Peripheral Plasma Concentrations Research Articles

Assessment of plasma soluble Tie-2 level to distinguish moyamoya disease from atherosclerotic cerebrovascular disease and predict postoperative neovascularization.

Moyamoya disease (MMD) is a chronic steno-occlusive cerebrovascular disease and features the formation of hazy collateral vessels at the base of the brain. Angiopoietin (Ang)-1 and -2, their receptor Tie-2, and vascular endothelial growth factor (VEGF) that regulate angiogenesis might be important in MMD pathophysiology and postoperative collateral formation. The goal of this study was to determine whether levels of these angiogenic factors could predict collateralization in patients with MMD. A total of 196 patients with MMD and 57 with atherosclerotic cerebrovascular disease serving as controls were enrolled. Ang-1, Ang-2, Tie-2, and VEGF mRNA levels were analyzed in middle cerebral artery (MCA) arterial wall specimens by using real-time quantitative polymerase chain reaction. MCA and peripheral plasma concentrations of Ang-1, Ang-2, soluble Tie-2 (sTie-2), and VEGF were examined by enzyme-linked immunosorbent assay. Cerebral arteriography was performed 6 months after bypass surgery to assess the postoperative collateralization. In MCA specimens, patients with MMD exhibited higher expression levels of Ang-1 and Ang-2 but lowered VEGF expression. The patients with MMD had significantly higher concentrations of Ang-1 and Ang-2 but lower levels of VEGF in MCA plasma. Peripheral plasma concentrations of these growth factors were not changed. MCA and peripheral plasma sTie-2 levels were both reduced in patients with MMD. The 6-month follow-up showed that patients with good collateral formation had lower sTie-2 levels in both MCA and peripheral plasma. Furthermore, the Suzuki stage and peripheral plasma sTie-2 level were significantly correlated with good postoperative collateral formation on multivariate analysis. Ang-1, Ang-2, Tie-2, and VEGF are involved in MMD pathogenesis. The peripheral plasma level of sTie-2 can differentiate MMD from atherosclerotic cerebrovascular disease and serve as a novel biomarker to predict postoperative collateral formation.

RENAL SYMPATHETIC DENERVATION AND NEURO-HORMONAL EVALUATION BY SELECTIVE VENOUS CATHETERIZATION: THE RESIDENCY STUDY

Objective: Renal denervation (RD) is effective in reducing blood pressure (BP) in hypertensive patients, but the pathophysiologic mechanisms underlying the variable individual response are unclear and early markers of procedural success are lacking. The aim of the study was to evaluate intra-/peri-operative effects of RD on the renin-angiotensin-aldosterone system (RAAS) either as determinants and markers of efficacy. Design and method: Patients with resistant hypertension and eGFR>45 ml/min underwent radiofrequency-RD (Medtronic Symplicity-Spyral) with simultaneous sampling of the renal veins (RV), left adrenal vein and pre-renal inferior vena cava (IVC). Samples were collected before (t0) and 15’ after RD from each sequentially-approached side. Additional peripheral blood samples collected on the morning before RD and 3h, 24h and 7 days after the procedure were tested for renin, aldosterone and cortisol. During this timeframe, antihypertensive therapy remained unchanged. Results: Ten patients were recruited (61±11yo; 30%F; BP = 149/84 ±11/12 mmHg on 4 (range 3-6) antihypertensive drugs). Peripheral renin concentration showed no significant changes during/immediately after RD and up to 7 days after, in spite of the observed BP reduction (136/78 ±17/15 mmHg; p<0.05). Similarly, RV renin was unaffected by ipsilateral RD compared to the corresponding t0 values (p = 0.899). Aldosterone and cortisol increased in the adrenal vein during RD (p<0.001), but their IVC-corrected ratio and their peripheral plasma concentrations at follow-up did not, likely reflecting a self-limiting stress reaction. Conclusions: These results do not suggest any direct blunting effect of RD on RAAS, and do not support the contention of monitoring acute RAAS changes as proxies for procedural effectiveness.

Spontaneous reperfusion enhances succinate concentration in peripheral blood from stemi patients but its levels does not correlate with myocardial infarct size or area at risk

Succinate is enhanced during initial reperfusion in blood from the coronary sinus in ST-segment elevation myocardial infarction (STEMI) patients and in pigs submitted to transient coronary occlusion. Succinate levels might have a prognostic value, as they may correlate with edema volume or myocardial infarct size. However, blood from the coronary sinus is not routinely obtained in the CathLab. As succinate might be also increased in peripheral blood, we aimed to investigate whether peripheral plasma concentrations of succinate and other metabolites obtained during coronary revascularization correlate with edema volume or infarct size in STEMI patients. Plasma samples were obtained from peripheral blood within the first 10 min of revascularization in 102 STEMI patients included in the COMBAT-MI trial (initial TIMI 1) and from 9 additional patients with restituted coronary blood flow (TIMI 2). Metabolite concentrations were analyzed by 1H-NMR. Succinate concentration averaged 0.069 ± 0.0073 mmol/L in patients with TIMI flow ≤ 1 and was significantly increased in those with TIMI 2 at admission (0.141 ± 0.058 mmol/L, p < 0.05). However, regression analysis did not detect any significant correlation between most metabolite concentrations and infarct size, extent of edema or other cardiac magnetic resonance (CMR) variables. In conclusion, spontaneous reperfusion in TIMI 2 patients associates with enhanced succinate levels in peripheral blood, suggesting that succinate release increases overtime following reperfusion. However, early plasma levels of succinate and other metabolites obtained from peripheral blood does not correlate with the degree of irreversible injury or area at risk in STEMI patients, and cannot be considered as predictors of CMR variables.Trial registration: Registered at www.clinicaltrials.gov (NCT02404376) on 31/03/2015. EudraCT number: 2015-001000-58.

Interferon Tau (IFNt) and Interferon-Stimulated Genes (ISGs) Expression in Peripheral Blood Leukocytes and Correlation with Circulating Pregnancy-Associated Glycoproteins (PAGs) during Peri-Implantation and Early Pregnancy in Buffalo Cows.

The objective of this study was to analyze interferon-stimulated genes (ISGs) and interferon tau (IFNt) gene expression in peripheral blood leukocytes during the peri-implantation period and until 40 days of pregnancy in buffalo cows. Relationships were also examined between the expression of ISGs and IFNt and pregnancy-associated glycoproteins (PAGs) peripheral plasma concentration. Buffalo cows were synchronized and artificially inseminated (d 0). Blood samples were collected on days 0, 18, 28 and 40 after artificial insemination (AI) for peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) isolation and PAGs radioimmunoassay analysis. The study was carried out on 21 buffalo cows divided ex post into Pregnant (n = 12) and Non-pregnant (n = 9) groups. Steady state levels of OAS1, MX2, ISG15 and IFNt mRNA were measured by RT-qPCR and their estimated marginal means (p &lt; 0.01 for all) were higher in pregnant than non-pregnant buffaloes, both in PBMCs and PMNs. In PBMCs, pairwise comparisons showed that OAS1 and MX2 expressions differed between pregnant and non-pregnant buffaloes on all the days of observation (p &lt; 0.001), while significant differences in ISG15 and IFNt started from day 28 post-AI (p &lt; 0.05). In PMNs, ISG15 expression differed between groups only at days 18 and 28 (p &lt; 0.001), while comparisons were always significant for IFNt (p &lt; 0.05). The expression of all genes, except ISG15 as determined in PMNs, was positively associated with PAGs plasma concentrations (p &lt; 0.05). This work showed a significant increase in ISGs and IFNt expressions in PBMCs and PMNs in buffalo during the peri-implantation period and early pregnancy, and their correlation with PAGs plasma concentration.

Increased Levels of Cortisol in Individuals With Suicide Attempt and Its Relation With the Number of Suicide Attempts and Depression.

BackgroundAbnormalities in the hypothalamic-pituitary-adrenal axis (HPA) have been reported in individuals with suicide behavior. The aim of the study was to evaluate cortisol levels in peripheral plasma of individuals with more than one suicide attempt.MethodsCortisol concentrations in peripheral plasma were measured using the ELISA technique. Suicide attempts were evaluated by the Columbia Suicide Severity Rating Scale, while depression was evaluated by the Hamilton Depression Rating Scale.ResultsWe found elevated cortisol levels in the suicide attempt group when compared with healthy controls (F = 7.26, p-value = 0.008), but no statistical differences with the psychiatric diseases group (F = 1.49, p-value = 0.22). Cortisol levels were higher in individuals with depression (F = 8.99, P = 0.004) and in individuals with two or more suicide attempts (F = 13.56, P < 0.001).ConclusionsCortisol levels are increased in individuals who attempt suicide and higher of cortisol concentrations in plasma regard to depression and more attempts of suicide.

247 The fully human antibody SRF617 is a potent inhibitor of ecto-enzyme CD39 in vivo

BackgroundThe purine nucleotide adenosine plays an important role in dampening both the innate and adaptive arms of the immune system. In contrast, extracellular adenosine triphosphate (ATP), which can be generated at high levels due to cell stress, initiates proinflammatory responses. Extracellular ATP can be produced in cancerous tumors, and its conversion to adenosine monophosphate by CD39 (ENTPD1) and subsequent degradation to free adenosine by CD73 (NT5E) in the tumor microenvironment (TME) is a major contributor to tumor immune evasion. Accordingly, CD39 serves as an important immune regulator. CD39 is highly expressed in the TME of various tumor types, including lung, prostate, colon, ovarian, and pancreatic cancers, making CD39 an attractive target for immune-based anticancer therapy.SRF617 is a fully human anti-CD39 antibody designed to target human CD39 and inhibit its enzymatic activity. SRF617 provides a dual mechanism of immune activation by increasing inflammatory responses through accumulation of free ATP and alleviating immune suppression by reducing adenosine production. In this way, SRF617 shifts the TME to a proinflammatory state leading to reactivation of the immune system and antitumor function.MethodsA pharmacokinetic/pharmacodynamic profile was determined in CT26 tumor-bearing human CD39 knock-in (hCD39 KI) mice by measuring plasma SRF617 concentration, peripheral blood target occupancy (TO), and ATP hydrolysis activity in frozen tissue sections. Wild-type mice bearing subcutaneous (s.c.) KPC1245 tumors were treated with anti-murine CD39 antibody, alone or in combination with gemcitabine.ResultsCirculating SRF617 was observed in the plasma after intravenous injection of CT26 tumor-bearing hCD39 KI mice, which correlated with CD39-bound SRF617 on B cells. The presence of SRF617 correlated with a decrease in ATPase activity and CD39 expression in tissues. In the KPC tumor model, a combination effect was observed for anti-CD39 treatment with gemcitabine. Treatment with anti-CD39 antibody resulted in an increase in CD8+ T cells in the TME as measured by flow cytometry.ConclusionsSRF617 treatment of hCD39 KI mice led to reduction of ATPase activity in the TME that correlated with peripheral TO and plasma concentration. Anti-CD39 treatment alone or in combination with gemcitabine led to tumor growth inhibition and increased tumor-infiltrating lymphocytes as measured by flow cytometry in s.c. KPC tumors. These studies demonstrate the therapeutic potential of targeting CD39 for the treatment of cancer. SRF617 is currently being evaluated in a Phase 1 clinical trial in patients with advanced solid tumors (NCT04336098) and in combination with chemotherapy in patients with pancreatic cancer.

The physiological basis of insulin therapy in people with diabetes mellitus

Insulin therapy has been in use now for 100 years, but only recently insulin replacement has been based on physiology. The pancreas secretes insulin at continuously variable rates, finely regulated by sensitive arterial glucose sensing. Pancreatic insulin is delivered directlyin the portal blood to insulinize preferentially the liver. In the fasting state, insulin is secreted at a low rate to modulate hepatic glucose output. After liver extraction (50%), insulin concentrations in peripheral plasma are 2.4–4 times lower than in portal, but still efficacious to restrain lipolysis. In the prandial condition, insulin is secreted rapidly in large amounts to increase portal and peripheral concentrations to peaks 10–20 times greater vs the values of fasting within 30–40 min from meal ingestion. The prandial portal hyperinsulinemia fully suppresses hepatic glucose production while peripheral hyperinsulinemia increases glucose utilization, thus limitating the post-prandial plasma glucose elevation. Physiology of insulin indicates that insulin should be replaced in people with diabetes mimicking the pancreas, i.e. in a basal-bolus mode, for fasting and prandial state, respectively. Despite the presently ongoing limitations (subcutaneous and peripheral rather than portal and intravenous insulin delivery), basal-bolus insulin allows people with diabetes to achieve A1c in the range with minimal risk of hypoglycaemia, to prevent vascular complications and to ensure good quality of life.

Fire needle plus cupping for acute herpes zoster: study protocol for a randomized controlled trial

BackgroundAcute herpes zoster (AHZ) is a common skin disease caused by invasion of the varicella zoster virus into the ganglia and skin, and the severe pain is the most complaint, which can seriously disturb the normal life of patients. Fire needle plus cupping is a special acupuncture treatment, which is widely used to treat AHZ for its better analgesic effect in China although it has not been fully verified by rigorous randomized controlled trial (RCT).Methods/designTo test the effect, a three-arm randomized parallel controlled trial protocol has been designed. A total of 105 AHZ patients suffering pain will be randomly divided into three groups in an equal proportion. The interventions are fire needle plus cupping (FC) in group A, famciclovir plus gabapentin (FG) in group B, and fire needle plus cupping plus famciclovir (FCF) in group C. The length for the trial is set for a week time frame. Precisely speaking, the A group (FC) is to carry out 1 treatment per day for the total of 7 treatment sessions within 1 week period. On the other hand, the B group (FG) will take drugs orally three times a day within the trial 1 week. Meanwhile, with its combination element, the C group (FCF) is due to undertake both treatments and drugs as prescribed for A and B groups within the trial week. As an intra-trial arrangement, analgesic medication will be carefully administered for temporary pain release if the sudden intolerable pain appeared. For the primary outcome, this study is due to apply visual analogue scale to identify pain intensity relief. As the secondary outcomes are concerned, this study is aiming to focus on the issues related to changes in substance P and beta-endorphin concentrations in peripheral plasma, as well as those issues of analgesic needs, side effects, symptoms, and physical signs including pain classification, local itching, burning sensation, fever, local lymphadenopathy, skin lesion area, blisters, herpes clusters, vesicular traits, ulcers, and pimples; all these are taken into account for evaluation. For the final stage, the participants are to be followed up for postherpetic neuralgia.DiscussionThe results of this trial aim to provide sufficient evidence on FC treatment over both FG and FCF treatments. It will then give a credible alternative treatment to cut down acute pain and to cure AHZ infection.Trial registrationChinese Clinical Trial Registry ChiCTR1800015372. Registered on 28 March 2018.

An investigation of plasma interleukin-6 in sport-related concussion.

Increasing evidence suggests inflammation is an important component of concussion pathophysiology. However, its etiology, restitution, and potential clinical repercussions remain unknown. The purpose of the current study was to compare the blood concentrations of interleukin (IL) -6, a prominent inflammatory cytokine, between healthy athletes and athletes with a sport-related concussion (SRC), while addressing the potential confounds of sex, recent physical activity, and the interacting effect of concussion history. A prospective, observational cohort study was conducted on athletes at a single academic institute participating across 13 interuniversity sports. Follow-up of 96 athletes who agreed to provide a blood sample was completed: 41 athletes with a physician diagnosed SRC, and 55 healthy athletes. Ella™, the high sensitivity immunoassay system by ProteinSimple was used to measure peripheral plasma concentrations of IL-6 within the first week (median = 4 days, range = 2-7) following injury. A resampled ordinary least squares regression was used to evaluate the relationship between IL-6 concentrations and concussion status, while partial least squares regression was used to evaluate the relationship between IL-6 and both symptom burden and time to clinical recovery. Regression analysis identified a negative relationship between plasma IL-6 concentrations and the interaction between an acute SRC and a history of concussion (β = -0.29, p = 0.029). IL-6 did not differ between healthy athletes and those with an acute SRC independent of concussion history, and was not correlated with either recovery time or symptom burden in athletes with SRC. Perturbations to circulating IL-6 concentrations, a key inflammatory cytokine, may be more pronounced following SRC in athletes who have a history of concussion. These results add to a growing body of evidence supporting the involvement of inflammation at all phases of recovery following SRC, and potentially support a concomitant effect of prior concussion on acute SRC pathophysiology.

Variation in chemokines plasma concentrations in primary care depressed patients associated with Internet-based cognitive-behavioral therapy

How the presence of inflammation has repercussions for brain function is a topic of active research into depression. Signals released from immune system-related cells, including chemokines, might be indicative of active depression and can, hypothetically, serve as biomarkers of response to interventions, both pharmacological and psychological. The objective of this study is to analyze the peripheral plasma concentrations of CXCL12, CCL11, CX3CL1 and CCL2 in a cohort of depressed primary-care patients, as well as their evolution after an internet-based cognitive-behavioral intervention. The concentrations of those chemokines were measured in 66 primary-care patients with mild and moderate depression, before and after the intervention, as well as 60 controls, using multiplex immunoassays. Concentrations of CXCL12 and CCL2 were significantly higher in the clinical sample in comparison with controls. A stable multivariate discriminative model between both groups was found. Concentrations of all chemokines decreased after the internet-based psychological intervention. These findings support the implication of chemokines in depression, even in a sample of patients with mild and moderate severity. Furthermore, they demonstrate the need for further multidisciplinary research that confirms how biomarkers such as plasma chemokines can serve as a marker for depression and are sensitive to non-pharmacological interventions.

Plasma Level of Interferon-γ Predicts the Prognosis in Patients With New-Onset Atrial Fibrillation

Patients with atrial fibrillation are at increased risk of stroke and mortality. It is not clear if inflammatory biomarkers are associated with stroke and mortality in patients with atrial fibrillation. We aimed to evaluate the predictive value of three inflammatory biomarkers (interleukin [IL]-9, IL-10, and interferon [IFN]-γ) for stroke and mortality in atrial fibrillation. A total of 232 patients with new-onset atrial fibrillation were enrolled and 217 patients were completely followed-up. Peripheral plasma concentrations of cytokines (IL-9, IL-10, and IFN-γ) were measured using Luminex xMAP assays. The association between dichotomous groups of cytokines and outcomes were evaluated by a Cox proportional hazards model. The incremental value of inflammatory biomarkers, in addition to the CHA2DS2-VASc score, was also assessed. Patients were followed-up for a median duration of 27 (interquartile range [IQR], 23-30) months. The elevated plasma level of IFN-γ was an independent risk factor for stroke (hazard ratio [HR], 4.02 [IQR, 1.06-15.34]; p=0.042) and all-cause mortality (HR, 3.93 [IQR, 1.43-10.78]; p=0.008) in patients with atrial fibrillation. Adding high IFN-γ to the CHA2DS2-VASc score showed improvement in discrimination and reclassification prediction for stroke and mortality. However, IL-9 and IL-10 had no statistically significant association with stroke and all-cause mortality in patients with atrial fibrillation. In this "real-world" cohort of patients with atrial fibrillation, we have shown for the first time that plasma levels of IFN-γ could provide incremental prognostic value supplementary to that obtained from the CHA2DS2-VASc scores for predicting of stroke and all-cause mortality.

Concurrent Mild Traumatic Brain Injury and Posttraumatic Stress Disorder Is Associated With Elevated Tau Concentrations in Peripheral Blood Plasma.

Concurrent mild traumatic brain injury (mTBI) and posttraumatic stress disorder (PTSD) are common in U.S. military service members and veterans. Tau and amyloid-beta-42 (Aβ42) are proteins that have been linked to cognitive impairment, neurological hallmarks of Alzheimer's disease, and may also relate to recovery from mTBI. However, the role of these proteins in the maintenance or resolution of chronic symptoms has not yet been determined. Participants in the current study were 102 service members and veterans who had sustained an mTBI (n = 84) or injured controls (IC) without TBI (n = 18). They were categorized into three groups based on the presence or absence of mTBI and PTSD: IC/PTSD-Absent (n = 18), mTBI/PTSD-Absent (n = 63), and mTBI/PTSD-Present (n = 21). Concentrations of tau and Aβ42 in peripheral blood plasma were measured using SimoaTM , an ultrasensitive technology, and compared across groups. Tau concentrations were highest in the mTBI/PTSD-Present group, F(2, 99) = 4.33, p = .016, compared to the other two groups. Linear multiple regression was conducted to determine the independent effects of PTSD and mTBI on tau concentrations, controlling for gender and sleep medication. PTSD was a significant and independent predictor of tau concentrations, β = .25, p = .009, ηp 2 = .26. Aβ42 concentrations did not differ between the groups. The results indicated that PTSD was associated with an elevation of tau in peripheral blood and suggest that there may be increased biological effects of PTSD in this young cohort of service members and veterans following mTBI.

Classification of microadenomas in patients with primary aldosteronism by steroid profiling

In primary aldosteronism (PA) the differentiation of unilateral aldosterone-producing adenomas (APA) from bilateral adrenal hyperplasia (BAH) is usually performed by adrenal venous sampling (AVS) and/or computed tomography (CT). CT alone often lacks the sensitivity to identify micro-APAs. Our objectives were to establish if steroid profiling could be useful for the identification of patients with micro-APAs and for the development of an online tool to differentiate micro-APAs, macro-APAs and BAH. The study included patients with PA (n = 197) from Munich (n = 124) and Torino (n = 73) and comprised 33 patients with micro-APAs, 95 with macro-APAs, and 69 with BAH. Subtype differentiation was by AVS, and micro- and macro-APAs were selected according to pathology reports. Steroid concentrations in peripheral venous plasma were measured by liquid chromatography-tandem mass spectrometry. An online tool using a random forest model was built for the classification of micro-APA, macro-APA and BAH. Micro-APA were classified with low specificity (33%) but macro-APA and BAH were correctly classified with high specificity (93%). Improved classification of micro-APAs was achieved using a diagnostic algorithm integrating steroid profiling, CT scanning and AVS procedures limited to patients with discordant steroid and CT results. This would have increased the correct classification of micro-APAs to 68% and improved the overall classification to 92%. Such an approach could be useful to select patients with CT-undetectable micro-APAs in whom AVS should be considered mandatory.

Oxidized Mitochondrial DNA Is a Catalyst and Biomarker of Pyroptotic Cell Death in Myelodysplastic Syndromes

Constitutive innate immune activation is a pathogenetic driver of Myelodysplastic Syndromes (MDS) that directs ineffective hematopoiesis by NLRP3 inflammasome (IFM) assembly and pyroptotic cell death. IFM activation involves recruitment of caspase-1 (casp1) through the adapter protein, ASC, to facilitate autocatalytic cleavage of the zymogen to its active form that is responsible for interleukin (IL)-1β maturation, membrane pore formation and pyroptosis. Oxidized mitochondrial DNA (ox-mtDNA) has been proposed to serve as an alarmin that can activate the IFM by interaction directly with NLRP3 or engagement by DNA sensors, Toll-like receptor 9 (TLR9) and Cyclic GMP-AMP synthase (cGAS). Upon cytolysis, ox-mtDNA is released, permitting interaction with pattern recognition receptors on neighboring cells (Grishman, Pediatric Research, 2012, Shimada, 2012, Immunity. Vollmer, 2004, Immunology). Here, we investigate ox-mtDNA as an IFM-activator and pyroptotic biomarker in MDS.Incubation of TLR9 expressing cell lines, SKM1 (high expresser) and U937 (moderate expresser) with 50ng/mL ox-mtDNA (ND1 gene, amplified with oxidized guanosine) induced IFM activation evidenced by increased p-NFkβ, casp1 and IL-1β cleavage, ASC oligomerization and liberation of ASC specks. Direct interaction of ox-mtDNA with NLRP3 was confirmed by NLRP3 immunoprecipitation followed by probing for mtDNA using ND1 and CYTB specific primers and GAPDH primers as negative genomic control; mtDNA oxidation status was confirmed by dot blot. Furthermore, significantly increased expression of interferon stimulated genes (ISG) was seen in MDS bone marrow (BM) specimens (p≤0.01) compared to normal donors indicating TLR9 and/or cGAS activation. Ox-mtDNA engagement of TLR9 and cGAS was confirmed in MDS specimens by IF colocalization with corresponding IFM activation, as well as in MDS somatic gene mutation murine models (Tet2, SRSF2, U2AF) vs. Wt controls. Evaluation of surface TLR9 by flow cytometry showed significantly increased membrane expression in MDS CD34+ BMMC (n=4) vs. healthy donors (n=13) (p<0.05), suggesting priming for enhanced sensitivity to ox-mtDNA. Concentrations of ox-mtDNA in supernatants from THP1 (N=5, p=0.0547), U937 (N=3, p=0.0637), and SKM1 (N=3, p=0.028) cells confirmed extracellular release after LPS/ATP/Nigericin induced pyroptosis. We then investigated ox-mtDNA concentrations in peripheral blood plasma from MDS patients by ELISA. Glucose adjusted, ox-mtDNA levels were significantly increased in 177 MDS cases compared to 29 healthy donors (p<0.0001), which was validated in an independent cohort of 154 MDS cases and 34 healthy donors (p<0.0001). Ox-mtDNA was higher in lower risk (LR) compared to higher risk MDS (p<0.0001) consistent with greater pyroptotic cell fraction in LR-MDS. Further, we found a direct correlation between ox-mtDNA (n=134) and known MDS IFM-activating alarmins S100A9 (r=0.395), S100A8 (r=0.618), and percentage of ASC specks (r=0.589) (p<0.0001 for all), demonstrating specificity of ox-mtDNA as a biomarker for the magnitude of medullary pyroptosis in MDS. Importantly, compared to other hematologic malignancies, we found that plasma ox-mtDNA was significantly increased in LR-MDS (n=162) compared to ALL (n=7), de novo (n=20) and secondary AML (n=18), CML (n=8), CMML (n=18), LGL (n=19), MF (n=12), ET (n=20), PV (n=20), MM (n=18), and type 2 diabetes (n=25) where IFM activation promotes insulin resistance (p ≤0.036). There was no significant difference in oxDNA concentrations between MDS and CLL (n=50), which is reported to have high ox-mtDNA levels associated with genomic abnormalities (Collado, 2014, Biomed Res Int.). ROC/AUC analysis demonstrated that ox-mtDNA is an MDS pyroptosis-specific biomarker when compared to healthy donors (AUC=0.929). Similarly, a 5-fold cross-validation (k=5) repeated 30 times to compare MDS cases to non-MDS hematological malignancies (excluding CLL), similarly confirmed biomarker specificity (AUC=0.877).Collectively, these data indicate that ox-mtDNA both directly engages NLRP3 and the DNA sensors TLR9/cGAS to induce IFM activation and pyroptosis, creating a feed forward inflammatory cascade that extends to neighboring cells. Ox-mtDNA may serve as a biomarker and companion diagnostic for pyroptosis execution in MDS. DisclosuresList:Celgene: Research Funding.

Saliva oxytocin measures do not reflect peripheral plasma concentrations after intranasal oxytocin administration in men

Oxytocin plays an important role in social behavior. Thus, there has been significant research interest for the role of the oxytocin system in several psychiatric disorders, and the potential of intranasal oxytocin administration to treat social dysfunction. Measurement of oxytocin concentrations in saliva are sometimes used to approximate peripheral levels of oxytocin; however, the validity of this approach is unclear. In this study, saliva and plasma oxytocin was assessed after two doses of Exhalation Delivery System delivered intranasal oxytocin (8 IU and 24 IU), intravenous oxytocin (1 IU) and placebo in a double-dummy, within-subjects design with men. We found that intranasal oxytocin (8 IU and 24 IU) administration increased saliva oxytocin concentrations in comparison to saliva oxytocin concentration levels after intravenous and placebo administration. Additionally, we found that saliva oxytocin concentrations were not significantly associated with plasma oxytocin concentrations after either intranasal or intravenous oxytocin administration. Altogether, we suggest that saliva oxytocin concentrations do not accurately index peripheral oxytocin after intranasal or intravenous oxytocin administration, at least in men. The data indicates that elevated oxytocin saliva levels after nasal delivery primarily reflect exogenous administered oxytocin that is cleared from the nasal cavity to the oropharynx, and is therefore a weak surrogate for peripheral blood measurements.

Correlation of Lactate Concentration in Peripheral Plasma and Cerebrospinal Fluid with Glasgow Outcome Scale for Patients with Tuberculous Meningitis Complicated by Acute Hydrocephalus Treated with Fluid Diversions.

Tuberculous meningitis (TBM) is an endemic infectious disease in developing countries, and it can become a serious illness in children. Treatment of TBM is more difficult and prone to failure than treatment of pulmonary tuberculosis. TBM causes hydrocephalus, cerebral edema, increased intracranial pressure, global ischemia, and neurologic deficits, which disturb cellular metabolism and increase lactate levels. A reliable, widely available clinical indicator of TBM severity is needed. Successful treatment of TBM is assessed using the Glasgow Outcome Scale (GOS). This prospective cohort study included 34patients with TBM and acute hydrocephalus who had undergone fluid diversions and were admitted to Dr. Hasan Sadikin Hospital in Bandung from 2014 to 2015. A portable machine for blood glucose measurement was used to measure lactate concentrations. Statistical significance was defined as P ≤ 0.05. Average levels of plasma and cerebrospinal fluid (CSF) lactate were 1.99 ± 0.70 mmol/L and 3.04±1.05mmol/L, respectively. A significantly higher level of lactate was observed in CSF compared with plasma. Preoperative plasma lactate was negatively correlated to GOS (r=-0.539; P= 0.013), and CSF lactate was negatively correlated to GOS (r=-0.412; P= 0.027). Average lactate levels in CSF (central) were higher than plasma (peripheral) levels. GOS scale of patients decreased with increased plasma and CSF lactate levels. Examination of plasma and CSF lactate levels should be included in routine examinations to determine extent of cellular damage and GOS score in patients with TBM and acute hydrocephalus who have undergone fluid diversions.

Caspase 3, 6, 8, and 9 Gene Expression in Peripheral Blood Leukocytes and Plasma Concentrations of IL-6 and TNF-α in Carriers of Different Polymorphic Marker -174G>C Genotypes of IL6 Gene Associated with the Risk of Nonalcoholic Steatohepatitis.

We revealed an association of IL6 gene -174G>C polymorphism with the development of nonalcoholic steatohepatitis in the Russian population. The risk is significantly higher in carriers of C allele: OR=1.77 (1.04; 3.02). The effects of -174G>C substitution in IL6 gene involving caspase 9 gene transcripts in peripheral blood leukocytes and on blood content of TNF-α in healthy individuals without clinical manifestations of nonalcoholic steatohepatitis were detected. The content of caspase 9 gene transcripts in peripheral blood leukocytes and plasma level of TNF-α were significantly higher in healthy subjects carrying C allele than in carriers of GG genotype. The levels of caspases 3, 6, 8, and 9 gene transcripts in peripheral blood leukocytes and plasma concentrations of TNF-α in patients with nonalcoholic steatohepatitis did not depend on IL6 genotype by -174G<C marker.

High leukocyte mtDNA content contributes to poor prognosis through ROS-mediated immunosuppression in hepatocellular carcinoma patients.

Compelling epidemiological evidences indicate a significant association between leukocyte mitochondrial DNA (mtDNA) content and incidence risk of several malignancies, including hepatocellular carcinoma (HCC). However, whether leukocyte mtDNA content affect prognosis of HCC patients and underlying mechanism has never been explored. In our study, leukocyte mtDNA content was measured in 618 HCC patients and its prognostic value was analyzed. Moreover, we detected the immunophenotypes of peripheral blood mononuclear cells (PBMCs) and plasma concentrations of several cytokines in 40 HCC patients and assessed the modulating effects of mtDNA content on immunosuppression in cell models. Our results showed that HCC patients with high leukocyte mtDNA content exhibited a significantly worse recurrence-free survival (RFS) and overall survival (OS) than those with low leukocyte mtDNA content. Leukocyte mtDNA content and TNM stage exhibited a notable joint effect in prognosis prediction. Furthermore, we found that patients with high leukocyte mtDNA content exhibited a higher frequency of CD4+CD25+FOXP3+ regulatory T (Treg) cells and lower frequency of NK cells in PBMCs and had higher TGF-β1 and lower TNF-α and IFN-γ plasma concentration when compared with those with low leukocyte mtDNA content, which suggests an immunosuppressive status. High leukocyte mtDNA content significantly enhanced the ROS-mediated secretion of TGF-β1, which accounted for higher Treg and lower NK frequency in PBMCs. In a conclusion, our study for the first time demonstrates that leukocyte mtDNA content is an independent prognostic marker complementing TNM stage and associated with an ROS-mediated immunosuppressive phenotype in HCC patients.

Mass Spectrometry-Based Adrenal and Peripheral Venous Steroid Profiling for Subtyping Primary Aldosteronism.

Differentiating patients with primary aldosteronism caused by aldosterone-producing adenomas (APAs) from those with bilateral adrenal hyperplasia (BAH), which is essential for choice of therapeutic intervention, relies on adrenal venous sampling (AVS)-based measurements of aldosterone and cortisol. We assessed the utility of LC-MS/MS-based steroid profiling to stratify patients with primary aldosteronism. Fifteen adrenal steroids were measured by LC-MS/MS in peripheral and adrenal venous plasma from AVS studies for 216 patients with primary aldosteronism at 3 tertiary referral centers. Ninety patients were diagnosed with BAH and 126 with APAs on the basis of immunoassay-derived adrenal venous aldosterone lateralization ratios. Among 119 patients confirmed to have APAs at follow-up, LC-MS/MS-derived lateralization ratios of aldosterone normalized to cortisol, dehydroepiandrosterone, and androstenedione were all higher (P < 0.0001) than immunoassay-derived ratios. The hybrid steroids, 18-oxocortisol and 18-hydroxycortisol, also showed lateralized secretion in 76% and 35% of patients with APAs. Adrenal venous concentrations of glucocorticoids and androgens were bilaterally higher in patients with BAH than in those with APAs. Consequently, peripheral plasma concentrations of 18-oxocortisol were 8.5-fold higher, whereas concentrations of cortisol, corticosterone, and dehydroepiandrosterone were lower in patients with APAs than in those with BAH. Correct classification of 80% of cases of APAs vs BAH was thereby possible by use of a combination of steroids in peripheral plasma. LC-MS/MS-based steroid profiling during AVS achieves higher aldosterone lateralization ratios in patients with APAs than immunoassay. LC-MS/MS also enables multiple measures for discriminating unilateral from bilateral aldosterone excess, with potential use of peripheral plasma for subtype classification.

High leukocyte mitochondrial DNA content contributes to poor prognosis in glioma patients through its immunosuppressive effect.

Background:Epidemiological studies have indicated significant associations of leukocyte mitochondrial DNA (mtDNA) copy number with risk of several malignancies, including glioma. However, whether mtDNA content can predict the clinical outcome of glioma patients has not been investigated.Methods:The mtDNA content of peripheral blood leukocytes from 336 glioma patients was examined using a real-time PCR-based method. Kaplan–Meier curves and Cox proportional hazards regression model were used to examine the association of mtDNA content with overall survival (OS) and progression-free survival (PFS) of patients. To explore the potential mechanism, the immune phenotypes of peripheral blood mononuclear cells (PBMCs) and plasma concentrations of several cytokines from another 20 glioma patients were detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively.Results:Patients with high mtDNA content showed both poorer OS and PFS than those with low mtDNA content. Multivariate Cox regression analysis demonstrated that mtDNA content was an independent prognostic factor for both OS and PFS. Stratified analyses showed that high mtDNA content was significantly associated with poor prognosis of patients with younger age, high-grade glioma or adjuvant radiochemotherapy. Immunological analysis indicated that patients with high mtDNA content had significantly lower frequency of natural killer cells in PBMCs and higher plasma concentrations of interleukin-2 and tumour necrosis factor-α, suggesting an immunosuppression-related mechanism involved in mtDNA-mediated prognosis.Conclusions:Our study for the first time demonstrated that leukocyte mtDNA content could serve as an independent prognostic marker and an indicator of immune functions in glioma patients.