Monitoring of Post-Brain Injuries By Measuring Plasma Levels of Neuron-Derived Extracellular Vesicles.

Background: Extracellular vesicles (EV) released from neurons into the blood can reflect the state of nervous tissue. Measurement of neuron derived EV (NDE) may serve as an indicator of brain injury. Methods: A sandwich immunoassay was established to measure plasma NDE using anti-neuron CD171 and anti-EV CD9 ([CD171 + CD9+]). Plasma samples were obtained from commercial sources, cross-country (n = 9), football (n = 22), soccer (n = 19), and rugby (n = 18) athletes over time. Plasma was also collected from patients undergoing total aortic arch replacement (TAR) with selective cerebral perfusion during cardiopulmonary bypass before and after surgery (n = 36). Results: The specificity, linearity, and reproducibility of NDE assay (measurement of [CD171 + CD9+]) were confirmed. By scanning electron microscopy and nanoparticle tracking, spherical vesicles ranging in size from 150 to 300 nm were confirmed. Plasma levels of NDE were widely spread over 2 to 3 logs in different individuals with a significant age-dependent decrease. However, NDE were very stable in each individual within a ± 50% change over time (cross-country, football, soccer), whereas rugby players were more variable over 4 years. In patients undergoing TAR, NDE increased rapidly in days post-surgery and were significantly (P = .0004) higher in those developing postoperative delirium (POD) (n = 13) than non-delirium patients (n = 23). Conclusions: The blood test to determine plasma levels of NDE was established by a sandwich immunoassay using 2 antibodies against neuron (CD171) and exosomes (CD9). NDE levels varied widely in different individuals and decreased with age, indicating that NDE levels should be considered as a normalizer of NDE biomarker studies. However, NDE levels were stable over time in each individual, and increased rapidly after TAR with greater increases associated with patients developing POD. This assay may serve as a surrogate for evaluating and monitoring brain injuries.

Neuron-related blood inflammatory markers as an objective evaluation tool for major depressive disorder: An exploratory pilot case-control study.

BACKGROUND: Neuroinflammation is suggested to be a crucial factor in the pathophysiology of major depressive disorder (MDD). Analysis of neuron-derived exosomes (NDE) in peripheral blood has recently been highlighted to reveal the pathophysiology of brain diseases without using brain biopsy. Currently, human NDE studies require a considerable amount of peripheral blood to measure multiple substances inside exosomes. Previously, NDE-based clinical studies focusing on MDD have not been reported. METHODS: As an exploratory pilot case-control study between healthy controls (HC) and drug-free MDD patients (each; N = 34), we searched for NDE-related blood biomarkers with a small amount of peripheral blood using a novel sandwich immunoassay between anti-neuron antibody and antibodies against CD81 (an exosome marker) and against other proteins related to neuroinflammation and synaptic functions. RESULTS: Most neuron-related blood biomarkers had moderately to strongly positive correlation with CD81 (NDE), thus we normalized the above biomarkers by CD81 (quantity of each biomarker/CD81) to predict NDE-related blood substances. Interleukin 34 (IL34)/CD81 levels were significantly higher in MDD group compared to HC group. Synaptophysin (SYP), SYP/CD81, and tumor necrosis factor receptor 1 (TNFR1)/CD81 were positively correlated with severities of depression and/or various sub-symptoms. LIMITATIONS: We did not actually extract NDE from peripheral blood. CONCLUSIONS: Using a small amount of peripheral blood, we have successfully detected possible NDE-related blood biomarkers. This is the first study to suggest that not only SYP and TNFR1 but also IL34 are important blood biomarkers for patients with MDD. Further studies are warranted to evaluate the present study.

A Preliminary Report on Brain-Derived Extracellular Vesicle as Novel Blood Biomarkers for Sport-Related Concussions.

The purpose of the study was to test the utility of unique panel of blood biomarkers as a means to reflect one's recovery process after sport-related neurotrauma. We established a panel of biomarkers that reacted positive with CD81 (extracellular vesicle marker) and various neuron- and glia-specific antigens [e.g., neurofilament light polypeptide (NF-L), tau, synaptosome-associated protein 25 (SNAP25), glial fibrillary acidic protein, and myelin basic protein]. We first evaluated test-retest reliabilities of brain-derived exosome markers, followed by an application of these markers in eight professional ice hockey players to detect cumulative neuronal burden from a single ice hockey season. During the season, two players were diagnosed with concussions by team physician based on an exhibition of symptoms as well as abnormality in balance and ocular motor testing. One player reached symptom-free status 7 days after the concussion, while the other player required 36 days for symptoms to completely resolve. Blood samples and clinical assessments including balance error scoring system and near point of convergence throughout recovery process were obtained. Biomarkers indicative of axonal damage, neuronal inflammation, and glial activation showed excellent test-retest reliabilities (intraclass correlation coefficient: 0.713-0.998, p's < 0.01). There was a statistically significant increase in the NF-L marker at post-season follow-up compared to pre-season baseline (Z = -2.100, P = 0.036); however the statistical significance did not withstand Bonferroni correction for multiple comparisons. In concussion cases, neuronal and microglia markers notably increased after concussions, with the unique expression patterns being similar to that of concussion recovery process. These longitudinal data coupled with excellent test-retest reliabilities of novel array of blood biomarkers potentially reflect the damage in neural cell structures and metabolic crisis due to concussion. However, future studies with larger sample size and appropriate control groups to evaluate sensitivity and specificity of these markers are needed. This preliminary case report suggests the potential utility of multimodal blood biomarkers for concussion prognosis and recovery assessment.

Luminal Extracellular Vesicles (EVs) in Inflammatory Bowel Disease (IBD) Exhibit Proinflammatory Effects on Epithelial Cells and Macrophages.

BACKGROUND: Extracellular vesicles (EVs) are membrane-enclosed particles released by cells as a means of intercellular communication. They are potential novel biomarkers, as they are readily isolated from body fluids, and their composition reflects disease pathways. Whether these particles are released from sites of intestinal inflammation in inflammatory bowel disease (IBD) has not previously been determined. METHODS: EVs were isolated by ultracentrifugation of colonic luminal fluid aspirates and characterized according to surface proteins, and constituent mRNA and proteins. The effects of EVs on colonic epithelial cells and macrophages in culture were assessed at the transcriptional, translational, and functional levels. RESULTS: Intestinal luminal aspirates contained abundant EVs, at a mean concentration of 4.3 × 10 particles/mL and with a mean diameter of 146 nm. EVs from patients with IBD with a high endoscopic score (≥1) contained significantly higher mRNA and protein levels of interleukin 6 (IL-6), IL-8, IL-10, and tumor necrosis factor α than EVs from healthy controls. EVs were absorbed by cultured colonic epithelial cells, leading to an increased translation of IL-8 protein by recipient cells when treated with EVs from patients with IBD. EVs and EV-treated epithelial cells induced migration of a significantly greater number of macrophages than epithelial cells alone. CONCLUSIONS: EVs shed from sites of intestinal inflammation in patients with IBD have a distinct mRNA and protein profile from those of healthy individuals. These EVs have proinflammatory effects on the colonic epithelium, in vitro. Their stability in luminal samples and their mRNA and protein content identify them as a potential fecal biomarker that reflects mucosal inflammatory pathways.

Tumor-derived exosomes regulate expression of immune function-related genes in human T cell subsets.

Tumor cell-derived exosomes (TEX) suppress functions of immune cells. Here, changes in the gene profiles of primary human T lymphocytes exposed in vitro to exosomes were evaluated. CD4(+) Tconv, CD8(+) T or CD4(+) CD39(+) Treg were isolated from normal donors' peripheral blood and co-incubated with TEX or exosomes isolated from supernatants of cultured dendritic cells (DEX). Expression levels of 24-27 immune response-related genes in these T cells were quantified by qRT-PCR. In activated T cells, TEX and DEX up-regulated mRNA expression levels of multiple genes. Multifactorial data analysis of ΔCt values identified T cell activation and the immune cell type, but not exosome source, as factors regulating gene expression by exosomes. Treg were more sensitive to TEX-mediated effects than other T cell subsets. In Treg, TEX-mediated down-regulation of genes regulating the adenosine pathway translated into high expression of CD39 and increased adenosine production. TEX also induced up-regulation of inhibitory genes in CD4(+) Tconv, which translated into a loss of CD69 on their surface and a functional decline. Exosomes are not internalized by T cells, but signals they carry and deliver to cell surface receptors modulate gene expression and functions of human T lymphocytes.

Exosomes isolated from plasma of glioma patients enrolled in a vaccination trial reflect antitumor immune activity and might predict survival.

Exosomes in plasma of glioma patients hold promise as biomarkers of prognosis. We aimed to determine whether changes in total exosomal protein and mRNA expression levels could serve as surrogate markers of immunological and clinical responses in glioma patients receiving antitumor vaccines. Exosomes were isolated from pre/post-vaccine plasma specimens in 20/22 patients enrolled in a phase I/II trial with the antitumor vaccine. Exosomal protein content was analyzed and mRNA expression levels for 24 genes were simultaneously assessed by qRT-PCR. Pre- to post-vaccination changes in exosomal protein and ΔCt values were correlated with immunological and clinical responses and survival using Spearman rank statistics and hazard ratios (HR). Exosomal protein levels positively correlated (p < 0.0043) with the WHO tumor grade at diagnosis. Protein levels were lower in post- vs. pre-vaccination exosome fractions. Post-therapy increases in tumor size were associated with elevations in exosome proteins in glioblastoma but not always in anaplastic astrocytoma (AA). Only exosomal ΔCt values for IL-8, TIMP-1, TGF-ß and ZAP70 were significant (p < 0.04 to p < 0.001). The ΔCt for IL-8 and TGF-ß mRNA positively correlated with post-vaccine immunologic responses to glioma antigens, while ΔCt for TIMP-1 mRNA was negatively correlated to ΔCt for IL-8 and TGF-ß. Only ΔCt for IL-8 weakly correlated with OS and time to progression (TTP). In post-vaccine exosomes of the longest surviving patient with AA, mRNA for PD-1 was persistently elevated. Protein and mRNA expression levels for immune-related genes in plasma exosomes were useful in evaluating glioma patients' response to vaccination therapy.

Lipopolysaccharide-Induced CXCL10 mRNA Level and Six Stimulant-mRNA Combinations in Whole Blood: Novel Biomarkers for Bortezomib Responses Obtained from a Prospective Multicenter Trial for Patients with Multiple Myeloma.

To identify predictive biomarkers for clinical responses to bortezomib treatment, 0.06 mL of each whole blood without any cell separation procedures was stimulated ex vivo using five agents, and eight mRNAs were quantified. In six centers, heparinized peripheral blood was prospectively obtained from 80 previously treated or untreated, symptomatic multiple myeloma (MM) patients with measurable levels of M-proteins. The blood sample was procured prior to treatment as well as 2-3 days and 1-3 weeks after the first dose of bortezomib, which was intravenously administered biweekly or weekly, during the first cycle. Six stimulant-mRNA combinations; that is, lipopolysaccharide (LPS)-granulocyte-macrophage colony-stimulating factor (GM-CSF), LPS-CXCL chemokine 10 (CXCL10), LPS-CCL chemokine 4 (CCL4), phytohemagglutinin-CCL4, zymosan A (ZA)-GMCSF and ZA-CCL4 showed significantly higher induction in the complete and very good partial response group than in the stable and progressive disease group, as determined by both parametric (t-test) and non-parametric (unpaired Mann-Whitney test) tests. Moreover, LPS-induced CXCL10 mRNA expression was significantly suppressed 2-3 days after the first dose of bortezomib in all patients, as determined by both parametric (t-test) and non-parametric (paired Wilcoxon test) tests, whereas the complete and very good partial response group showed sustained suppression 1-3 weeks after the first dose. Thus, pretreatment LPS-CXCL10 mRNA and/or the six combinations may serve as potential biomarkers for the response to bortezomib treatment in MM patients.

Posttransplantation bone marrow assessment by quantifying hematopoietic cell-derived mRNAs in plasma exosomes/microvesicles.

BACKGROUND: Bone marrow (BM) aspiration often can be a painful medical procedure. It is unavoidable, however, because hematopoietic precursor cells (HPC) exist only in BM and few escape to peripheral blood (PB). We hypothesized that HPCs might release exosomes and microvesicles (EMV) in BM, and the resulting EMV would penetrate into PB. Such BM-derived EMV might be identified in PB by measuring specific mRNAs produced by HPC. METHODS: Human plasma was applied to an EMV-capture filter plate. After centrifugation, captured EMV were lysed on the filter plate. Resulting lysates were transferred to an oligo(dT)-immobilized microplate for mRNA isolation followed by reverse transcription PCR (RT-PCR). Using this system, myeloid-, erythroid-, and megakaryocyte-lineage-specific poly(A)(+) mRNAs were quantified in plasma obtained from 18 patients who had undergone hematopoietic stem cell transplantation (HSCT). RESULTS: When fluorescent liposomes were applied to the filter plate, more than 95% of applied liposomes were absorbed. When human plasma was applied, a scanning electron microscope showed EMV-like particles on the membrane of the filter plate. After RT-PCR, various HPC-specific mRNAs were detected, and the results were equivalent to those derived from the standard ultracentrifugation method. The levels of these mRNAs were undetectable after HSCT and became detectable 1-2 weeks after HSCT, a substantially earlier time point than with traditional hematological analysis. The recovery of EMV mRNA at day 15 corresponded to the final clinical outcome at day 180. CONCLUSIONS: HPC-derived mRNAs in plasma EMV may represent new biomarkers for the assessment of BM condition and could reduce the necessity for frequent BM aspiration.

Aging enhances release of exosomal cytokine mRNAs by Aß1-42-stimulated macrophages.

Amyloid-ß1-42 (Aß) peptide effects on human models of central nervous system (CNS)-patrolling macrophages (Ms) and CD4 memory T-cells (CD4-Tms) were investigated to examine immune responses to Aß in Alzheimer's disease. Aß and lipopolysaccharide (LPS) elicited similar M cytokine and exosomal mRNA (ex-mRNA) responses. Aß- and LPS-stimulated Ms from 20 ≥65-yr-old subjects generated significantly more IL-1, TNF-α, and IL-6, but not IL-8 or IL-12, and significantly more ex-mRNAs for IL-6, TNF-α, and IL-12, but not for IL-8 or IL-1, than Ms from 20 matched 21- to 45-yr-old subjects. CD4-Tm generation of IL-2, IL-4, and IFN-γ and, for young subjects, IL-10, but not IL-6, evoked by Aß was significantly lower than with anti-T-cell antigen receptor antibodies (Abs). Abs significantly increased all CD4-Tm ex-mRNAs, but only IL-2 and IL-6 ex-mRNAs were increased by Aß. There were no significant differences between cytokine and ex-mRNA responses of CD4-Tms from the old compared to the young subjects. M-derived serum exosomes from the old subjects had significantly higher IL-6 and IL-12 ex-mRNA levels than those from the young subjects, whereas there were no differences for CD4-Tm-derived serum exosomes. An Aß level relevant to neurodegeneration elicited broad M cytokine and ex-mRNA responses that were significantly greater in the old subjects, but only narrow and age-independent CD4-Tm responses.

A phase II trial of dasatinib in patients with metastatic castration-resistant prostate cancer treated previously with chemotherapy.

There is a need for efficacious therapies for metastatic castration-resistant prostate cancer (mCRPC) after disease progression on docetaxel. The SRC tyrosine kinase and its related family members may be important drivers of prostate cancer and can be inhibited by dasatinib. mCRPC patients, after one previous chemotherapy, started dasatinib at 70 mg twice daily, amended to 100 mg daily. The primary endpoint was the disease control (DC) rate, defined as complete response (CR), partial response (PR), or stable disease (SD) in prostate specific antigen (PSA), RECIST, bone scan, and FACT-P score. Up to 41 patients were to be accrued (two-stage design, 21+20) to rule out a null-hypothesized effect of 5 versus 20% (α=0.05, ß=0.1). Secondary endpoints included progression-free survival, toxicity, and pharmacokinetic and pharmacodynamic correlatives. Of 38 patients, 27 were evaluable for response or toxicity. The median duration of therapy was 55 days (6-284). Five patients showed DC after 8 weeks of therapy (18.5% DC, 95% CI: 6.3-38.1%). One PR (3.7% response rate, 95% CI: 0.1-19.0%) was observed in a patient treated for 284 days. Twelve patients (43%) discontinued treatment for toxicity. Dasatinib induced a decrease in phytohemagglutinin-stimulated CSF2, CD40L, GZMB, and IL-2 mRNAs in blood cells, indicating target engagement. Decreases in plasma IL-6 and bone alkaline phosphatase, and in urinary N-telopeptide, were associated with DC. Dasatinib has definite but limited activity in advanced mCRPC, and was poorly tolerated. The observation of a patient with prolonged, objective, clinically significant benefit warrants molecular profiling to select the appropriate patient population.

[Prediction of WT1/MUC1 peptide dendritic cell therapy responders by quantification of ex vivo induced mRNA in whole blood].

Cancer immunotherapy has shown much potential, but is not yet beneficial for all patients. Acquired immunity after immunotherapy can be assessed by a variety of methods; however, the methods for the prediction of such responses before treatment are quite limited. To challenge this classic problem, we quantified 17 different leukocyte function-associated mRNAs( IFN-γ,TNFSF1, TNFSF2, TNFSF5, IL-10, TGF ß,CTLA4, PD1, FOXP3, GMCSF, VEGF, IL-8, CCL8, CXCL3, and IL-2) in whole blood after ex vivo stimulation with 7 different agents(PHA, HAG, zymosan, IFN-γ,rIL-2, aTCR, and picibanil) to activate various subsets of leukocytes. The mRNAs were quantified by the Hitachi Chemical Research Center, Inc. Irvine, CA. The clinical outcomes for WT1 peptide-and/or MUC1 peptide-pulsed dendritic cell therapy for advanced cancer (n=26) were CR+PR, 4 cases; SD, 9 cases; and PD, 13 cases. The accuracy of prediction was found to be 100% using the formula developed by multivariate discriminant analysis of the values for ex vivo induced mRNAs. Because the volume of blood needed for this test is less than 1.5 mL, and because cell isolation and culture are not necessary, this method will become a model of personalized medicine diagnostics for cancer immunotherapy.

Phosphatase inhibitor, sodium stibogluconate, in combination with interferon (IFN) alpha 2b: phase I trials to identify pharmacodynamic and clinical effects.

Since sodium stibogluconate (SSG) inhibited phosphatases including SHP-1 and augmented anti-tumor actions of IFN-α2b in vitro and in mice, two Phase I trials of SSG/IFN-α2b combination were undertaken to evaluate safety and target inhibition. Escalating doses of SSG (200-1200 mg/m2) and fixed doses of IFN-α2b (3x106 units/m2) with or without chemotherapy (dacarbazine, vinblastine, cisplatin) were evaluated for side effects and impact on SHP-1 phospho-substrates and IFNα-stimulated-genes (ISGs) in peripheral blood in 40 patients with metastatic melanoma, soft tissue sarcomas, gastrointestinal stromal tumors, and breast or colorectal carcinomas who did not have other established treatment options. Common adverse events were bone marrow suppression, fatigue, gastrointestinal upset, and asymptomatic lipase elevation (n=13); the latter was dose related and mostly after 10d of SSG/IFN-α2b in combination. Levels of SHP-1 substrates (pSTAT1, pSTAT3, pLck and pSlp76) were increased (up to 3x) in peripheral blood cells following SSG with no potentiation by combination with IFN-α2b. Representative ISGs in peripheral blood were induced after IFN-α2b at 4 and 24 hrs with selective modulations by combination. The median time on trials was 2.3 months (10-281d) with no objective regression of disease. Alive at 1y were 17/40 (43%) patients and after 2y were 8/40 (20%) following treatment initiation. These data demonstrate that SSG impacted signal molecules consistent with PTP inhibition and was tolerated in combination with IFN-α2b. Phase II investigations of SSG could safely utilize doses of up to 1200 mg/m2 of SSG for up to 10d alone or in combination with IFN-α2b with or without chemotherapy.

Moving on up: Second-Line Agents as Initial Treatment for Newly-Diagnosed Patients with Chronic Phase CML.

The treatment of chronic myelogenous leukemia (CML) was revolutionized by the development of imatinib mesylate, a small molecule inhibitor of several protein tyrosine kinases, including the ABL1 protein tyrosine kinase. The current second generation of FDA-approved ABL tyrosine kinase inhibitors, dasatinib and nilotinib, are more potent inhibitors of BCR-ABL1 kinase in vitro. Originally approved for the treatment of patients who were refractory to or intolerant of imatinib, dasatinib and nilotinib are now also FDA approved in the first-line setting. The choice of tyrosine kinase inhibitor (ie, standard or high dose imatinib, dasatinib, nilotinib) to use for initial therapy in chronic-phase CML (CML-CP) will not always be obvious. Therapy selection will depend on both clinical and molecular factors, which we will discuss in this review.

Ex vivo simulation of leukocyte function: stimulation of specific subset of leukocytes in whole blood followed by the measurement of function-associated mRNAs.

In order to characterize a wide spectrum of leukocyte functions with clinically applicable procedures, 0.06 ml each of heparinized whole blood was stimulated in triplicate for 4h with phytohemagglutinin (T cell stimulator), heat aggregated IgG (IgG Fc receptor stimulator), lipopolysaccharide (toll-like receptor (TLR)-4 stimulator), zymosan (TLR-2 stimulator), monoclonal antibody against T-cell receptor alpha/beta chain, recombinant interleukin-2, and solvent controls, then 32 different leukocyte function-associated mRNAs were quantified by the method reported previously (Mitsuhashi et al. Clin. Chem. 2006). Two control genes (beta-actin, beta-2-microglobulin) were not affected by these stimulations, whereas the induction of CCL chemokines-2, 4, 8, 20, CXCL chemokines-3, 10, interleukin (IL)-8 (markers of leukocyte accumulation/recruit), granzyme B, perforin 1, tumor necrosis factor superfamily-1, 2, 5, 14, 15, CD16 (markers of cell killing), IL10, transforming growth factor beta 1 (humoral factors of immune suppression), forkhead box P3, CD25, arginase (cellular markers of immune suppression), IL2, IL4, interferon-gamma, IL17 (markers of various subsets of T helper cells), granulocyte-macrophage colony-stimulating factor (marker of antigen presenting cells), immunoglobulin heavy locus (marker of B-cells), vascular endothelial growth factor (marker of angiogenesis), pro-opiomelanocortin (marker of local pain), and CD11a mRNA (marker of leukocyte adherence to endothelium) were identified by these stimulations. The blood volume in this assay was 1.44 ml, and 4 h' incubation in whole blood was physiological. Using triplicate aliquots of whole blood for both stimulant and solvent control, statistical conclusion was drawn for each stimulant for each mRNA. The method introduced in this study will be a new paradigm for clinical cellular immunology.

A dried tofu-supplemented diet affects mRNA expression of inflammatory cytokines in human blood.

In order to develop a new model of diet research, blood was drawn from 12 adult volunteers for 3 wk on regular diets as controls, and for a subsequent 3 wk supplemented with 18.5 g of freeze-dried tofu (Koya tofu) every day. Triplicate aliquots of 0.06 mL each of whole blood were stimulated ex vivo with phytohemagglutinin (PHA)-P, heat aggregated human IgG (HAG), lipopolysaccharide (LPS), zymosan A, and anti-T cell receptor (TCR) monoclonal antibody to activate specific subsets of leukocytes, then the levels of various inflammatory cytokine mRNA were quantified by real time PCR. Koya tofu significantly (p<0.05) augmented the fold increase of PHA-induced tumor necrosis factor superfamily (TNFSF) 15, IL6, and IL8, HAG-induced TNFSF15 and IL8, LPS-induced IL6 and IL8, zymosan-induced TNFSF15, IL6 and IL8, and TCR-induced TNFSF2 in comparison to the regular diet. Such increase was due to the reduction of baseline mRNA expression, not the enhancement of mRNA induction after specific stimulations. Six (TNFSF15), 4 (IL6), and 3 (IL10) subjects showed significant reduction of baseline mRNA during the Koya tofu diet compared to that of the control diet. Despite large individual-to-individual and day-to-day variation of mRNA, the method employed in this study was sensitive enough to identify statistically significant results as a group as well as on an individual basis, which will be a foundation for tailored diet in the future. The results also indicated that Koya tofu had a power to alter mRNA expression in leukocytes, and TNFSF15, IL6, and IL10 would be biomarkers for soy.

Ex vivo TCR-induced leukocyte gene expression of inflammatory mediators is increased in type 1 diabetic patients but not in overweight children.

BACKGROUND: Abnormal systemic concentrations of proinflammatory cytokines/chemokines have been implicated in the development of long-term cardiovascular complications in type 1 diabetes (T1DM) and obesity. Whether leukocyte white blood cell (WBC) gene expression of these proinflammatory mediators contributes to their increased systemic levels, however, remains unclear, especially in the pediatric patient populations. This study examines mRNA changes of 9 cytokines and chemokines in WBCs following ex vivo immunostimulation from 9 T1DM (13.4 +/- 0.5 year, 4F/5 M), 23 overweight (OW, 12.3 +/- 0.5 year, 10F/13M, BMI% 97.1 +/- 0.5 and > 90.0), and 21 healthy (CL, 13.8 +/- 0.7 year, 9F/12 M, BMI% 59.6 +/- 4.6 and < 85.0) children. METHODS: All subjects had been maintained in euglycemic conditions for at least 90 min before blood draws. Whole blood was then sampled and incubated with anti-T-cell receptor (TCR) antibody or heat-aggregated IgG (HAG) to stimulate T-cell and Fc receptors (FcR), respectively. After lysis of leukocytes, mRNA levels of six tumor necrosis factor superfamily cytokines (TNFSF2, 5, 6, 7, 9, 14) and three chemokines (CCL8, 20, and CXCL10) were measured using RT-PCR. RESULTS: Following TCR stimulation, T1DM displayed significantly greater mRNA responses than CL for TNFSF5, 7, 9, and CCL8, and CXCL10; TNFSF9, CCL8, and CXCL10 were also significantly higher in T1DM than OW; no difference was observed between OW and CL. FcR stimulation induced similar responses across groups. CONCLUSIONS: Leukocytes of T1DM children displayed exaggerated gene expression in response to ex vivo TCR induction of five key proinflammatory cytokines/chemokines. This elevated leukocyte gene expression may be one of the pathophysiological contributors to the development of vascular complications in T1DM.

Enhanced Expression of Radiation-induced Leukocyte CDKN1A mRNA in Multiple Primary Breast Cancer Patients: Potential New Marker of Cancer Susceptibility.

This study was designed to discover blood biomarkers of cancer susceptibility using invasive multiple (n = 21), single primary breast cancer (n = 21), and control subjects (n = 20). Heparinized whole blood was incubated at 37 degrees C for 2 hours after 0-10 Gy of radiation, then cell cycle arrest marker CDKN1A and apoptosis marker BBC3 mRNA were quantified. This epidemiological study was practically feasible because radiation-induced mRNA was preserved for at least 1 day whenever blood was stored at 4 degrees C (r(2) = 0.901). Moreover, blood could be stored frozen after radiation treatment (r(2) = 0.797). Radiation-induced CDKN1A and BBC3 mRNA were dose dependent, and the degree of induction of CDKN1A was correlated with that of BBC3 (r(2) = 0.679). Interestingly, multiple primary cases showed higher induction of CDKN1A mRNA than single primary and control groups, whereas BBC3 did not show such differences. The results suggested that cancer susceptibility represented by the multiple primary breast cancer cases was related to over-reaction of CDKN1A mRNA, not BBC3. The study also suggests that ex vivo gene expression analysis could potentially be used as a new tool in epidemiological studies for cancer and radiation sensitivity research.

Altered kinetics of interleukin-6 and other inflammatory mediators during exercise in children with type 1 diabetes.

BACKGROUND: Leukocyte mobilization and secretions of cytokines, chemokines, and growth factors in children during exercise are necessary biochemical signals for physiological growth and long-term cardiovascular protection. Because of glycemic instability, altered exercise responses, particularly the proinflammatory cytokine interleukin (IL)-6, may occur in type 1 diabetes mellitus (T1DM) that could influence the onset/progression of diabetic vascular complications. Relatively little is known, however, on most molecular aspects of immunomodulatory adaptation to exercise in diabetic children. METHODS: We therefore studied 21 children (age, 13.4 +/- 0.3 years; 13 boys/8 girls) with T1DM and 21 age-matched healthy controls during 30 minutes of intense and intermittent cycling exercise. Euglycemia was maintained during and for greater than 90 minutes before exercise; blood samples for IL-6 and other cytokines/chemokines were drawn before, during (every 6 minutes), and after (every 15 minutes) exercise. RESULTS: In T1DM, exercise-induced IL-6 peak occurred earlier and with greater magnitude than that in controls; an exploratory analysis of additional inflammatory mediators displayed a similarly accelerated/exaggerated pattern in T1DM, including the kinetic profiles of tumor necrosis factor alpha, IL-4, IL-12p70, IL-17, granulocyte-monocyte colony-stimulating factor, monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and eotaxin (interferon-inducible protein-10 was the only measured variable essentially indistinguishable between groups). CONCLUSION: Therefore, during intense and intermittent exercise, significant alterations in the immunologic pattern of inflammatory regulation occurred in children with T1DM as compared with healthy controls. Our findings underscore how the understanding of all the underlying molecular mechanisms is a necessary prerequisite for achieving effective use of exercise and the full manifestation of its health benefits, particularly in understudied populations such as children with T1DM who are at increased risk for cardiovascular complications.

Ex vivo simulation of IgG Fc and T-cell receptor functions: an application to inflammatory bowel disease.

BACKGROUND: Human leukocyte IgG Fc receptors (FcgammaR) and T-cell receptors (TCR) are primary molecules involved in inflammatory and immune pathways. METHODS: These 2 receptors were stimulated in whole blood for 2-4 hours with immune complex and specific agonistic antibody, respectively, and various mRNAs were quantified by a method we developed previously. RESULTS: FcgammaR stimulation induced tumor necrosis factor superfamily 2 (TNFSF2), TNFSF8, TNFSF15, interleukin 1B (IL1B), IL8, CCL chemokine 2 (CCL2), CCL3, CCL4, CCL11, CCL20, CXCL chemokine 1 (CXCL1), CXCL2, and CXCL3 mRNA, whereas TCR stimulation induced different subsets of mRNA such as TNFSF1, TNFSF2, TNFSF5, TNFSF6, TNFSF9, TNFSF14, IL6, CCL2, CCL8, CCL20, and CXCL10. Interestingly, respondents and nonrespondents were identified for each mRNA. When we applied this method to inflammatory bowel disease, the respondent populations of TCR-induced TNFSF2 (= TNFalpha), TNFSF5, TNFSF14, CCL2, CCL8, and CCL20 mRNA were significantly higher in Crohn's disease (CD) patients than in healthy controls or those with ulcerative colitis (UC). No difference was found for FcgammaR-mediated responses. The respondent population of TCR-induced TNFSF2 showed significantly (P = 0.05) higher incidence of multiple surgeries than did nonrespondents. CONCLUSIONS: These data demonstrate an underlining hyperfunction of TCR in peripheral-blood leukocytes in CD patients. The ex vivo simulation demonstrates an underlining hyperfunction of TCR in peripheral-blood leukocytes in CD patients and may form the basis of a relatively noninvasive test for distinguishing these IBDs.

Ex vivo simulation of the action of antileukemia drugs by measuring apoptosis-related mRNA in blood.

BACKGROUND: In conventional bioassays, isolated cells are suspended in culture media, incubated in vitro for several days, and then characterized with respect to any cellular changes. In developing new molecular tests under physiological ex vivo conditions, we quantified the production of mRNAs for p21 and PUMA (p53 up-regulated modulator of apoptosis), which are involved in cell cycle arrest and apoptosis, respectively. METHODS: We stimulated human whole blood with a chemotherapeutic drug (cytarabine, daunorubicin, mitoxantrone, aclarubicin, etoposide, or idarubicin) for 4 h and then quantified mRNA by assessing mRNA recovery and cDNA-synthesis efficiency in each sample. We also used immunoassay and flow cytometry to investigate nucleosome and annexin V, respectively, as apoptosis markers. RESULTS: Ex vivo mRNA analysis yielded more positive results than nucleosome and annexin V analyses. The concentrations of cytarabine- and daunorubicin-induced p21 and PUMA mRNAs were significantly lower in acute myelogenous leukemia (AML) patients than in healthy controls (P <0.0001), whereas idarubicin induced significantly greater responses in AML patients than in controls (P = 0.01). The patients had different mRNA-response patterns, which were largely classifiable into 4 groups. Prednisone enhanced cytarabine or mitoxantrone induction of p21 and PUMA mRNAs in 3 (2.6%) of 114 reactions. All 15 patients who achieved complete remission had received at least one drug that produced positive mRNA responses, whereas we observed a lack of mRNA response to the clinically used drugs in all 3 cases in which the therapy failed to induce any hematologic improvement. CONCLUSION: This study introduced ex vivo mRNA analysis as a candidate platform for drug-sensitivity tests in leukemia.