An approach for the analysis of axonal neuroinflammation by measuring dual biomarkers of oligodendrocytes and inflammatory cytokine in human plasma.

The myelin sheath surrounding axons is vulnerable to mechanical stresses after head injuries, as well as autoimmune attacks and degeneration in neurological disorders. Unfortunately, there is currently no effective method to assess these axonal conditions in individual patients. We have developed a sandwich immunoassay detecting dual signals of myelin oligodendrocyte glycoprotein (MOG) and interleukin 1B (IL1B) in human plasma ([IL1B on MOG]). While IL1B is one of common inflammation markers, its lack of tissue specificity is addressed by identifying IL1B on extracellular vesicles from oligodendrocytes isolated using anti-MOG, suggesting inflammation around axons. In 77 control subjects, plasma levels of [IL1B on MOG] did not increase more than 2 fold from baseline. During the sports season, 14% (151 football players) and 22% (18 rugby players) exhibited a substantial 2-17 fold increase, despite the absence of traumatic brain injuries. This elevation demonstrated a non-random pattern, with some individuals gradually rising towards the season's end, followed by a decline. [IL1B on MOG] levels also correlated with the clinical course of a post-concussion syndrome case. These data indicate that [IL1B on MOG] blood test is a potential marker for assessing mild axonal neuroinflammation.

Elevation of astrocyte-derived extracellular vesicles over the first month post-stroke in humans.

We sought to identify alterations in the quantity of plasma brain-derived extracellular vesicles (EV) over the first month post-stroke to shed light on related injury and repair mechanisms. We assessed plasma levels of presumed neuron-derived EVs (NDEs), astrocyte-derived EVs (ADEs), and oligodendrocyte-derived EVs (ODEs) in 58 patients 5, 15, and 30 days post-ischemic stroke and 46 controls matched for cardiovascular risk factors using sandwich immunoassays. Subsets of brain-derived EVs were identified by co-expression of the general EV marker CD9 and markers for neurons (L1CAM, CD171), astrocytes (EAAT1), and oligodendrocytes (MOG) respectively. Clinical MRIs assessed lesion volume and presence of hemorrhagic transformation. ADE levels were elevated 5, 15, and 30 days post-stroke compared to controls (p = 0.002, p = 0.002, and p = 0.005 respectively) with no significant change for NDE or ODE. ADEs were increased 15 days post-stroke in patients with hemorrhagic transformation (p = 0.04) compared to patients with no hemorrhage. We conclude that ADE levels are preferentially increased over the first month post-stroke in humans, possibly to provide trophic support to injured neurons following ischemia. ADEs hold potential as biomarkers of blood-brain barrier breakdown and hemorrhagic transformation, but this requires further study at earlier time points post-stroke.

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.

Quantification of brain-derived extracellular vesicles in plasma as a biomarker to diagnose Parkinson's and related diseases.

INTRODUCTION: There is still a substantial unmet need for blood-based biomarkers to make an objective diagnosis of Parkinson's disease (PD) and the parkinsonism-plus syndromes. This study is aimed to determine whether enumeration of brain-derived exosomes (BDEs) in plasma is informative in the diagnosis of those diseases. METHODS: We have developed a specific method to enumerate the plasma levels of neuron-derived, astrocyte-derived, and oligodendrocyte-derived exosomes (NDEs, ADEs and ODEs, respectively), and quantified them individually in patients with PD (n = 15), multiple system atrophy (MSA, n = 15), progressive supranuclear palsy (PSP, n = 7) and disease controls (n = 15). Our assays employ specific antibodies against molecules expressed by neurons, astrocytes and oligodendrocytes, respectively, combined with an antibody to the exosome common marker CD81. RESULTS: The plasma levels of NDEs showed significant increase in PD compared to control (p < 0.01) and MSA (p < 0.05) (one-way ANOVA, Bonferroni post hoc test). The plasma levels of ODEs and the ratio of ODE/NDE showed a significant correlation with UPDRS part III scores in the patients with MSA with predominant parkinsonism (MSA-P) (r2 = 0.57, n = 6, p = 0.048) and in the patients with PD (r2 = 0.51, n = 14, p = 0.0041), respectively. CONCLUSIONS: This is the first paper that enumerated NDE, ADE, and ODE in human plasma and showed the usefulness of those levels as biomarkers for PD. Our results suggest the capability of the plasma levels of NDE and ODE as a diagnostic and surrogate biomarker for PD and MSA-P, respectively.

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.

Uromodulin mRNA from Urinary Extracellular Vesicles Correlate to Kidney Function Decline in Type 2 Diabetes Mellitus.

BACKGROUND: Extracellular vesicles (EVs) enclose mRNA derived from their cell of origin and are considered a source of potential biomarkers. We examined urinary EV mRNA from individuals with diabetic kidney disease (DKD), chronic kidney disease, type 2 diabetes (T2DM), and obese and healthy controls to determine if such biomarkers had the potential to classify kidney disease and predict patients at higher risk of renal function decline. METHODS: A total of 242 participants enrolled in this study. Urinary EV mRNA from all subjects were isolated by a filter-based platform, and the expression of 8 target genes were determined by quantitative polymerase chain reaction (qPCR). Changes in estimated glomerular filtration rate (eGFR) in 161 T2DM patients were evaluated for 2 consecutive years and compared with EV RNA profiles at baseline. RESULTS: We observe that mild and severe DKD groups show a significant 3.2- and -4.4-fold increase in UMOD compared to healthy controls and expression increases linearly from healthy, diabetic, and DKD subjects. UMOD expression is significantly correlated to albumin creatinine ratio (ACR), eGFR, and HbA1c. Using linear discriminant analyses with mRNA from severe DKD and T2DM as training data, a multi-gene signature classified DKD and -non-DKD with a sensitivity of 93% and specificity of 73% with area under the receiver operating characteristic (ROC) curve (AUC) = 0.90. Although 6% of T2DM were determined to have a > 80% posterior probability of developing DKD based on this mRNA profile, eGFR changes observed within the 2-year follow-up did not reveal a decline in kidney function. CONCLUSION: Urinary EV UMOD mRNA levels are progressively elevated from T2DM to DKD groups and correlate with widely used eGFR and ACR diagnostic criteria. An EV mRNA signature could identify DKD with greater than 90% sensitivity and 70% specificity.

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.

Development of glomerulus-, tubule-, and collecting duct-specific mRNA assay in human urinary exosomes and microvesicles.

Urinary exosomes and microvesicles (EMV) are promising biomarkers for renal diseases. Although the density of EMV is very low in urine, large quantity of urine can be easily obtained. In order to analyze urinary EMV mRNA, a unique filter device to adsorb urinary EMV from 10 mL urine was developed, which is far more convenient than the standard ultracentrifugation protocol. The filter part of the device is detachable and aligned to a 96-well microplate format, therefore multiple samples can be processed simultaneously in a high throughput manner following the isolation step. For EMV mRNA quantification, the EMV on the filter is lysed directly by adding lysis buffer and transferred to an oligo(dT)-immobilized microplate for mRNA isolation followed by cDNA synthesis and real-time PCR. Under the optimized assay condition, our method provided comparable or even superior results to the standard ultracentrifugation method in terms of mRNA assay sensitivity, linearity, intra-assay reproducibility, and ease of use. The assay system was applied to quantification of kidney-specific mRNAs such as NPHN and PDCN (glomerular filtration), SLC12A1 (tubular absorption), UMOD and ALB (tubular secretion), and AQP2 (collecting duct water absorption). 12-hour urine samples were collected from four healthy subjects for two weeks, and day-to-day and individual-to-individual variations were investigated. Kidney-specific genes as well as control genes (GAPDH, ACTB, etc.) were successfully detected and confirmed their stable expressions through the two-week study period. In conclusion, this method is readily available to clinical studies of kidney diseases.

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.

Microassay for glucose-induced preproinsulin mRNA expression to assess islet functional potency for islet transplantation.

BACKGROUND: The capacity for insulin synthesis in islets is important for islet transplantation to succeed. We developed a microassay that evaluates the potency of human islets by measuring changes in glucose-induced human insulin gene (INS) expression using a single islet in octuplicate samples. METHODS: Poly (A) messenger RNA (mRNA) was purified from a set of single handpicked human islets. Glucose-induced mature (postspliced) and premature (prespliced) insulin mRNA were quantified by reverse-transcriptase polymerase chain reaction using several insulin mRNA primers designed at different locations including, intron, exon, and an exon-intron junction. RESULTS: The synthesis of premature INS mRNA was significantly increased in islets exposed to high glucose for 16 vs. 4 hr (P<0.01), whereas mature INS mRNA showed no difference. Glucose-induced premature INS mRNA synthesis was attenuated in heat-damaged islets. Stimulation index (SI) calculated by normalizing premature by mature INS mRNA (SI_INS mRNA) positively correlated with SI of insulin release (SI_16h insulin) from the same set of islets during 16-hr incubation in high or low glucose media, and SI of glucose-mediated insulin release obtained from the same islet lot in a perifusion system (n=12). Furthermore, linear multiple regression analysis using SI_INS mRNA and SI_16h insulin predicted islet transplantation outcome in nonobese diabetic (NOD) scid mice (n=8). CONCLUSION: The measurement of glucose-induced premature INS mRNA normalized by mature INS mRNA can be used to assess the functional quality of human islets and may predict islet function after transplantation in type 1 diabetic patients.