receptor CXCR5 and its ligand CXCL13. Affymetrix microarrays were used to identify transcription factors, cytokines, and cell surface molecules that underlie the differentiation pathways and functional properties of the T(FH) subset. The transcriptional profile of human CXCR5(+) T(FH) cells was compared with that of Th1 and Th2 cells, which enabled the identification of numerous genes expressed preferentially by T(FH) cells, over the other effector subsets. Certain T(FH) genes were also expressed by B cells and thus appear to be particularly relevant for humoral immunity. Abs were used to confirm the expression of several factors. In particular, CD84 and CD200, the cytokine IL-21, and the transcription factor BCL6 were all strongly associated with T(FH) cells. Gene microarrays reveal a highly distinctive transcriptional profile for a third subset of effector T cells that differs markedly from Th1 and Th2 cells.

Antigens, CD↗

PubMed2003

Interleukin-4-mediated downregulation of cytotoxic T lymphocyte activity is associated with reduced proliferation of antigen-specific CD8+ T cells.

During virus infection, exogenous IL-4 strongly downregulates expression of antiviral cytokines and cytotoxic T lymphocyte (CTL) responses. In this study, we have employed a T cell receptor (TCR) transgenic system to more closely investigate the effect of IL-4 on CTL activity. This system involves mice transgenic for an H2-Kb restricted TCR recognising an ovalbumin (OVA)-specific peptide (OT-I mice), and recombinant vaccinia viruses expressing the gene for OVA (VV-OVA), or OVA together with IL-4 (VV-OVA-IL-4). Spleen cells from OT-I mice were adoptively transferred to irradiated C57BL/6 mice infected with VV-OVA or VV-OVA-IL-4. Five days following transfer, markedly stronger CTL activity was detected in VV-OVA- than in VV-OVA-IL-4-infected recipients. The reduction in CTL activity was associated with a reduction in the number of OVA-specific CD8+ T cells. Proliferation of cells from VV-OVA-IL-4-infected recipients was dramatically reduced, and this is a likely explanation for the IL-4-mediated reduction in the total number of OVA-specific cells and the reduced cytotoxic activity. On a per cell basis, the production of IFNgamma and cytotoxic activity of OVA-specific CD8+ cells was not influenced by IL-4. Taken together, our results indicate that the reduction in CTL activity by exogenous IL-4 is due to a reduced number of antigen-specific effectors, and does not involve a downregulation of effector function of these cells.

Adoptive Transfer↗

PubMed2002

Macrophage migration inhibitory factor (MIF) plays a pivotal role in immunity against Salmonella typhimurium.

The cytokine macrophage migration inhibitory factor (MIF) exerts a multitude of biological functions. Notably, it induces inflammation at the interface between the immune system and the hypothalamus-pituitary-adrenal stress axis. The role of MIF in infectious diseases is not understood completely. Here, we show that MIF-deficient (MIF(-/-)) knockout mice fail to control an infection with wild-type Salmonella typhimurium. Increased susceptibility was accompanied by a reduced Th1 response, demonstrated by decreased levels of IL-12, IFNgamma, and tumor necrosis factor alpha. In Salmonella-infected MIF(-/-) mice, levels of IL-1beta were markedly increased. Additionally, infected MIF(-/-) mice showed elevated serum levels of nitric oxide and corticosterone as compared with control mice. Our results point to MIF as a key mediator in the host response to S. typhimurium. MIF not only promotes development of a protective Th1 response but ameliorates disease by altering levels of reactive nitrogen intermediates and corticosteroid hormones, which both exert immunosuppressive functions.

Animals↗

PubMed2002

Production of the long pentraxin PTX3 in advanced atherosclerotic plaques.

Elevated plasma levels of the pentraxin protein family member C-reactive protein (CRP) are associated with increased risk of cardiovascular disease in both healthy and high-risk subjects. The long pentraxin family member, pentraxin 3 (PTX3), was recently described. Like CRP, PTX3 is induced by acute inflammatory stimuli and is increased in the blood of patients with acute myocardial infarction. Unlike CRP, it is expressed in a wide range of cell types, but not in hepatocytes. In this study, we have investigated the expression of PTX3 in atherosclerosis. Immunohistochemical staining of advanced atherosclerotic lesions revealed strong expression of PTX3. In contrast, no PTX3 expression was observed in nonatherosclerotic internal mammary arteries. By staining serial sections with cell type- and PTX3-specific antibodies, we observed that PTX3 was produced principally by macrophages and endothelial cells. Infrequent expression by smooth muscle cells was also observed. Our results suggest that PTX3 may contribute to the pathogenesis of atherosclerosis.

Acute-Phase Proteins↗

PubMed2002

MIP-1alpha, MIP-1beta, RANTES, and ATAC/lymphotactin function together with IFN-gamma as type 1 cytokines.

We analyzed for the first time the expression of chemokines in subpopulations of the murine immune system at the single-cell level. We demonstrate in vitro and in a model of murine listeriosis that macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, regulated on activation normal T cell expressed and secreted (RANTES), and activation-induced, T cell-derived, and chemokine-related cytokine (ATAC)/lymphotactin are cosecreted to a high degree with IFN-gamma by activated individual natural killer (NK), CD8(+) T, and CD4(+) T helper 1 (Th1) cells. Functionally, ATAC and the CC chemokines cooperate with IFN-gamma in the up-regulation of CD40, IL-12, and tumor necrosis factor-alpha, molecules playing a central role in the effector phase of macrophages. Our data indicate that (i) MIP-1alpha, MIP-1beta, RANTES, and ATAC are not only chemoattractants but also coactivators of macrophages, (ii) MIP-1alpha, MIP-1beta, RANTES, and ATAC constitute together with IFN-gamma a group of "type 1 cytokines," and (iii) these cytokines act together as a functional unit that is used by NK cells in the innate phase and then "handed over" to CD8(+) T cells in the antigen-specific phase of the immune defense, thus bridging the two components of a Th1 immune reaction.

Animals↗