Mark Barton Frank

Novel approaches to gene expression analysis of active polyarticular juvenile rheumatoid arthritis

Juvenile rheumatoid arthritis (JRA) has a complex, poorly characterized pathophysiology. Modeling of transcriptosome behavior in pathologic specimens using microarrays allows molecular dissection of complex autoimmune diseases. However, conventional analyses rely on identifying statistically significant differences in gene expression distributions between patients and controls. Since the principal aspects of disease pathophysiology vary significantly among patients, these analyses are biased. Genes with highly variable expression, those most likely to regulate and affect pathologic processes, are excluded from selection, as their distribution among healthy and affected individuals may overlap significantly. Here we describe a novel method for analyzing microarray data that assesses statistically significant changes in gene behavior at the population level. This method was applied to expression profiles of peripheral blood leukocytes from a group of children with polyarticular JRA and healthy control subjects. Results from this method are compared with those from a conventional analysis of differential gene expression and shown to identify discrete subsets of functionally related genes relevant to disease pathophysiology. These results reveal the complex action of the innate and adaptive immune responses in patients and specifically underscore the role of IFN-γ in disease pathophysiology. Discriminant function analysis of data from a cohort of patients treated with conventional therapy identified additional subsets of functionally related genes; the results may predict treatment outcomes. While data from only 9 patients and 12 healthy controls was used, this preliminary investigation of the inflammatory genomics of JRA illustrates the significant potential of utilizing complementary sets of bioinformatics tools to maximize the clinical relevance of microarray data from patients with autoimmune disease, even in small cohorts.

Variants within MECP2, a key transcription regulator, are associated with increased susceptibility to lupus and differential gene expression in patients with systemic lupus erythematosus

OBJECTIVE Both genetic and epigenetic factors play an important role in the pathogenesis of lupus. The aim of this study was to examine methyl-CpG-binding protein 2 gene (MECP2) polymorphisms in a large cohort of patients with lupus and control subjects, and to determine the functional consequences of the lupus-associated MECP2 haplotype. METHODS We genotyped 18 single-nucleotide polymorphisms within MECP2, located on chromosome Xq28, in a large cohort of patients with lupus and control subjects of European descent. We studied the functional effects of the lupus-associated MECP2 haplotype by determining gene expression profiles in B cell lines in female lupus patients with and those without the lupus-associated MECP2 risk haplotype. RESULTS We confirmed, replicated, and extended the genetic association between lupus and genetic markers within MECP2 in a large independent cohort of lupus patients and control subjects of European descent (odds ratio 1.35, P = 6.65 x 10(-11)). MECP2 is a dichotomous transcription regulator that either activates or represses gene expression. We identified 128 genes that are differentially expressed in lupus patients with the disease-associated MECP2 haplotype; most ( approximately 81%) were up-regulated. Genes that were up-regulated had significantly more CpG islands in their promoter regions compared with genes that were down-regulated. Gene ontology analysis using the differentially expressed genes revealed significant association with epigenetic regulatory mechanisms, suggesting that these genes are targets for MECP2 regulation in B cells. Furthermore, at least 13 of the 104 up-regulated genes are regulated by interferon. The disease-risk MECP2 haplotype was associated with increased expression of the MECP2 transcription coactivator CREB1 and decreased expression of the corepressor histone deacetylase 1. CONCLUSION Polymorphism in the MECP2 locus is associated with lupus and, at least in part, contributes to the interferon signature observed in lupus patients.

Erroneous augmentation of multiplex assay measurements in patients with rheumatoid arthritis due to heterophilic binding by serum rheumatoid factor

OBJECTIVE Serum rheumatoid factor (RF) and other heterophilic antibodies potentially interfere with antibody-based immunoassays by nonspecifically binding detection reagents. The purpose of this study was to assess whether these factors confound multiplex-based immunoassays, which are used with increasing frequency to measure cytokine and chemokine analytes in patients with rheumatoid arthritis (RA). METHODS We performed multiplex immunoassays using different platforms to measure analyte concentrations in RA patient samples. Samples were depleted of RF by column-based affinity absorption or were exposed to agents that block heterophilic binding activity. RESULTS In RA patients with high-titer RF, 69% of analytes demonstrated at least a 2-fold stronger multiplex signal in non-RF-depleted samples as compared to RF-depleted samples. This degree of erroneous signal amplification was less frequent in low-titer RF samples (17% of analytes; P < 0.0000001). Signal amplification by heterophilic antibodies was blocked effectively by HeteroBlock (≥ 150 μg/ml). In 35 RA patients, multiplex signals for 14 of 22 analytes were amplified erroneously in unblocked samples as compared to blocked samples (some >100-fold), but only in patients with high-titer RF (P < 0.002). Two other blocking agents, heterophilic blocking reagent and immunoglobulin-inhibiting reagent, also blocked heterophilic activity. CONCLUSION All multiplex protein detection platforms we tested exhibited significant confounding by RF or other heterophilic antibodies. These findings have broad-reaching implications in the acquisition and interpretation of data derived from multiplex immunoassay testing of RA patient serum and possibly also in other conditions in which RF or other heterophilic antibodies may be present. Several available blocking agents effectively suppressed this erroneous signal amplification in the multiplex platforms tested.

Unique Patterns of Molecular Profiling between Human Prostate Cancer LNCaP and PC-3 Cells

Human prostate cancer LNCaP and PC‐3 cell lines have been extensively used to study prostate cancer progression and to develop therapeutic agents. Although LNCaP and PC‐3 cells are generally assumed to represent early and late stages of prostate cancer, respectively, there is limited information regarding gene expression patterns between these two cell lines and its relationship to prostate cancer.

The canonical Wnt pathway shapes niches supportive of hematopoietic stem/progenitor cells

Considerable information has accumulated about components of BM that regulate the survival, self-renewal, and differentiation of hematopoietic cells. In the present study, we investigated Wnt signaling and assessed its influence on human and murine hematopoiesis. Hematopoietic stem/progenitor cells (HSPCs) were placed on Wnt3a-transduced OP9 stromal cells. The proliferation and production of B cells, natural killer cells, and plasmacytoid dendritic cells were blocked. In addition, some HSPC characteristics were maintained or re-acquired along with different lineage generation potentials. These responses did not result from direct effects of Wnt3a on HSPCs, but also required alterations in the OP9 cells. Microarray, PCR, and flow cytometric experiments revealed that OP9 cells acquired osteoblastic characteristics while down-regulating some features associated with mesenchymal stem cells, including the expression of angiopoietin 1, the c-Kit ligand, and VCAM-1. In contrast, the production of decorin, tenascins, and fibromodulin markedly increased. We found that at least 1 of these extracellular matrix components, decorin, is a regulator of hematopoiesis: upon addition of this proteoglycan to OP9 cocultures, decorin caused changes similar to those caused by Wnt3a. Furthermore, hematopoietic stem cell numbers in the BM and spleen were elevated in decorin-knockout mice. These findings define one mechanism through which canonical Wnt signaling could shape niches supportive of hematopoiesis.

Estrogen receptor signaling promotes dendritic cell differentiation by increasing expression of the transcription factor IRF4

During inflammation, elevated granulocyte macrophage-colony-stimulating factor (GM-CSF) directs the development of new dendritic cells (DCs). This pathway is influenced by environmental factors, and we previously showed that physiologic levels of estradiol, acting through estrogen receptor alpha (ERalpha), promote the GM-CSF-mediated differentiation of a CD11b(+) DC subset from myeloid progenitors (MPs). We now have identified interferon regulatory factor 4 (IRF4), a transcription factor induced by GM-CSF and critical for CD11b(+) DC development in vivo, as a target of ERalpha signaling during this process. In MPs, ERalpha potentiates and sustains GM-CSF induction of IRF4. Furthermore, retroviral delivery of the Irf4 cDNA to undifferentiated ERalpha(-/-) bone marrow cells restored the development of the estradiol/ERalpha-dependent DC population, indicating that an elevated amount of IRF4 protein substitutes for ERalpha signaling. Thus at an early stage in the MP response to GM-CSF, ERalpha signaling induces an elevated amount of IRF4, which leads to a developmental program underlying CD11b(+) DC differentiation.

Inflammation and neurological disease-related genes are differentially expressed in depressed patients with mood disorders and correlate with morphometric and functional imaging abnormalities.

Depressed patients show evidence of both proinflammatory changes and neurophysiological abnormalities such as increased amygdala reactivity and volumetric decreases of the hippocampus and ventromedial prefrontal cortex (vmPFC). However, very little is known about the relationship between inflammation and neuroimaging abnormalities in mood disorders. A whole genome expression analysis of peripheral blood mononuclear cells yielded 12 protein-coding genes (ADM, APBB3, CD160, CFD, CITED2, CTSZ, IER5, NFKBIZ, NR4A2, NUCKS1, SERTAD1, TNF) that were differentially expressed between 29 unmedicated depressed patients with a mood disorder (8 bipolar disorder, 21 major depressive disorder) and 24 healthy controls (HCs). Several of these genes have been implicated in neurological disorders and/or apoptosis. Ingenuity Pathway Analysis yielded two genes networks, one centered around TNF with NFKβ, TGFβ, and ERK as connecting hubs, and the second network indicating cell cycle and/or kinase signaling anomalies. fMRI scanning was conducted using a backward-masking task in which subjects were presented with emotionally-valenced faces. Compared with HCs, the depressed subjects displayed a greater hemodynamic response in the right amygdala, left hippocampus, and the ventromedial prefrontal cortex to masked sad versus happy faces. The mRNA levels of several genes were significantly correlated with the hemodynamic response of the amygdala, vmPFC and hippocampus to masked sad versus happy faces. Differentially-expressed transcripts were significantly correlated with thickness of the left subgenual ACC, and volume of the hippocampus and caudate. Our results raise the possibility that molecular-level immune dysfunction can be mapped onto macro-level neuroimaging abnormalities, potentially elucidating a mechanism by which inflammation leads to depression.

Evidence for chronic, peripheral activation of neutrophils in polyarticular juvenile rheumatoid arthritis

Although strong epidemiologic evidence suggests an important role for adaptive immunity in the pathogenesis of polyarticular juvenile rheumatoid arthritis (JRA), there remain many aspects of the disease that suggest equally important contributions of the innate immune system. We used gene expression arrays and computer modeling to examine the function in neutrophils of 25 children with polyarticular JRA. Computer analysis identified 712 genes that were differentially expressed between patients and healthy controls. Computer-assisted analysis of the differentially expressed genes demonstrated functional connections linked to both interleukin (IL)-8- and interferon-γ (IFN-γ)-regulated processes. Of special note is that the gene expression fingerprint of children with active JRA remained essentially unchanged even after they had responded to therapy. This result differed markedly from our previously reported work, in which gene expression profiles in buffy coats of children with polyarticular JRA reverted to normal after disease control was achieved pharmacologically. These findings suggest that JRA neutrophils remain in an activated state even during disease quiescence. Computer modeling of array data further demonstrated disruption of gene regulatory networks in clusters of genes modulated by IFN-γ and IL-8. These cytokines have previously been shown to independently regulate the frequency (IFN-γ) and amplitude (IL-8) of the oscillations of key metabolites in neutrophils, including nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and superoxide ion. Using real-time, high-speed, single-cell photoimaging, we observed that 6/6 JRA patients displayed a characteristic defect in 12% to 23% of the neutrophils tested. Reagents known to induce only frequency fluctuations of NAD(P)H and superoxide ion induced both frequency and amplitude fluctuations in JRA neutrophils. This is a novel finding that was observed in children with both active (n = 4) and inactive (n = 2) JRA. A subpopulation of polyarticular JRA neutrophils are in a chronic, activated state, a state that persists when the disease is well controlled pharmacologically. Furthermore, polyarticular JRA neutrophils exhibit an intrinsic defect in the regulation of metabolic oscillations and superoxide ion production. Our data are consistent with the hypothesis that neutrophils play an essential role in the pathogenesis of polyarticular JRA.

Frankincense oil derived from Boswellia carteri induces tumor cell specific cytotoxicity

BackgroundOriginating from Africa, India, and the Middle East, frankincense oil has been important both socially and economically as an ingredient in incense and perfumes for thousands of years. Frankincense oil is prepared from aromatic hardened gum resins obtained by tapping Boswellia trees. One of the main components of frankincense oil is boswellic acid, a component known to have anti-neoplastic properties. The goal of this study was to evaluate frankincense oil for its anti-tumor activity and signaling pathways in bladder cancer cells.MethodsFrankincense oil-induced cell viability was investigated in human bladder cancer J82 cells and immortalized normal bladder urothelial UROtsa cells. Temporal regulation of frankincense oil-activated gene expression in bladder cancer cells was identified by microarray and bioinformatics analysis.ResultsWithin a range of concentration, frankincense oil suppressed cell viability in bladder transitional carcinoma J82 cells but not in UROtsa cells. Comprehensive gene expression analysis confirmed that frankincense oil activates genes that are responsible for cell cycle arrest, cell growth suppression, and apoptosis in J82 cells. However, frankincense oil-induced cell death in J82 cells did not result in DNA fragmentation, a hallmark of apoptosis.ConclusionFrankincense oil appears to distinguish cancerous from normal bladder cells and suppress cancer cell viability. Microarray and bioinformatics analysis proposed multiple pathways that can be activated by frankincense oil to induce bladder cancer cell death. Frankincense oil might represent an alternative intravesical agent for bladder cancer treatment.

Gene expression in systemic lupus erythematosus: Bone marrow analysis differentiates active from inactive disease and reveals apoptosis and granulopoiesis signatures

OBJECTIVE The cells of the immune system originate from the bone marrow, where many of them also mature. This study was undertaken to examine gene expression in the bone marrow of patients with systemic lupus erythematosus (SLE), in order to better understand the aberrant immune response in this disease. METHODS Bone marrow mononuclear cells (BMMCs) from 20 SLE patients (11 with active disease and 9 with inactive disease) and peripheral blood mononuclear cells (PBMCs) from 27 patients (16 with active disease and 11 with inactive disease) were studied; BMMCs and PBMCs from 7 healthy individuals and 3 osteoarthritis patients were studied as controls. Samples were analyzed on genome-scale DNA microarrays, with 21,329 genes represented. RESULTS We identified 102 genes involved in various biologic processes that were differentially expressed between patient and control BMMCs; 53 of them are genes that are involved in major networks, including cell death, growth, signaling, and proliferation. Comparative analysis revealed 88 genes that were differentially expressed between bone marrow and blood, the majority of which are involved in cell growth and differentiation, cellular movement and morphology, immune response, and other hematopoietic cell functions. Unsupervised clustering of highly expressed genes revealed 2 major SLE patient clusters (active disease and inactive disease) based on gene expression in bone marrow, but not in peripheral blood. The up-regulated genes in the bone marrow of patients with active disease included genes involved in cell death and granulopoiesis. CONCLUSION Microarray analysis of the bone marrow differentiated active from inactive SLE and provided further evidence of the role of apoptosis and granulocytes in the pathogenesis of the disease.