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PubMed2004

Comparison of snap freezing versus ethanol fixation for gene expression profiling of tissue specimens.

Frozen tissue specimens are the gold standard for molecular analysis. However, snap freezing presents several challenges regarding collection and storage of tissue, and preservation of histological detail. We evaluate an alternative preservation method, ethanol fixation followed by paraffin embedding, by analyzing expression profiles of microdissected cells on Affymetrix oligonucleotide arrays of three matched benign prostatic hyperplasia (BPH) and tumor samples processed with each preservation method. Frozen samples generated an average present call of 26% of the probe sets, compared to 4.5% in ethanol-paraffin samples. Eighty-eight percent of the probe sets called present in the ethanol-paraffin samples were also present in the frozen specimens. Comparing ethanol-paraffin BPH to tumor, 52 probe sets showed a twofold differential expression or higher in at least two cases, 23 of which were also differentially expressed in at least one frozen case. Despite a significant drop in the number of transcripts detectable, the data suggests that the obtainable information in ethanol-fixed samples may be useful for molecular profiling where frozen tissue is not available. However, ethanol fixation and paraffin embedding of tissue specimens is not optimal for high-throughput mRNA expression analysis. Improved methods for transcript profiling of archival samples, and/or tissue processing are still required.

Ethanol↗

PubMed2006

Lymphovascular invasion is associated with poor survival in gastric cancer: an application of gene-expression and tissue array techniques.

OBJECTIVES: To examine a population-based cohort for the association between clinicopathologic predictors of survival and immunohistochemical markers (IHC), and to assess changes in gene expression that are associated with lymphovascular invasion (LVI). SUMMARY BACKGROUND DATA: LVI has been associated with poor survival and aggressive tumor behavior. The molecular changes responsible for the behavior of gastric cancer have yet to be determined. Characterization of IHC markers and gene expression profiles may identify molecular alterations governing tumor behavior. METHODS: : Clinicopathologic and survival data of 114 patients were reviewed. Archival specimens were used to construct a multitumor tissue array that was subjected to IHC of selected protein targets. Correlation of IHC with tumor thickness (T status), LVI and prognosis was studied. Microarray analysis of fresh gastric cancer tissue was conducted to examine the gene expression profile with respect to LVI. RESULTS: In a multivariate analysis, nodal status (N), metastasis (M), and LVI were independent predictors of survival. LVI was associated with a 5-year survival of 13.9% versus 55.9% in patients in whom it was absent. LVI correlated with advancing T status (P = 0.001) and N status (P < 0.001). IHC staining of cyclooxygenase-2 (COX-2) correlated with T status, tumor grade, lymph node positivity, and IHC staining of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Microarray analyses suggested differential expression of oligophrenin-1 (OPHN1) and ribophorin-II (RPNII) with respect to LVI. CONCLUSION: LVI was an independent predictor of survival in gastric cancer. Expression of COX-2 may facilitate tumor invasion through MMP-2 and MMP-9 activation. OPHN1 and RPN II appeared to be differentially expressed in gastric cancers exhibiting LVI. The reported function of OPHN1 and RPN II makes these gene products promising candidates for future studies involving LVI in gastric cancer.

Adenocarcinoma↗

PubMed2005

Interpreting expression profiles of cancers by genome-wide survey of breadth of expression in normal tissues.

A critical and difficult part of studying cancer with DNA microarrays is data interpretation. Besides the need for data analysis algorithms, integration of additional information about genes might be useful. We performed genome-wide expression profiling of 36 types of normal human tissues and identified 2503 tissue-specific genes. We then systematically studied the expression of these genes in cancers by reanalyzing a large collection of published DNA microarray datasets. We observed that the expression level of liver-specific genes in hepatocellular carcinoma (HCC) correlates with the clinically defined degree of tumor differentiation. Through unsupervised clustering of tissue-specific genes differentially expressed in tumors, we extracted expression patterns that are characteristic of individual cell types, uncovering differences in cell lineage among tumor subtypes. We were able to detect the expression signature of hepatocytes in HCC, neuron cells in medulloblastoma, glia cells in glioma, basal and luminal epithelial cells in breast tumors, and various cell types in lung cancer samples. We also demonstrated that tissue-specific expression signatures are useful in locating the origin of metastatic tumors. Our study shows that integration of each gene's breadth of expression (BOE) in normal tissues is important for biological interpretation of the expression profiles of cancers in terms of tumor differentiation, cell lineage, and metastasis.

Algorithms↗

PubMed2006

Direct and sensitive miRNA profiling from low-input total RNA.

We have developed a sensitive, accurate, and multiplexed microRNA (miRNA) profiling assay that is based on a highly efficient labeling method and novel microarray probe design. The probes provide both sequence and size discrimination, yielding in most cases highly specific detection of closely related mature miRNAs. Using a simple, single-vial experimental protocol, 120 ng of total RNA is directly labeled using Cy3 or Cy5, without fractionation or amplification, to produce precise and accurate measurements that span a linear dynamic range from 0.2 amol to 2 fmol of input miRNA. The results can provide quantitative estimates of the miRNA content for the tissues studied. The assay is also suitable for use with formalin-fixed paraffin-embedded clinical samples. Our method allows rapid design and validation of probes for simultaneous quantitative measurements of all human miRNA sequences in the public databases and to new miRNA sequences as they are reported.

Animals↗

PubMed2003

Laser capture microdissection-based in vivo genomic profiling of wound keratinocytes identifies similarities and differences to squamous cell carcinoma.

Keratinocytes undergo a dramatic phenotypic conversion during reepithelialization of skin wounds to become hyperproliferative, migratory, and invasive. This transient healing response phenotypically resembles malignant transformation of keratinocytes during squamous cell carcinoma progression. Here we present the first analysis of global changes in keratinocyte gene expression during skin wound healing in vivo, and compare these changes to changes in gene expression during malignant conversion of keratinized epithelium. Laser capture microdissection was used to isolate RNA from wound keratinocytes from incisional mouse skin wounds and adjacent normal skin keratinocytes. Changes in gene expression were determined by comparative cDNA array analyses, and the approach was validated by in situ hybridization. The analyses identified 48 candidate genes not previously associated with wound reepithelialization. Furthermore, the analyses revealed that the phenotypic resemblance of wound keratinocytes to squamous cell carcinoma is mimicked at the level of gene expression, but notable differences between the two tissue-remodeling processes were also observed. The combination of laser capture microdissection and cDNA array analysis provides a powerful new tool to unravel the complex changes in gene expression that underlie physiological and pathological remodeling of keratinized epithelium.

Animals↗

PubMed2005

Identification of protein pattern in kidney cancer using ProteinChip arrays and bioinformatics.

Tumor biology of renal cell carcinoma (RCC) is not very well understood, although many studies on molecular and cellular biology have been performed. It is accepted now that cancer research has to be performed also with proteomic tools, because proteins are the real actors in the genesis and progression of cancer. Therefore, we used a ProteinChip System(R) (SELDI) which is able to detect minute amounts of protein and moreover to analyze a complex protein pattern. We analyzed 37 cases of clear cell RCC as a training set including corresponding normal tissue. From all samples protein lysates were made and spotted directly on different chip surfaces (SAX2, WCX). After a washing procedure the arrays were analyzed in the ProteinChip Reader. All profiles were subjected to a bioinformatical analysis including normalization, clustering, rule extraction and rating. Defined rules (markers) were evaluated using a test set of 24 samples (13 tumor tissues and 11 normal kidney tissues). The generated rule base for the SAX2 surface showed a sensitivity of 100% and a specificity of 97.3%. For the WCX arrays the optimal rule base showed worse results. A combined rule base for SAX2 and WCX did not result in a higher sensitivity or specificity. Using the optimal rule base for the SAX2 chip in the test set, sensitivity and specificity reached 76.9% and 100%, respectively. The ProteinChip System represents a key technology for the rapid detection of cancer specific proteomic patterns. It is possible to identify clear cell renal cancer with high sensitivity and specificity from minimal amounts of cells.

Cell Line, Tumor↗

PubMed2002

Detection of genes expressed in primary colon cancers by in situ hybridisation: overexpression of RACK 1.

AIMS: The isolation of various genes that are expressed in a region specific manner is considered useful for research in molecular pathology. In situ hybridisation (ISH) was used in a screening procedure to isolate these genes efficiently, using colon cancer as a model. METHODS: Suppression subtractive hybridisation (SSH) between colon cancer tissue samples and corresponding non-cancerous tissues was performed. Genes showing high expression in the cancers were selected using macro-DNA array analysis. As a final screening procedure, conventional ISH was performed to isolate genes expressed specifically in colon cancers. RESULTS: Sixty nine clones were selected by SSH and macro-DNA array analyses. These clones were then analysed by ISH to examine their expression patterns. ISH screening revealed that all the clones screened showed more intense signals in colon cancers than in non-cancerous tissues. Among them, RACK 1, which is a protein kinase C receptor and a homologue of the G protein beta subunit, was expressed intensely in colon cancer cells. RACK 1 expression was evaluated in multiple samples by ISH, and the results confirmed that RACK 1 was universally overexpressed in cells of all 11 colon cancers examined. CONCLUSIONS: Many genes, including RACK 1, expressed in colon cancer cells can be isolated efficiently by this method, and their precise expression pattern can be evaluated. These results indicate that ISH is an excellent technique for systemic screening of genes expressed in a region specific manner.

Blotting, Northern↗

PubMed2004

A bayesian hierarchical model for the analysis of Affymetrix arrays.

An area of active research in DNA microarray analysis focuses on identifying differentially expressed genes between normal and malignant tissues. The analysis is complicated by the presence of several unreliable expression readings. Here, we illustrate a methodology where the expression estimates are modeled as censored data and discriminating genes are selected using ANOVA-based criteria.

Analysis of Variance↗

PubMed2004

Expression of toll-like receptors 2 and 4 in rheumatoid synovial tissue and regulation by proinflammatory cytokines interleukin-12 and interleukin-18 via interferon-gamma.

OBJECTIVE: To study the expression of Toll-like receptor 2 (TLR-2) and TLR-4 and its association with proinflammatory cytokines in synovial tissue from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and healthy individuals. METHODS: Synovial tissue specimens from 29 RA patients were stained for TLR-2, TLR-4, and proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-12, IL-17, IL-18, and tumor necrosis factor alpha [TNFalpha]). The expression of TLR-2, TLR-4, and cytokines as well as the degree of inflammation in synovial tissue were compared between patients with RA, patients with OA (n = 5), and healthy individuals (n = 3). Peripheral blood mononuclear cells (PBMCs) were incubated with IL-12 and IL-18, and TLR expression was assessed using fluorescence-activated cell sorter analysis. Production of TNFalpha and IL-6 was measured using Luminex bead array technology. RESULTS: In RA synovial tissue, the expression of TLR-2 was slightly higher than that of TLR-4. Interestingly, both TLR-2 and TLR-4 were expressed at higher levels in moderately inflamed synovium, as compared with synovial tissue with no or severe inflammation. TLR expression in both the lining and the sublining was associated with the presence of IL-12 and IL-18, but no other cytokines, in the lining. The expression of both TLRs was low in synovial tissue from OA patients and healthy donors. Stimulation of PBMCs with IL-12 and IL-18 resulted in increased expression of both TLR-2 and TLR-4; this could be blocked with anti-interferon-gamma (anti-IFNgamma) antibodies, suggesting a role for IFNgamma. Lipopolysaccharide- or lipoteichoic acid-mediated triggering of PBMCs incubated with IL-12/IL-18 or IFNgamma led to an increased production of both TNFalpha and IL-6, indicating the functionality of TLR-2 and TLR-4. CONCLUSION: TLR-2 and TLR-4 are expressed in synovial tissue of patients with clinically active disease and are associated with the levels of both IL-12 and IL-18. The synergistic effect of IL-12 and IL-18 on T cell IFNgamma production seems to regulate expression of TLR-2 and TLR-4 in the synovial tissue of RA patients.

Arthritis, Rheumatoid↗

PubMed2000

Validation of tissue microarray technology in breast carcinoma.

The recent development of tissue microarray technology has potentiated large-scale retrospective cohort studies using archival formalin-fixed, paraffin-embedded tissues. A major obstacle to broad acceptance of microarrays is that they reduce the amount of tissue analyzed from a whole tissue section to a disk, 0.6 mm in diameter, that may not be representative of the protein expression patterns of the entire tumor. In this study, we examine the number to disks required to adequately represent the expression of three common antigens in invasive breast carcinoma--estrogen receptor, progesterone receptor, and the Her2/neu oncogene--in 38 cases of invasive breast carcinoma. We compared the staining of 2 to 10 microarray disks and the whole tissue sections from which they were derived and determined that analysis of two disks is comparable to analysis of a whole tissue section in more than 95% of cases. To evaluate the potential for using archival tissue in such arrays, we created a breast cancer microarray of 8 to 11 cases from each decade beginning in 1932 to the present day and evaluated the antigenicity of these markers and others. This array demonstrates that many proteins retain their antigenicity for more than 60 years, thus validating their study on archival tissues. We conclude that the tissue microarray technique, with 2-fold redundancy, is a valuable and accurate method for analysis of protein expression in large archival cohorts.

Biomarkers, Tumor↗

PubMed2001

Effects of exercise on gene-expression profile in the rat hippocampus.

Exercise has beneficial effects on brain function, including the promotion of plasticity and the enhancement of learning and memory performance. Previously we found that exercise increases the expression of certain neurotrophic factors including brain derived neurotrophic factor in the rat hippocampus. To further explore the molecular mechanisms underlying these changes, we used high-density oligonucleotide microarrays containing probe sets representing approximately 5000 genes to analyze the level of gene transcripts in the hippocampus of rats voluntary running for 3 weeks in comparison with sedentary animals. An improved statistical approach for the analysis of DNA microarray data, Cyber-T, was utilized in data analysis. Here we show that exercise leads to changes in the level of a large number of gene transcripts, many of which are known to be associated with neuronal activity, synaptic structure, and neuronal plasticity. Our data indicate that exercise elicits a differential gene expression pattern with significant changes in genes of relevance for brain function.

Aging↗

PubMed2003

Distinct gene expression of osteopontin in patients with ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is a multifactorial disorder of unknown etiology. Few studies have applied genome-wide gene expression analysis in colon tissue samples of UC. We report the analysis of mucosal gene expression in UC and noninflamed control specimens. MATERIALS AND METHODS: This study included 7 UC patients who received a total colectomy because of severe total colitis. Normal control colon tissues were obtained at least 10 cm from the area of pathology in 3 colon cancer patients. Ten colonic tissue samples (7 UC and 3 normal control samples) were subjected to high-density oligonucleotide array analysis. To compare differences in the level of gene expression between UC and control samples, Mann-Whitney U-test was used, with significance set at P < 0.05. RESULTS: Twenty-five genes had a 3.0 approximately 23.4-fold higher mRNA expression in UC samples compared with normal samples, whereas three genes had a 3.0 approximately 3.4-fold lower expression in UC samples compared with normal samples. Two genes showing more than a 10-fold increase expression in UC samples were a macrophage metalloelastase (L23808) and a osteopontin (AF052124). It has been said that macrophage metalloelastase is related to ulcer formation of the intestine, whereas osteopontin plays an important role in the pathogenesis of systemic lupus erythematosus and rheumatoid arthritis. CONCLUSION: Our present study supports the previous report that macrophage metalloelastase is related to ulcer formation of UC, and it also indicates the possibility that osteopontin plays an important role in the pathogenesis of UC via increased immune activity.

Adult↗

PubMed2002

An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis.

Archival formalin-fixed, paraffin-embedded and ethanol-fixed tissues represent a potentially invaluable resource for gene expression analysis, as they are the most widely available material for studies of human disease. Little data are available evaluating whether RNA obtained from fixed (archival) tissues could produce reliable and reproducible microarray expression data. Here we compare the use of RNA isolated from human archival tissues fixed in ethanol and formalin to frozen tissue in cDNA microarray experiments. Since an additional factor that can limit the utility of archival tissue is the often small quantities available, we also evaluate the use of the tyramide signal amplification method (TSA), which allows the use of small amounts of RNA. Detailed analysis indicates that TSA provides a consistent and reproducible signal amplification method for cDNA microarray analysis, across both arrays and the genes tested. Analysis of this method also highlights the importance of performing non-linear channel normalization and dye switching. Furthermore, archived, fixed specimens can perform well, but not surprisingly, produce more variable results than frozen tissues. Consistent results are more easily obtainable using ethanol-fixed tissues, whereas formalin-fixed tissue does not typically provide a useful substrate for cDNA synthesis and labeling.

Animals↗

PubMed2004

Gene expression profiling of cultured human islet preparations.

The expression of functional and regulatory genes by islet cells is a key determinant for the success of islet transplantation. The aim of this study is twofold: first, to characterize the cluster of genes expressed in human islet isolations; and second, to validate the capability of gene array technology to assess with accuracy the expression of various transcripts. RNA from isolated islet preparations obtained from three independent donors was converted to cDNA and then transcribed to cRNA. Individual cRNA preparations were then hybridized to U133A microarrays carrying approximately 23,000 genes, and analyzed using GeneSpring (SiliconGenetics, Redwood City, CA) software. Real-time reverse transcription-polymerase chain reaction was performed to validate results obtained by microarray analysis. Microarray analysis identified the expression of about 7,000 genes transcribed in cultured human islet preparations. Enzymes represented the most abundant class of genes identified, followed by nuclear binding proteins, signal transduction molecules, transport proteins, and growth factor receptors and their ligands. Real-time polymerase chain reaction confirmed the identification of various islet-specific genes detected by microarray analysis, but also showed that such genes as pancreatic duodenal homeobox 1 protein and glucagon-like peptide 1 receptor, which were not detected by gene array, can be readily identified and quantified. In addition, gene array produced a suboptimal quantification of genes expressed in large amounts by islet cells. Indeed, the abundance of mRNA for insulin when compared with the level of somatostatin mRNA was not as different as one would have predicated based on the classic knowledge of islet physiology. Gene array analysis appears to be a valuable tool to obtain preliminary information of genes expressed by a given tissue. The expression levels of transcripts expressed in very low or very high quantities need to be confirmed by an independent technique.

Base Sequence↗

PubMedDate not supplied

Differential gene expression between visceral and subcutaneous fat depots.

Abdominal obesity has been linked to the development of insulin resistance and Type 2 diabetes mellitus (DM2). By surgical removal of visceral fat (VF) in a variety of rodent models, we prevented insulin resistance and glucose intolerance, establishing a cause-effect relationship between VF and the metabolic syndrome. To characterize the biological differences between visceral and peripheral fat depots, we obtained perirenal visceral (VF) and subcutaneous (SC) fat from 5 young rats. We extracted mRNA from the fat tissue and performed gene array hybridization using Affymetrix technology with a platform containing 9 000 genes. Out of the 1 660 genes that were expressed in fat tissue, 297 (17.9 %) genes show a two-fold or higher difference in their expression between the two tissues. We present the 20 genes whose expression is higher in VF fat (by 3 - 7 fold) and the 20 genes whose expression is higher in SC fat (by 3 - 150 fold), many of which are predominantly involved in glucose homeostasis, insulin action, and lipid metabolism. We confirmed the findings of gene array expression and quantified the changes in expression in VF of genes involved in insulin resistance (PPARgamma leptin) and its syndrome (angiotensinogen and plasminogen activating inhibitor-1, PAI-1) by real-time PCR (qRT-PCR) technology. Finally, we demonstrated increased expression of resistin in VF by around 12-fold and adiponectin by around 4-fold, peptides that were not part of the gene expression platform. These results indicate that visceral fat and subcutaneous fat are biologically distinct.

Adipocytes↗

PubMed2004

Gene expression analysis of the pro-oestrous-stage rat uterus reveals neuroligin 2 as a novel steroid-regulated gene.

In the present study, differential gene expression in the uteri of ovariectomised (OVX) and pro-oestrous rats (OVX v. pro-oestrus pair) was investigated using cDNA expression array analysis. Differential uterine gene expression in OVX rats and progesterone (P(4))-injected OVX rats (OVX v. OVX + P(4) pair) was also examined. The uterine gene expression profiles of these two sets of animals were also compared for the effects of P(4) treatment. RNA samples were extracted from uterine tissues and reverse transcribed in the presence of [alpha(32)P]-dATP. Membrane sets of rat arrays were hybridised with cDNA probe sets. Northern blot analysis was used to validate the relative gene expression patterns obtained from the cDNA array. Of the 1176 cDNAs examined, 23 genes showed significant (>two-fold) changes in expression in the OVX v. pro-oestrus pair. Twenty of these genes were upregulated during pro-oestrus compared with their expression in the OVX rat uterus. In the OVX v. OVX + P(4) pair, 22 genes showed significant (>two-fold) changes in gene expression. Twenty of these genes were upregulated in the OVX + P(4) animals. The genes for nuclear factor I-XI, afadin, neuroligin 2, semaphorin Z, calpain 4, cyclase-associated protein homologue, thymosin beta-4X and p8 were significantly upregulated in the uteri of the pro-oestrus and OVX + P(4) rats of both experimental pairs compared with the OVX rat uteri. These genes appear to be under the control of P(4). One of the most interesting findings of the present study is the unexpected and marked expression of the neuroligin 2 gene in the rat uterus. This gene is expressed at high levels in the central nervous system and acts as a nerve cell adhesion factor. According to Northern blot analysis, neuroligin 2 gene expression was higher during the pro-oestrus and metoestrus stages than during the oestrus and dioestrus stages of the oestrous cycle. In addition, neuroligin 2 mRNA levels were increased by both 17beta-oestradiol (E(2)) and P(4), although P(4) administration upregulated gene expression to a greater extent than injection of E(2). These results indicate that neuroligin 2 gene expression in the rat uterus is under the control of both E(2) and P(4), which are secreted periodically during the oestrous cycle.

Animals↗

PubMed2002

Evaluation of the tissue microarray technique for immunohistochemical analysis in rectal cancer.

BACKGROUND: Immunohistochemical staining for tumor-associated proteins is widely used for the identification of novel prognostic markers. Recently, a tissue-conserving, high-throughput technique, tissue microarray, has been introduced. This technique uses 0.6-mm tissue core biopsy specimens, 500 to 1000 of which are brought into a new paraffin array block, which can be sectioned up to 100 times. METHODS: We evaluated the tissue microarray technique for immunohistochemical analysis in 20 rectal cancers. Immunohistochemical staining was performed for the proliferation marker Ki-67 and the tumor suppressor protein p53 in whole tissue sections and in tissue core biopsy specimens. RESULTS: The whole tissue sections were assessed by counting all cells in 10 high-power fields (x40), which resulted in a mean fraction of Ki-67-expressing tumor cells of 0.81 (range, 0.54-1.0). p53 expression assessed in whole tissue sections showed nuclear staining in 15 (75%) of 20 rectal carcinomas. For the tissue microarray technique, a median of 3 (range, 3-5) 0.6-mm tissue core biopsy specimens were studied from each of the 20 tumor specimens. The tissue microarray method gave a mean Ki-67 expression of 0.85 (range, 0.50-1.0) in tumor cell nuclei and showed p53 protein expression in the same 15 of 20 tumors as in the whole tissue sections. CONCLUSION: We conclude that the tissue microarray technique for immunohistochemical staining in rectal cancer yields staining of good quality and expression data for Ki-67 and p53 comparable to those obtained with whole tissue staining. The feasibility of tissue microarray thus enables time- and tissue-preserving studies of multiple markers in large tumor series.

Adult↗

PubMed2006

Effects of a novel tylophorine analog on collagen-induced arthritis through inhibition of the innate immune response.

OBJECTIVE: To test the effects of a novel tylophorine analog, DCB 3503, on the prevention and treatment of collagen-induced arthritis (CIA) and to elucidate its underlying mechanisms. METHODS: DBA/1J mice were immunized with type II collagen, and in some cases, lipopolysaccharide (LPS) was used to boost the development of arthritis. DCB 3503 was injected intraperitoneally before or after the onset of CIA. Mice were monitored to assess the effects of DCB 3503 on the clinical severity of the disease, and pathologic changes in the joints were examined histologically. Levels of tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) in serum and joint tissues were measured by enzyme-linked immunosorbent assay and by cytometric bead array analysis. The effect of DCB 3503 on LPS-induced proinflammatory cytokines from bone marrow-derived dendritic cells was determined by flow cytometry. RESULTS: DCB 3503 significantly suppressed the development and progression of CIA. Moreover, DCB 3503 completely blocked the LPS-triggered acceleration of joint inflammation and destruction. Consistent with its effects in vivo, DCB 3503 significantly suppressed the synthesis of proinflammatory cytokines in inflamed joints as well as cytokine synthesis by macrophages examined ex vivo. Treatment also reduced the levels of inflammatory cytokines (IL-6, IL-12, TNFalpha, and monocyte chemotactic protein 1) produced by bone marrow-derived dendritic cells in vitro. However, DCB 3503 showed no direct effects on T cell proliferation and B cell antibody response. CONCLUSION: Because of its ability to specifically suppress innate immune responses, DCB 3503 may be a novel therapeutic agent for inflammatory arthritis in humans.

Alkaloids↗