Unique immunologic patterns in fibromyalgia

BACKGROUND: Fibromyalgia (FM) is a clinical syndrome characterized by chronic pain and allodynia. The diagnosis of FM has been one of exclusion as a test to confirm the diagnosis is lacking. Recent data highlight the role of the immune system in FM. Aberrant expressions of immune mediators, such as cytokines, have been linked to the pathogenesis and traits of FM. We therefore determined whether cytokine production by immune cells is altered in FM patients by comparing the cellular responses to mitogenic activators of stimulated blood mononuclear cells of a large number of patients with FM to those of healthy matched individuals. METHODS: Plasma and peripheral blood mononuclear cells (PBMC) were collected from 110 patients with the clinical diagnosis of FM and 91 healthy donors. Parallel samples of PBMC were cultured overnight in medium alone or in the presence of mitogenic activators; PHA or PMA in combination with ionomycin. The cytokine concentrations of IFN-γ, IL-5, IL-6, IL-8, IL-10, MIP-1β , MCP-1, and MIP1-α in plasma as well as in cultured supernatants were determined using a multiplex immunoassay using bead array technology. RESULTS: Cytokine levels of stimulated PBMC cultures of healthy control subjects were significantly increased as compared to matched non-stimulated PBMC cultures. In contrast, the concentrations of most cytokines were lower in stimulated samples from patients with FM compared to controls. The decreases of cytokine concentrations in patients samples ranged from 1.5-fold for MIP-1β to 10.2-fold for IL-6 in PHA challenges. In PMA challenges, we observed 1.8 to 4-fold decreases in the concentrations of cytokines in patient samples. CONCLUSION: The cytokine responses to mitogenic activators of PBMC isolated from patients with FM were significantly lower than those of healthy individuals, implying that cell-mediated immunity is impaired in FM patients. This novel cytokine assay reveals unique and valuable immunologic traits, which, when combined with clinical patterns, can offer a diagnostic methodology in FM

Oligo- and Polymetastatic Progression in Lung Metastasis(es) Patients Is Associated with Specific MicroRNAs

<div><h3>Rationale</h3><p>Strategies to stage and treat cancer rely on a presumption of either localized or widespread metastatic disease. An intermediate state of metastasis termed oligometastasis(es) characterized by limited progression has been proposed. Oligometastases are amenable to treatment by surgical resection or radiotherapy.</p> <h3>Methods</h3><p>We analyzed microRNA expression patterns from lung metastasis samples of patients with ≤5 initial metastases resected with curative intent.</p> <h3>Results</h3><p>Patients were stratified into subgroups based on their rate of metastatic progression. We prioritized microRNAs between patients with the highest and lowest rates of recurrence. We designated these as high rate of progression (HRP) and low rate of progression (LRP); the latter group included patients with no recurrences. The prioritized microRNAs distinguished HRP from LRP and were associated with rate of metastatic progression and survival in an independent validation dataset.</p> <h3>Conclusion</h3><p>Oligo- and poly- metastasis are distinct entities at the clinical and molecular level.</p> </div

Prioritized microRNAs between lung metastasis patients with a high rate or progression (HRP) and low rate of progression (LRP).

<p>LRP patient n = 32; HRP patient n = 16. Fold change values for microRNAs were calculated using the delta-delta CT method, and p-values were calculated using a Student's t test (two-tailed p≤5%, <b>Methods</b>). A positive fold change represents elevated expression in patient samples with a high rate of progression as compared to a low rate of progression (LRP) progression (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050141#pone.0050141.s004" target="_blank">Tables S1</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050141#pone.0050141.s005" target="_blank">S2</a></b> for clinical information). Further details on the quality control for TaqMan microRNA expression is presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050141#pone.0050141.s002" target="_blank">Figure S2</a></b>.</p

Lung oligometastatic and polymetastatic progression samples are differentiated through hierarchical clustering with prioritized microRNA features derived from the independent validation dataset GSE25552.

<p>29 microRNAs differentially expressed between polymetastatic vs. oligometastatic progression (GSE25552) were used to stratify lung metastatic samples, described in the current report (n = 63) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050141#pone.0050141-Lussier1" target="_blank">[22]</a>. As shown above, 17 of 39 oligometastatic progression patients cluster together (left branch) and 20 of 24 polymetastatic progression patients cluster separately (right branch; odds ratio = 3.86; Fisher's Exact Test, two-tailed p = 0.032; <b>Methods</b>). See <b>Methods</b> for definitions of polymetastatic and oligometastatic progression.</p

Unsupervised hierarchical clustering of microRNAs derived from lung metastasis samples distinguishes patients with high vs. low rates of metastatic progression.

<p>Expression of 384 microRNAs in metastatic lung tumor samples above, at, and below mean level are represented by red, black, and green TaqMan qPCR Ct values across all patients (n = 63) and were used to cluster oligo- and poly-metastatic patients. As shown above, 13 of 16 HRP patients (red on color-coded bar above the dendrogram) cluster together (left dendrogram branch) and 20 of 32 LRP patients (blue color-coded bar) cluster together from these HRP patients (right dendrogram branch), resulting in a divergence of these diametric sub-phenotypes (odds ratio = 7.22; Fisher's Exact Test, two-tailed p = 0.006). We also observed significant differences between patients classified as polymetastatic (P) vs. oligometastatic (O) (OR = 3.89, FET p = 0.019; <b>Methods</b>).</p

Family and -3p/-5p co-expression analysis of microRNAs prioritized between metastatic samples of patients with high vs. low rates of progression.

<p>Three microRNA families and -3p/-5p expression pairs were prioritized by enrichment in congruently co-expressed microRNA (empirical permutation resampling p = 0.0385, <b>Methods</b>).</p

Stratification of metastatic phenotypes in lung metastasis patient samples reveals three distinct subgroups and a difference in survival outcome between patients with high vs. low rates of progression.

<p><b>A</b>, lung metastasis patients were stratified according to the time (years) to first recurrent metastasis following pulmonary metastectomy (x-axis) and rate of recurrent metastases (per year) over follow-up after surgery (y-axis). Three distinct subgroups emerged using cutoffs for the rate of recurrent metastases: patients with a high rate of progression develop at least 3.6 new tumors/year after surgery and tend to exhibit their first recurrence in the first year following surgery (HRP, red circles); patients with a low rate of progression develop less than 0.6 new tumors/year after surgery (LRP, blue circles); remaining patients that do not meet the criteria for high or low rate of progression status are classified with an intermediate rate of progression (IRP, white circles). Green dotted lines represent the aforementioned rate thresholds. LRP patients exhibiting no recurrence after metastectomy were also plotted here for convenience as yielding 0.0 metastases/month on the y-axis. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050141#pone.0050141.s004" target="_blank">Tables S1</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050141#pone.0050141.s005" target="_blank">S2</a></b> for further clinical information. <b>B</b>, lung metastasis patients of the LRP (n = 32) and HRP (n = 16) subgroups were compared for their survival outcome using log-rank Mantel-Cox analysis. The median survival of LRP and HRP patients is 63.5 months and 18 months, respectively. A log-rank Mantel-Cox p<0.0001 was obtained when comparing LRP vs. HRP survival outcome.</p