Ethidium bromide-stained polyacrylamide gel showing polymerase chain reaction products derived from gene rearrangements in patients with rheumatoid arthritis and T-cell large granular lymphocyte (T-LGL) proliferations

Polyclonal expansion of T-LGLs in patient 10. Lane 1: gene rearrangement–negative, polyclonal smear (tube A); lane 2: gene rearrangement-negative, polyclonal smear (tube B); lane 3: gene rearrangement-negative, polyclonal smear (tube C); lane 4: standard 50 base pairs (bp); lane 5: gene rearrangement-negative, polyclonal smear (tube A); lane 6: gene rearrangement-negative, polyclonal smear (tube B); and lane 7: gene rearrangement-negative, polyclonal smear. Monoclonal expansion in polyclonal background in patient 7. Lane 1: gene rearrangement: monoclonal product 180 bp (i) in tube A; lane 2: gene rearrangement: monoclonal product 210 bp (ii) in polyclonal background (tube B); lane 3: gene rearrangement: monoclonal product 160 bp (iii); lane 4: (tube A) gene rearrangement-negative, polyclonal smear; lane 5: standard 50 bp; lane 6: (tube B) gene rearrangement-negative, polyclonal smear; and lane 7: (tube C) gene rearrangement-negative, polyclonal smear. Monoclonal gene rearrangements in patient 1 with T-LGL leukemia. Lane 1: gene rearrangement-negative (tube A); lane 2: gene rearrangement-positive, monoclonal product 250 bp (iv) in tube B; lane 3: (tube C): gene rearrangement-negative, polyclonal smear; lane 4: standard 50 bp; lane 5: gene rearrangement-positive, monoclonal product 230 bp (v) in tube A; lane 6: gene rearrangement-positive, monoclonal product 180 bp (vi) in tube B; and lane 7: gene rearrangement-negative, polyclonal smear. TCR, T-cell receptor.<p><b>Copyright information:</b></p><p>Taken from "Characteristics of T-cell large granular lymphocyte proliferations associated with neutropenia and inflammatory arthropathy"</p><p>http://arthritis-research.com/content/10/3/R55</p><p>Arthritis Research & Therapy 2008;10(3):R55-R55.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2483444.</p><p></p

Histopathological features of bone marrow in patients with arthritis and T-cell large granular lymphocyte (T-LGL) lymphocytosis

Patient 1 with rheumatoid arthritis (RA) and T-LGL leukemia. Staining for CD57 demonstrates intrasinusoidal linear arrays and interstitial clusters of T cells (EnVision stain, ×100). Granzyme B highlights cytotoxic granules in these cells (EnVision stain, ×200). Patient 10 with polyclonal T-LGL lymphocytosis. Staining for CD8 shows dispersed T cells (EnVision stain, ×200). Patient 9 with unclassified arthritis, T-LGL leukemia, and and gene rearrangements. CD3 staining shows interstitial and nodular infiltration of T cells (EnVision stain, ×100). Patient 9. The lymphoid nodule contains few CD20B cells (EnVision stain, ×200). Patient 7 with RA and T-LGL leukemia. A decreased count of granulocytic precursors (myeloperoxydase) is shown (EnVision stain, ×200). IGKV, immunoglobulin kappa variable; IGLV, immunoglobulin lambda variable<p><b>Copyright information:</b></p><p>Taken from "Characteristics of T-cell large granular lymphocyte proliferations associated with neutropenia and inflammatory arthropathy"</p><p>http://arthritis-research.com/content/10/3/R55</p><p>Arthritis Research & Therapy 2008;10(3):R55-R55.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2483444.</p><p></p

Modeling the Role of Peroxisome Proliferator-Activated Receptor γ and MicroRNA-146 in Mucosal Immune Responses to <em>Clostridium difficile</em>

<div><p><em>Clostridium difficile</em> is an anaerobic bacterium that has re-emerged as a facultative pathogen and can cause nosocomial diarrhea, colitis or even death. Peroxisome proliferator-activated receptor (PPAR) γ has been implicated in the prevention of inflammation in autoimmune and infectious diseases; however, its role in the immunoregulatory mechanisms modulating host responses to <em>C. difficile</em> and its toxins remains largely unknown. To characterize the role of PPARγ in <em>C. difficile</em>-associated disease (CDAD), immunity and gut pathology, we used a mouse model of <em>C. difficile</em> infection in wild-type and T cell-specific PPARγ null mice. The loss of PPARγ in T cells increased disease activity and colonic inflammatory lesions following <em>C. difficile</em> infection. Colonic expression of IL-17 was upregulated and IL-10 downregulated in colons of T cell-specific PPARγ null mice. Also, both the loss of PPARγ in T cells and <em>C. difficile</em> infection favored Th17 responses in spleen and colonic lamina propria of mice with CDAD. MicroRNA (miRNA)-sequencing analysis and RT-PCR validation indicated that miR-146b was significantly overexpressed and nuclear receptor co-activator 4 (NCOA4) suppressed in colons of <em>C. difficile</em>-infected mice. We next developed a computational model that predicts the upregulation of miR-146b, downregulation of the PPARγ co-activator NCOA4, and PPARγ, leading to upregulation of IL-17. Oral treatment of <em>C. difficile</em>-infected mice with the PPARγ agonist pioglitazone ameliorated colitis and suppressed pro-inflammatory gene expression. In conclusion, our data indicates that miRNA-146b and PPARγ activation may be implicated in the regulation of Th17 responses and colitis in <em>C. difficile</em>-infected mice.</p> </div

Effect of <i>Clostridium difficile</i> infection on the colonic expression of miR-146b and target genes NCOA4, CD36 and GLUT4 mRNA in mice.

<p>Colonic expression of miRNA-146b (A) as well as NCOA4 (B), CD36 (C) and GLUT4 (D) were assessed by real-time quantitative RT-PCR in mice infected with <i>C. difficile</i> (n = 10). Data are represented as mean ± standard error. Points with an asterisk are significantly different when compared to the control group (<i>P</i><0.05).</p

<i>Clostridium difficile</i> infection modulates colonic gene expression in mice.

<p>Colonic expression of monocyte chemotactic Protein 1 (MCP-1) (A), interleukin 6 (IL-6) (B), interleukin 17 (IL-17) (C) and interleukin 1β (IL-1β) (D were assessed by real-time quantitative RT-PCR in mice infected with <i>C. difficile</i> (n = 10). Data are represented as mean ± standard error. Points with an asterisk are significantly different when compared to the control group (<i>P</i><0.05).</p

The loss of PPARγ in T cells regulates colonic cytokine expression of mice infected with <i>Clostridium difficile</i>.

<p>Colonic expression of interleukin 10 (IL-10) (A), interleukin 17 (IL-17) (B), monocyte chemoattractant protein 1 (MCP-1) (C) and tumor necrosis factor (TNF-α) (D) were assessed by real-time quantitative RT-PCR in wild type and T cell PPARγ null mice infected with <i>C. difficile</i> (n = 8). Data are represented as mean ± standard error. Points with an asterisk are significantly different when compared to the wild type control group (<i>P</i><0.05).</p

The loss of PPARγ in T cells and <i>Clostridium difficile</i> infection enhances Th17 responses in spleen and lamina propria of mice.

<p>Splenocytes and lamina propria lymphocytes from wild type and T cell PPARγnull mice infected with <i>C. difficile</i> (n = 6) were immunophenotyped to identify immune cell subsets by flow cytometry. Data are represented as mean ± standard error. Points with an asterisk are significantly different when compared to the control group (<i>P</i><0.05).</p

Computational modeling of mucosal immune responses to <i>Clostridium difficile</i> infection.

<p>CellDesigner-based illustration of the Complex Pathway SImulator model of the model for Clostridium difficile immune response (A). The model represents the interaction between <i>C. difficile</i>, miRNA-146, nuclear receptor coactivator 4 (NCOA4), peroxisome proliferator-activated receptor γ (PPAR γ), interleukin 10 (IL-10) and interleukin 17 (IL-17) in Systems Biology Markup Language format. Inhibition is represented in red and activation in green. COPASI steady state scan showing the variation on the species concentrations with increasing computational concentration of <i>C. difficile</i> (B). In silico simulations show how increasing concentrations of <i>C. difficile</i> increase miRNA-146b levels, thus decreasing NCOA4 and PPAR γ. In line with the experimental data, IL-17 expression also increases with the infection.</p

Model fitting performed by using COPASI's global parameter estimation.

<p>A species is fitted computationally using experimental data and simulation algorithms. The objective value is the value that the modeling software targets based on the experimental data and the computational simulation.</p

Impact of the loss of PPARγ in T cells and pharmacological activation of PPARγ colonic inflammatory lesions in <i>Clostridium difficile</i>-infected mice.

<p>Representative photomicrographs of colons of uninfected (A and E), infected wild type mice (B and F), infected CD4cre+ mice (C and G) and infected wild-type mice treated orally with pioglitazone (70 mg/kg) (D and H) (n = 8). Original magnification at 40× (top panel) and 100× (bottom panel).</p