Response In Healthy Volunteers Research Articles

A randomized, placebo-controlled phase I study assessing the safety and immunogenicity of a Pseudomonas aeruginosa hybrid outer membrane protein OprF/I vaccine (IC43) in healthy volunteers

Introduction: IC43 is a recombinant outer membrane protein-based vaccine against Pseudomonas aeruginosa (P. aeruginosa) consisting of OprF- and OprI- epitopes (Opr, outer membrane protein; OprF/I, OprF/OprI hybrid vaccine) with an N-terminal His 6 tag (Met-Ala-(His)6-OprF190–342-OprI21–83).Objectives: The study aimed to confirm the optimal dose of IC43 in adults with regard to immunogenicity, safety, and tolerability after vaccination with three different dosages of IC43, compared with placebo, and to investigate a potential immune-enhancing effect of the adjuvant, aluminum hydroxide. Subjects were randomly allocated in a 1:1:1:1:1 ratio to one of five treatment groups: 50, 100, or 200 µg IC43 with adjuvant, 100 µg IC43 without adjuvant, or placebo (0.9% sodium chloride) and two intramuscular injections were given in the deltoid region 7 d apart.Results: The primary immunogenicity analysis of OprF/I-specific IgG antibody titers on day 14 demonstrated statistically significant differences among treatment groups (P < 0.0001), with a significantly higher immune response detected in each IC43 treatment group compared with placebo. From day 0 to day 14, a ≥4-fold increase in OprF/I-specific immunoglobulin G (IgG) antibody titers were observed in >90% of subjects in all IC43 treatment groups in the per-protocol (PP) and intention-to-treat (ITT) populations; a ≥ 50-fold titer increase was observed in 42.6% subjects including all IC43 treatment groups. On day 90, OprF/I-specific IgGs started to decline in all IC43 treatment groups but remained significantly higher until 6 mo compared with placebo. Assessment of functional antibody induction by opsonophagocytic assay (OPA) followed a similar pattern compared with OprF/I-specific IgG kinetics. At day 14, a ≥2-fold increase in OPA titer was observed in 54.5% subjects within all IC43 treatment groups. An increase in antibody avidity index was observed after the second vaccination. At day 14, >96% of subjects in each IC43 treatment group had detectable OprF/I-specific IgG antibodies. Anti-histidine IgG antibody titers peaked on day 14 and were reduced on day 90 in all IC43 treatment groups. OprF/I-specific IgG secreted by antibody-secreting cell (ASC) was detected in all IC43 groups by B-cell ELIspot after the second vaccination and up to 6 mo. All vaccinations were safe and well tolerated up to the maximum cumulative dosage of 400 µg IC43.Conclusion: IC43 doses equal to or greater than 50 µg were sufficient to induce a plateau of IgG antibody responses in healthy volunteers. Higher doses, whether adjuvanted or non-adjuvanted, were not more effective.Methods: In this phase I, randomized, placebo-controlled, observer-blinded, multicenter clinical trial, 163 healthy volunteers (18−65 y) were randomly assigned to five treatment groups (1:1:1:1:1). Three groups received IC43 with adjuvant: 50 µg (n = 32), 100 µg (n = 33), or 200 µg (n = 33). One group received IC43 100 µg without adjuvant (n = 32), and one group received placebo (0.9% sodium chloride) (n = 33). Each subject received two intramuscular vaccinations, separated by a 7-d interval (days 0 and 7) (Fig. 1). Humoral immune response was assessed by measurement of outer membrane protein F/I (OprF/I)-specific antibodies determined by enzyme-linked immunosorbent assay (ELISA), anti-histidine antibodies determined by ELISA, and functional antibody activity determined by opsonophagocytic assay (OPA), up to 6 mo post-vaccination. Antibody avidity was measured on days 7 and 14 from samples that had detectable vaccine antibody-specific immunoglobulin G (IgG) antibody titers. At the Austrian site only, the B-cell ELIspot assay was used to determine specific ASC responses. Safety was assessed using adverse event monitoring and clinical laboratory tests. Local and systemic tolerability was recorded in a subject diary for 7 d after each vaccination and by investigators up to 6 mo post-vaccination.Clinical trial registration number: NCT00778388

Potential enhancing effects of histamine H1 agonism/H3 antagonism on working memory assessed by performance and bold response in healthy volunteers

Schizophrenia is a highly debilitating disorder characterized by hallucinations and delusions, but also impaired cognition such as memory. While hallucinations and delusions are the main target for pharmacological treatment, cognitive impairments are rarely treated. Evidence exists that histamine has a role in the cognitive deficits in schizophrenia, which could be the basis of the development of a histamine-type treatment. Histamine H₃ antagonists have been shown to improve memory performance in experimental animals, but these effects have been little investigated in humans within the context of impaired cognition in schizophrenia and using sensitive measures of brain activity. In the present study, the effects of betahistine (H₃ antagonist/H₁ agonist) on learning and memory, and associated brain activity were assessed. Sixteen healthy volunteers (eight female) aged between 18 and 50 years received two p.o. doses of betahistine (48 mg) or placebo separated by 30 min, on separate days according to a two-way, double-blind, crossover design. Volunteers performed an N-back working memory task and a spatial paired associates learning task while being scanned using a MRI scanner. Task-related activity changes in well-defined networks and performance were observed. No betahistine-induced changes in brain activity were found in these networks. Alternatively, liberal whole-brain analyses showed activity changes in areas outside task networks, like the lateral geniculate nucleus. Clear effects of betahistine on working memory could not be established. Future studies should use higher doses and explore the role of histamine in visual information processing.

Safety, Pharmacokinetic, Immunogenicity, and Pharmacodynamic Responses in Healthy Volunteers Following a Single Intravenous Injection of Purified Staphylococcal Protein A

A single-dose study was conducted to characterize the safety, pharmacokinetic, immunogenicity, and pharmacodynamic activity of highly purified Staphylococcal protein A (SPA), a native bacterial protein with immune-modulatory activity. Twenty healthy adults received a single intravenous dose of either 0.3 µg/kg (n = 8) or 0.45 µg/kg (n = 8) of SPA or placebo (n = 4). Changes in C-reactive protein and neopterin were used as markers of immune activation. All treatment-related AEs were of mild severity. Twelve of 16 active-dosed subjects developed detectable anti-protein A antibodies after dosing. These subjects had notably more rapid plasma clearance of SPA even prior to development of detectable titers. A transient post-dose decrease in circulating lymphocytes was observed as a notable pharmacodynamic effect, but was not correlated with plasma clearance or AUC. In peripheral blood mononuclear cells, SPA dosing increased transcription of multiple genes regulated by type-1 interferons, and up-regulation of several of these genes correlated with the degree of lymphopenia seen 24 hours after dosing. This study demonstrates the safety and tolerability of small intravenous doses of SPA and delineates acute and transient pharmacodynamic effects not previously reported.

Simultaneous measurement of tissue oxygen level-dependent (TOLD) and blood oxygenation level-dependent (BOLD) effects in abdominal tissue oxygenation level studies.

To assess oxygenation in abdominal organs with magnetic resonance imaging (MRI), a novel approach is presented to simultaneously measure both T1 - and T2*-maps serially during a single dynamic MRI scan in response to an oxygen challenge. The proposed acquisition scheme consists of a multishot multiecho gradient echo planar imaging sequence (ms-GEPI) interleaved with a multishot inversion recovery echo planar imaging (ms-IR-EPI) sequence. Respiratory motion compensation was accomplished with standard belt triggering and by acquiring all image data at the same phase of expiration. This respiratory-triggered, free-breathing, interleaved tissue oxygenation level-dependent (TOLD) and blood oxygenation level-dependent (BOLD) acquisition technique was validated on phantoms and seven healthy volunteers in response to an oxygen challenge. Measurements of relaxation times both in vitro and in vivo were in good agreement with those obtained using conventional pulse sequences and reported in the literature. The interleaved sequence was able to measure oxygen-induced relaxation time changes in human abdominal organs. The free-breathing respiratory-triggered interleaved T1 and T2* sequence successfully provided relaxation time maps of abdominal organs in a dynamic scan without the need for image registration. The simultaneous monitoring of tissue and blood oxygenation improves time efficiency and should enhance studies comparing dynamic T1 and T2* data within the abdomen.

Polyfunctional CD4+ T cell responses in HIV-1-infected viral controllers compared with those in healthy recipients of an adjuvanted polyprotein HIV-1 vaccine

A recombinant fusion protein (F4) consisting of HIV-1 p17, p24, reverse transcriptase (RT) and Nef, adjuvanted with AS01, induced strong and broad CD4+ T cell responses in healthy volunteers. Here we compare these vaccine-induced CD4+ T cell responses with the ones induced by natural infection in patients with varying disease courses.Thirty-eight HIV-infected, antiretroviral treatment-naïve subjects were classified into four categories: 8 long-term non-progressors (infection ≥7 years; CD4+ T cells ≥500/μL), 10 recently infected individuals (infection ≤2 years; CD4+ T cells ≥500/μL), 10 typical early progressors (CD4+ T cells ≤350/μL), and 10 viral controllers (plasma HIV-1 RNA <1000copies/mL). Peripheral blood mononuclear cells were stimulated in vitro with p17, p24, RT and Nef peptide pools and analyzed by flow cytometry for expression of IL-2, IFN-γ, TNF-α and CD40L. CD4+ T cell responses were compared to those measured with the same method in 50 HIV-uninfected subjects immunized with the F4/AS01 candidate vaccine (NCT00434512).After in vitro stimulation with p17, p24 and RT antigen viral controllers had significantly more CD4+ T cells co-expressing IL-2, IFN-γ and TNF-α than other HIV patient categories. The magnitude and quality of these responses in viral controllers were comparable to those observed in F4/AS01 vaccine recipients. In contrast with viral controllers, triple cytokine producing CD4+ T cells in vaccinees also expressed CD40L.Subjects who spontaneously control an HIV infection display polyfunctional CD4+ T cell responses to p17, p24, RT and Nef, with similar magnitude and qualities as those induced in healthy volunteers by an adjuvanted HIV candidate vaccine (F4/AS01).

Comparison of effects of 5 and 10 mg oral desloratadine and levocetirizine on histamine-induced wheal and flare response in healthy volunteers

Background: Levocetirizine and desloratadine are mostly used as H1-antihistamines in the treatment of allergic disease in 5 and 10 mg doses. Objective: In this study, the efficacy of single oral dosages of 5 and 10 mg desloratadine and levocetirizine were compared by using histamine-induced wheal and flare reactions. Methods: Eighty healthy volunteers were randomized for four double-blinded treatment with desloratadine 5 and 10 mg and levocetirizine 5 and 10 mg. Wheal and flare responses were produced by histamine. Measurements were performed just before the ingestion of antihistamines (baseline) and afterward at 30, 60, 240 min and 24 h. The values obtained for each antihistamine were compared with baseline values. Results: It was found that except the flare reactions at 30th min, levocetirizine 5 and 10 mg suppressed histamine-induced wheal and flare reactions more than desloratadine 5 and 10 mg did. There were not any significant differences between desloratadine 5 and 10 mg in all periods. Levocetirizine 10 mg suppressed wheal and flare reactions significantly more than levocetirizine 5 mg only at 24th h. Conclusion: In this study, it was observed that levocetirizine 5 and 10 mg had a higher activity than desloratadine 5 and 10 mg.

Sa1961 Effect of Low Dose Aspirin on Gastric Mucus Secretion in Relation to Gastric Adaptive Response in Healthy Volunteers

Sa1961 Effect of Low Dose Aspirin on Gastric Mucus Secretion in Relation to Gastric Adaptive Response in Healthy Volunteers

Can itch-related visual stimuli alone provoke a scratch response in healthy individuals?

The sensation of itch and the scratch response elicited by application of histamine are enhanced by itch-related visual cues in people with existing skin conditions and, to a lesser extent, in healthy controls. We tested whether visual cues alone could generate feelings of itch and provoke a scratch response in healthy volunteers. A secondary aim was to assess whether the content of some pictures evoked these sensations more effectively. Thirty participants viewed static images that were either itch-related (e.g. ants, fleas or skin conditions) or neutral (e.g. butterflies or healthy skin). These were further separated by picture type into 'skin contact' (e.g. ants crawling on the hand or a butterfly on a finger), 'skin response' (e.g. scratching an insect bite or washing the hands) or 'context only' (e.g. viewing midges or birds flying). The sensation of itch was successfully generated using itch-related pictures, with higher self-reports of itch in answer to the questions 'How itchy do you feel?' and 'How itchy do you think the person in the picture feels?', compared with viewing neutral pictures (P<0·001), and these measures correlated (P≤ 0·003). Participants also scratched themselves more when viewing itch-related pictures than when viewing neutral ones (P<0·001). The interaction with picture type was significant, with more scratching behaviour recorded when viewing pictures depicting others scratching (P=0·01). This study demonstrates the impact of visual cues in eliciting sensations of itch and provoking a scratch response, and may provide behavioural evidence linking contagious itch to the mirror neuron system.

Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel

AbstractLight transmission aggregometry (LTA) is used worldwide for the investigation of heritable platelet function disorders (PFDs), but interpretation of results is complicated by the feedback effects of ADP and thromboxane A2 (TxA2) and by the overlap with the response of healthy volunteers. Over 5 years, we have performed lumi-aggregometry on 9 platelet agonists in 111 unrelated research participants with suspected PFDs and in 70 healthy volunteers. Abnormal LTA or ATP secretion test results were identified in 58% of participants. In 84% of these, the patterns of response were consistent with defects in Gi receptor signaling, the TxA2 pathway, and dense granule secretion. Participants with defects in signaling to Gq-coupled receptor agonists and to collagen were also identified. Targeted genotyping identified 3 participants with function-disrupting mutations in the P2Y12 ADP and TxA2 receptors. The results of the present study illustrate that detailed phenotypic analysis using LTA and ATP secretion is a powerful tool for the diagnosis of PFDs. Our data also enable subdivision at the level of platelet-signaling pathways and in some cases to individual receptors. We further demonstrate that most PFDs can be reliably diagnosed using a streamlined panel of key platelet agonists and specified concentrations suitable for testing in most clinical diagnostic laboratories.

Pharmacodynamic Effects of Steady‐State Fingolimod on Antibody Response in Healthy Volunteers: A 4‐Week, Randomized, Placebo‐Controlled, Parallel‐Group, Multiple‐Dose Study

Fingolimod, a first-in-class oral sphingosine 1-phosphate receptor (S1PR) modulator, is approved in many countries for relapsing-remitting multiple sclerosis, at a once-daily 0.5-mg dose. A reduction in peripheral lymphocyte count is an expected consequence of the fingolimod mechanism of S1PR modulation. The authors investigated if this pharmacodynamic effect impacts humoral and cellular immunogenicity. In this double-blind, parallel-group, 4-week study, 72 healthy volunteers were randomized to steady state, fingolimod 0.5 mg, 1.25 mg, or to placebo. The authors compared T-cell dependent and independent responses to the neoantigens, keyhole limpet hemocyanin (KLH), and pneumococcal polysaccharides vaccine (PPV-23), respectively, and additionally recall antigen response (tetanus toxoid [TT]) and delayed-type hypersensitivity (DTH) to KLH, TT, and Candida albicans. Fingolimod caused mild to moderate decreases in anti-KLH and anti-PPV-23 IgG and IgM levels versus placebo. Responder rates were identical between placebo and 0.5-mg groups for anti-KLH IgG (both > 90%) and comparable for anti-PPV-23 IgG (55% and 41%, respectively). Fingolimod did not affect anti-TT immunogenicity, and DTH response did not differ between placebo and fingolimod 0.5-mg groups. Expectedly, lymphocyte count reduced substantially in the fingolimod groups versus placebo but reversed by study end. Fingolimod was well tolerated, and the observed safety profile was consistent with previous reports.

Cognitive Effects of a Single Dose of Atypical Antipsychotics in Healthy Volunteers Compared With Placebo or Haloperidol

Cognitive dysfunction in patients with schizophrenia is a strong correlate of poor outcome than any other symptom domain. To have greater knowledge about the effects of antipsychotics on cognitive function, subjects of this study were healthy volunteers who had no confounding variables typically found in patients with schizophrenia. The cognitive function of healthy volunteers in response to single doses of haloperidol, risperidone, aripiprazole, and amisulpride in a double-blind placebo-controlled trial was investigated. Assessments for the computerized neurocognitive test, mental and physical sedation, and extrapyramidal symptoms were performed within 1 week before (baseline) and approximately 4 hours after drug administration. Compared to the placebo, single administration of amisulpride at 400 mg in healthy volunteers enhanced word fluency test performance and remained intact after controlling for sedation and extrapyramidal symptoms. Significant improvement in some measurements of the computerized neurocognitive test was also observed in each antipsychotic-treated group but may have been related to practice effect. These findings suggest that amisulpride may have cognitive-enhancing effects in healthy volunteers.

The Effect of Novel Promoter Variants in MATE1 and MATE2 on the Pharmacokinetics and Pharmacodynamics of Metformin

Interindividual variation in response to metformin, first-line therapy for type 2 diabetes, is substantial. Given that transporters are determinants of metformin pharmacokinetics, we examined the effects of promoter variants in both multidrug and toxin extrusion protein 1 (MATE1) (g.-66T → C, rs2252281) and MATE2 (g.-130G → A, rs12943590) on variation in metformin disposition and response. The pharmacokinetics and glucose-lowering effects of metformin were assessed in healthy volunteers (n = 57) receiving metformin. The renal and secretory clearances of metformin were higher (22% and 26%, respectively) in carriers of variant MATE2 who were also MATE1 reference (P < 0.05). Both MATE genotypes were associated with altered post-metformin glucose tolerance, with variant carriers of MATE1 and MATE2 having an enhanced (P < 0.01) and reduced (P < 0.05) response, respectively. Consistent with these results, patients with diabetes (n = 145) carrying the MATE1 variant showed enhanced metformin response. These findings suggest that promoter variants of MATE1 and MATE2 are important determinants of metformin disposition and response in healthy volunteers and diabetic patients.

Different safety profiles of risperidone and paliperidone extended‐release: a double‐blind, placebo‐controlled trial with healthy volunteers

The present study investigated subjective experiences related to secondary negative symptoms and cognitive performance in healthy volunteers in response to the repeated administration of paliperidone extended-release (ER) and risperidone in a double-blind, placebo-controlled trial. Participants (n = 32) received a fixed dose of one of three study medications for three consecutive days: 6 mg of paliperidone ER, 3 mg of risperidone, or placebo. Subjects were evaluated at baseline and after the first and third administrations of the medications by using the Neuroleptic-Induced Deficit Syndrome Scale and the Scale for the Assessment of Negative Symptoms. Cognitive function was measured at baseline and after the third administration of the medications by using the computerized neuropsychological test. Risperidone was associated with more detrimental subjective experiences compared with paliperidone ER and placebo (p < .05), and these differences persisted after controlling for mental and physical sedation. Analysis of computerized neuropsychological test variables revealed significant differences in the changes in Stroop word-color test results from baseline between the paliperidone ER and risperidone groups (p < .005) and between the placebo and risperidone groups (p < .005). These results suggest that paliperidone ER may have a better safety profile than risperidone in terms of negative subjective experiences and cognitive function among normal volunteers.

Sa1414 Rectal Sensitivity, but Not Drinking Capacity, is Associated With Stress Response in Healthy Volunteers and Patients With Functional Gastrointestinal Disorders

Sa1414 Rectal Sensitivity, but Not Drinking Capacity, is Associated With Stress Response in Healthy Volunteers and Patients With Functional Gastrointestinal Disorders

PGE2 reverses Gs-mediated inhibition of platelet aggregation by interaction with EP3 receptors, but adds to non-Gs-mediated inhibition of platelet aggregation by interaction with EP4 receptors

Prostaglandin E2 (PGE2) has intriguing effects on platelet function in the presence of agents that raise cyclic adenosine 3′5′-monophosphate (cAMP). PGE2 reverses inhibition of platelet aggregation by agents that stimulate cAMP production via a Gs-linked receptor, but adds to the inhibition of platelet function brought about by agents that raise cAMP through other mechanisms. Here, we used the EP receptor antagonists DG-041 (which acts at the EP3 receptor) and ONO-AE3-208 (which acts at the EP4 receptor) to investigate the role of these receptors in mediating these effects of PGE2. Platelet aggregation was measured in platelet-rich plasma obtained from healthy volunteers in response to adenosine diphosphate (ADP) using single platelet counting. The effects of a range of concentrations of PGE2 were determined in the presence of (1) the prostacyclin mimetic iloprost, which operates through Gs-linked IP receptors, (2) the cAMP PDE inhibitor DN9693 and (3) the direct-acting adenylate cyclase stimulator forskolin. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation was also determined as a measure of cAMP. PGE2 reversed the inhibition of aggregation brought about by iloprost; this was prevented in the presence of the EP3 antagonist DG-041, indicating that this effect of PGE2 is mediated via the EP3 receptor. In contrast, PGE2 added to the inhibition of aggregation brought about by DN9693 or forskolin; this was reversed by the EP4 antagonist ONO-AE3-208, indicating that this effect of PGE2 is mediated via the EP4 receptor. Effects on aggregation were accompanied by corresponding changes in VASP phosphorylation. The dominant role of EP3 receptors circumstances where cAMP is increased through a Gs-linked mechanism may be relevant to the situation in vivo where platelets are maintained in an inactive state through constant exposure to prostacyclin, and thus the main effect of PGE2 may be prothrombotic. If so, the results described here further support the potential use of an EP3 receptor antagonist in the control of atherothrombosis.

Prediction of psilocybin response in healthy volunteers.

Responses to hallucinogenic drugs, such as psilocybin, are believed to be critically dependent on the user's personality, current mood state, drug pre-experiences, expectancies, and social and environmental variables. However, little is known about the order of importance of these variables and their effect sizes in comparison to drug dose. Hence, this study investigated the effects of 24 predictor variables, including age, sex, education, personality traits, drug pre-experience, mental state before drug intake, experimental setting, and drug dose on the acute response to psilocybin. The analysis was based on the pooled data of 23 controlled experimental studies involving 409 psilocybin administrations to 261 healthy volunteers. Multiple linear mixed effects models were fitted for each of 15 response variables. Although drug dose was clearly the most important predictor for all measured response variables, several non-pharmacological variables significantly contributed to the effects of psilocybin. Specifically, having a high score in the personality trait of Absorption, being in an emotionally excitable and active state immediately before drug intake, and having experienced few psychological problems in past weeks were most strongly associated with pleasant and mystical-type experiences, whereas high Emotional Excitability, low age, and an experimental setting involving positron emission tomography most strongly predicted unpleasant and/or anxious reactions to psilocybin. The results confirm that non-pharmacological variables play an important role in the effects of psilocybin.

The effect of meal size distribution and timing on interstitial glucose responses in healthy volunteers

The effect of meal size distribution and timing on interstitial glucose responses in healthy volunteers

Can itch-related visual stimuli provoke an itch response?

Itching is a common subjective sensation experienced on the skin and is associated with the desire and impulse to scratch. We tested whether visual cues could generate feelings of itch and provoke a scratch response in healthy volunteers. A secondary aim was to assess whether certain pictures were more effective in evoking sensations of itch. Thirty participants viewed static images that could either be itch-related (i.e., viewing ants or skin conditions) or neutral (viewing butterflies or healthy skin). These were further separated by picture type: ‘skin contact’ (i.e., ants crawling on the skin or a butterfly sitting on the hand); ‘skin response’ (i.e., scratching an insect bite or washing the hands) or ‘no skin’ (simply viewing midges or birds flying). The results indicate that the sensation of itch was successfully generated using itch-related pictures in terms of significantly higher self-reports of itch in answer to the questions ‘how itchy do you feel?’ and ‘how itchy do you think the person in the picture feels?’ compared to viewing neutral pictures (). In addition, participants scratched themselves significantly more when viewing itch-related pictures compared to neutral (). The picture type also had an effect upon these measures with more scratching behaviour recorded when viewing pictures depicting others scratching (). This study demonstrates the impact of visual cues on the sensation of itch and the scratch response and may provide preliminary evidence linking contagious itching to the mirror neuron system and the effectiveness of itch-inducing stimuli as a way to probe social communication.

The Mutated Region of Cytoplasmatic Nucleophosmine 1 (NPM1) Elicits Both CD4+ and CD8+ T Cell Responses

Abstract 2569 IntroductionIn AML, mutations in the nucleophosmin (NPM1) gene are one of the most frequent molecular alterations and predominantly occur in AML with normal cytogenetics. Patients with NPM1 mutation without FLT3-ITD mutation show a favourable prognosis of their disease. The functional role of mutated NPM1 for the improved clinical outcome is under evaluation. Immune responses might be involved in the clinical outcome of the disease. In this work, we demonstrate both CD4+ and CD8+ T cell responses against the mutated region of NPM1. MethodsThe entire amino acid sequences of the NPM1 wild type protein as well as of the mutated cytoplasmic NPM1 types A, B, C and D were screened for HLA-A*0201 binding T cell epitopes using the algorithms of the SYFPEITHI, the Rankpep and the HLA-Bind software programs. Ten peptides with most favourable characteristics were subjected to ELISpot analysis for interferon-γ and granzyme B in 22 healthy volunteers and 27 AML patients to test specific T cell responses of CD8+ T cells. Tetramer assays against the two most interesting epitopes have been performed and chromium release assays have been used to show the cytotoxicity of peptide-specific T cells to lyse T2 cells and leukemic blasts. Moreover, HLA-DR binding epitopes were screened in algorithmic analysis and HLA-DR*0701 binding peptides were exploited to stimulate CD4+ T cells. In the presence of overlapping peptide stimulated CD4+ T cells, NPM1-A specific CD8+ T cells revealed augmented interferon-γ and granzyme B secretion and up-regulation of intracellular interferon-γ. CD4+, CD4-CD8+, CD4-CD8- cell fractions were separated from PBMCs of HLA-A2+DR*0701+ healthy volunteers using a combination of CD4 and CD8 MicroBeads. ResultsTwo epitopes (P3 and P9) derived from the NPM1-mutated protein showed specific T cell responses in healthy volunteers and AML patients. In NPM1-mutated AML patients 33% showed immune responses of CD8+ T cells against peptide P3 and 42% against peptide P9. Specific lysis was detected in chromium release assays NPM1 peptide-primed effector T cells generated from NPM1-mutated AML patients. Tetramer assays showed peptide-specific T cells. To obtain a robust and effective immune response against tumor cells, the activation of CD4 + helper T cells is crucial. Thus NPM1-peptide-A overlapping MHC class II epitopes were searched by primary structure analysis program. Based on plenary search, eight favourable overlapping peptides OL 1–8 were synthesized and exploited for CD4+ T cell stimulation. In granzyme B ELISPOT assay, OL8 co-pulsed NPM1-A CD8+ T cells indicated notable S.I., in contrast other OL1-7 disabled to increase granzyme B secretion. To ensure that Th1 cytokine secretion, under the condition of CD8+ and CD4+ T cells mixed culture, was resulted from NPM1-A CD8+ T cells but not HLA-DR epitope stimulated CD4+ T cells activation, HLA-A2 blocking effect was confirmed in ELISPOT assay. NPM1-A CD8+ T cells co-pulsed with OL6, 7 and 8 showed lesser interferon-γ secretion after HLA-A2 blocking antibody exposure as 73, 35 and 57%. Of note, 83–94% of granzyme B secretion levels were reduced by HLA-A2 blockade administration, and by which NPM1-A CD8+ T cells seemed to be the most probable IFN-gamma and granzyme B producers and CD4+ T cells to interfere with CD8+ T cells. ConclusionTaken together, mutated NPM1 is a promising target structure for specific immunotherapies in AML patients. Disclosures:No relevant conflicts of interest to declare.

Application of Nexfin noninvasive beat-to-beat arterial blood pressure monitoring in autonomic function testing

Evaluation of autonomic function responses is increasingly important for risk prediction and hemodynamic evaluation in the ambulant and perioperative setting, but requires a noninvasive arterial blood pressure measurement device. This study describes whether a novel noninvasive beat-to-beat arterial blood pressure measurement device (Nexfin HD) is able to reproducibly reflect autonomic function responses in healthy volunteers. Noninvasive beat-to-beat arterial blood pressure measurements (Nexfin HD) were performed in 20 healthy men of 22 ± 3 years. Measurements were performed during supine steady state, controlled breathing (0.125 Hz), passive leg raising, a controlled Valsalva maneuver, and a quick stand test. Finally, relative changes in pulse pressure during autonomic function testing and the test-retest reproducibility were determined. Autonomic function tests induced beat-to-beat arterial blood pressure changes that were accurately monitored by the Nexfin device. The intraclass correlation coefficients for systolic and diastolic arterial blood pressure measurements during supine steady state were agreeable [0.91 (0.82-0.96) and 0.84 (0.69-0.93), respectively]. The reproducibility of blood pressure changes during controlled breathing, passive leg raising, and Valsalva maneuver averaged 0.92 (0.82-0.96), 0.76 (0.50-0.90), and 0.94 (0.89-0.97), respectively. The reproducibility of the pulse pressure variation (PPV) as calculated from controlled breathing-induced changes in the arterial blood pressure (13 ± 5%) was high [0.96 (0.93-0.98)]. This study shows that noninvasive beat-to-beat Nexfin HD arterial blood pressure measurements reproducibly reflect autonomic function responses in healthy volunteers.