Ex vivo analysis of human antigen-specific CD8+ T-cell responses: quality assessment of fluorescent HLA-A2 multimer and interferon-gamma ELISPOT assays for patient immune monitoring.

The authors developed a standardized approach for immune monitoring of antigen-specific CD8+ T cells within peripheral blood lymphocytes (PBLs) that combines direct ex vivo analysis of Melan-A/MART-1 and influenza-specific CD8+ T cells with HLA-A2/peptide multimers and interferon-gamma ELISPOT assays. Here the authors assessed the quality of results obtained with 180 PBLs from healthy donors and melanoma patients. Reproducibility of the multimer assay was good (average of 15% variation). In the absence of in vivo antigen-specific T-cell responses, physiologic fluctuations of multimer-positive T cells was low, with variation coefficients of 20% for Melan-A and 28% for influenza-specific T cells. In contrast, patients with vaccination-induced T-cell responses had significantly increased T-cell frequencies clearly exceeding physiologic fluctuations. Comparable results were obtained with ELISPOT assays. In conclusion, this approach is well suited to assess T-cell responses as biologic endpoints in clinical vaccine studies.

Intracranial hypertension: what additional information can be derived from ICP waveform after head injury?

OBJECTIVE: Although intracranial hypertension is one of the important prognostic factors after head injury, increased intracranial pressure (ICP) may also be observed in patients with favourable outcome. We have studied whether the value of ICP monitoring can be augmented by indices describing cerebrovascular pressure-reactivity and pressure-volume compensatory reserve derived from ICP and arterial blood pressure (ABP) waveforms. METHOD: 96 patients with intracranial hypertension were studied retrospectively: 57 with fatal outcome and 39 with favourable outcome. ABP and ICP waveforms were recorded. Indices of cerebrovascular reactivity (PRx) and cerebrospinal compensatory reserve (RAP) were calculated as moving correlation coefficients between slow waves of ABP and ICP, and between slow waves of ICP pulse amplitude and mean ICP, respectively. The magnitude of 'slow waves' was derived using ICP low-pass spectral filtration. RESULTS: The most significant difference was found in the magnitude of slow waves that was persistently higher in patients with a favourable outcome (p<0.00004). In patients who died ICP was significantly higher (p<0.0001) and cerebrovascular pressure-reactivity (described by PRx) was compromised (p<0.024). In the same patients, pressure-volume compensatory reserve showed a gradual deterioration over time with a sudden drop of RAP when ICP started to rise, suggesting an overlapping disruption of the vasomotor response. CONCLUSION: Indices derived from ICP waveform analysis can be helpful for the interpretation of progressive intracranial hypertension in patients after brain trauma.

Barbiturate infusion for intractable intracranial hypertension and its effect on brain oxygenation.

OBJECTIVE: Barbiturate-induced coma can be used in patients to treat intractable intracranial hypertension when other therapies, such as osmotic therapy and sedation, have failed. Despite control of intracranial pressure, cerebral infarction may still occur in some patients, and the effect of barbiturates on outcome remains uncertain. In this study, we examined the relationship between barbiturate infusion and brain tissue oxygen (PbtO2). METHODS: Ten volume-resuscitated brain-injured patients who were treated with pentobarbital infusion for intracranial hypertension and underwent PbtO2 monitoring were studied in a neurosurgical intensive care unit at a university-based Level I trauma center. PbtO2, intracranial pressure (ICP), mean arterial pressure, cerebral perfusion pressure (CPP), and brain temperature were continuously monitored and compared in settings in which barbiturates were or were not administered. RESULTS: Data were available from 1595 hours of PbtO2 monitoring. When pentobarbital administration began, the mean ICP, CPP, and PbtO2 were 18 +/- 10, 72 +/- 18, and 28 +/- 12 mm Hg, respectively. During the 3 hours before barbiturate infusion, the maximum ICP was 24 +/- 13 mm Hg and the minimum CPP was 65 +/- 20 mm Hg. In the majority of patients (70%), we observed an increase in PbtO2 associated with pentobarbital infusion. Within this group, logistic regression analysis demonstrated that a higher likelihood of compromised brain oxygen (PbtO2 < 20 mm Hg) was associated with a decrease in pentobarbital dose after controlling for ICP and other physiological parameters (P < 0.001). In the remaining 3 patients, pentobarbital was associated with lower PbtO2 levels. These patients had higher ICP, lower CPP, and later initiation of barbiturates compared with patients whose PbtO2 increased. CONCLUSION: Our preliminary findings suggest that pentobarbital administered for intractable intracranial hypertension is associated with a significant and independent increase in PbtO2 in the majority of patients. However, in some patients with more compromised brain physiology, pentobarbital may have a negative effect on PbtO2, particularly if administered late. Larger studies are needed to examine the relationship between barbiturates and cerebral oxygenation in brain-injured patients with refractory intracranial hypertension and to determine whether PbtO2 responses can help guide therapy.

Construction and Quantitative Evaluation of a Dual Specific Promoter System for Monitoring the Expression Status of Stra8 and c-kit Genes.

Applications of genetic constructs with multiple promoters, which are fused with reporter genes and simultaneous monitoring of various events in cells, have gained special attention in recent years. Lentiviral vectors, with their distinctive characteristics, have been considered to monitor the developmental changes of cells in vitro. In this study, we constructed a novel lentiviral vector (FUM-M), containing two germ cell-specific promoters (Stra8 and c-kit), fused with ZsGreen and DsRed2 reporter genes, and evaluated its efficiency in different cells following treatments with retinoic acid and DMSO. Several cell lines (P19, GC-1 spg and HEK293T) were transduced with this vector, and functional capabilities of the promoters were verified by flow cytometry and quantitative RT-PCR. Our results indicate that FUM-M shows dynamic behavior in the presence and absence of extrinsic factors. A correlation was also observed between the function of promoters, present in the lentiviral construct and the endogenous level of the Stra8 and c-kit mRNAs in the cells. In conclusion, we recommend this strategy, which needs further optimization of the constructs, as a beneficial and practical way to screen chemical inducers involved in cellular differentiation toward germ-like cells.

Multiplex liquid chromatography-tandem mass spectrometry assay for simultaneous therapeutic drug monitoring of ribavirin, boceprevir, and telaprevir.

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.

Front-crawl instantaneous velocity estimation using a wearable inertial measurement unit.

Monitoring the performance is a crucial task for elite sports during both training and competition. Velocity is the key parameter of performance in swimming, but swimming performance evaluation remains immature due to the complexities of measurements in water. The purpose of this study is to use a single inertial measurement unit (IMU) to estimate front crawl velocity. Thirty swimmers, equipped with an IMU on the sacrum, each performed four different velocity trials of 25 m in ascending order. A tethered speedometer was used as the velocity measurement reference. Deployment of biomechanical constraints of front crawl locomotion and change detection framework on acceleration signal paved the way for a drift-free integration of forward acceleration using IMU to estimate the swimmers velocity. A difference of 0.6 ± 5.4 cm · s(-1) on mean cycle velocity and an RMS difference of 11.3 cm · s(-1) in instantaneous velocity estimation were observed between IMU and the reference. The most important contribution of the study is a new practical tool for objective evaluation of swimming performance. A single body-worn IMU provides timely feedback for coaches and sport scientists without any complicated setup or restraining the swimmer's natural technique.

Clinical usefulness of therapeutic concentration monitoring for imatinib dosage individualization: results from a randomized controlled trial.

PURPOSE: This study assessed whether a cycle of "routine" therapeutic drug monitoring (TDM) for imatinib dosage individualization, targeting an imatinib trough plasma concentration (C min) of 1,000 ng/ml (tolerance: 750-1,500 ng/ml), could improve clinical outcomes in chronic myelogenous leukemia (CML) patients, compared with TDM use only in case of problems ("rescue" TDM). METHODS: Imatinib concentration monitoring evaluation was a multicenter randomized controlled trial including adult patients in chronic or accelerated phase CML receiving imatinib since less than 5 years. Patients were allocated 1:1 to "routine TDM" or "rescue TDM." The primary endpoint was a combined outcome (failure- and toxicity-free survival with continuation on imatinib) over 1-year follow-up, analyzed in intention-to-treat (ISRCTN31181395). RESULTS: Among 56 patients (55 evaluable), 14/27 (52 %) receiving "routine TDM" remained event-free versus 16/28 (57 %) "rescue TDM" controls (P = 0.69). In the "routine TDM" arm, dosage recommendations were correctly adopted in 14 patients (median C min: 895 ng/ml), who had fewer unfavorable events (28 %) than the 13 not receiving the advised dosage (77 %; P = 0.03; median C min: 648 ng/ml). CONCLUSIONS: This first target concentration intervention trial could not formally demonstrate a benefit of "routine TDM" because of small patient number and surprisingly limited prescriber's adherence to dosage recommendations. Favorable outcomes were, however, found in patients actually elected for target dosing. This study thus shows first prospective indication for TDM being a useful tool to guide drug dosage and shift decisions. The study design and analysis provide an interesting paradigm for future randomized TDM trials on targeted anticancer agents.

Clinical Experience with Immune Monitoring for Cytomegalovirus in Solid-Organ Transplant Recipients.

Novel strategies are needed to further reduce the burden of cytomegalovirus (CMV) disease in solid-organ transplant (SOT) recipients. Measurement of the specific cell-mediated immunity against CMV can identify the actual risk for the development of CMV disease in a given patient. Thus, immune monitoring is an attractive strategy for individualizing the management of CMV after transplantation. A growing number of observational studies on immune monitoring for CMV have been published over recent years, although there is a lack of data coming from interventional trials. In high-risk patients, measurement of CMV-specific T-cell responses appropriately stratifies the risk of CMV disease after discontinuation of antiviral prophylaxis. Immune monitoring may also help to identify patients followed by the preemptive approach at low risk for progression to CMV disease. Pretransplant assessment of cell-mediated immunity in seropositive patients may predict the development of posttransplant CMV infection. Overall, these studies indicate that the use of cell-mediated immunity assays has the potential to improve the management of CMV disease in SOT recipients.

Inpatient diabetes care in a Swiss multi-site hospital: is there room for improvement?

Background and objective: Optimal care of diabetic patients (DPs) decreases the risk of complications. Close blood glucose monitoring can improve patient outcomes and shorten hospital stay. The objective of this pilot study was to evaluate the treatment of hospitalized DPs according to the current standards, including their diabetic treatment and drugs to prevent diabetes related complications [=guardian drugs: angiotensin converting enzyme inhibitors (ACEI) or Angiotensin II Receptor Blockers (ARB), antiplatelet drugs, statins]. Guidelines of the American Diabetes Association (ADA) [1] were used as reference as they were the most recent and exhaustive for hospital care. Design: Observational pilot study: analysis of the medical records of all DPs seen by the clinical pharmacists during medical rounds in different hospital units. An assessment was made by assigning points for fulfilling the different criteria according to ADA and then by dividing the total by the maximum achievable points (scale 0-1; 1 = all criteria fulfilled). Setting: Different Internal Medicine and Geriatric Units of the (multi-site) Ho^pital du Valais. Main outcome measures: - Completeness of diabetes-related information: type of diabetes, medical history, weight, albuminuria status, renal function, blood pressure, (recent) lipid profile. - Management of blood glucose: Hb1Ac, glycemic control, plan for treating hyper-/hypoglycaemia. - Presence of guardian drugs if indicated. Results: Medical records of 42 patients in 10 different units were analysed (18 women, 24 men, mean age 75.4 ± 11 years). 41 had type 2 diabetes. - Completeness of diabetes-related information: 0.8 ± 0.1. Information often missing: insulin-dependence (43%) and lipid profile (86%). - Management of blood glucose: 0.5 ± 0.2. 15 patients had suboptimal glycemic balance (target glycaemia 7.2-11.2 mmol/ l, with values[11.2 or\3.8 mmol/l, or Hb1Ac[7%), 10 patients had a deregulated balance (more than 10 values[11.2 mmol/l or \3.8 mmol/l and even values[15 mmol/l). - Presence of guardian drugs if indicated: ACEI/ARB: 19 of 23 patients (82.6%), statin: 16 of 40 patients (40%), antiplatelet drug: 16 of 39 patients (41%). Conclusions: Blood glucose control was insufficient in many DPs and prescription of statins and antiplatelet drugs was often missing. If confirmed by a larger study, these two points need to be optimised. As it is not always possible and appropriate to make those changes during hospital stay, a further project should assess and optimise diabetes care across both inpatient and outpatient settings.

Prospective monitoring of cefepime in intensive care unit adult patients

Rapport de synthèse :Le céfépime a été associé à un taux de mortalité supérieur à celui des autres bêta-lactamines chez les patients traités pour un sepsis sévère. Une des hypothèses avancées pour expliquer ces échecs thérapeutiques sont de possibles effets secondaires cachés (par ex. neurologiques) ou des paramètres pharmacocinétiques/pharmacodynamiques (PK/PD) inadaptés. Le présent travail a étudié cette problématique en mesurant prospectivement la pharmacocinétique du céfépime chez 21 patients consécutifs hospitalisés aux soins intensifs adultes (SIAD) pour une pneumonie nosocomiale. La population étudiée avec un âge médian 55,1 ans, a reçu par voie intraveineuse du céfépime à raison de 2 g toutes les 12 heures pour une clairance de la créatinine (Clcr)>50 mI/ min, et 2 g toutes les 24 heures ou 36 heures pour une Clcr<50 ml /min. Les taux plasmatiques de céfépime ont été mesurés à plusieurs reprises avant et après administration du médicament après la lèoe dose et à l'état d'équilibre par chromatographie en phase liquide à haute pression. Les taux plasmatiques ont considérablement varié entre les patients. Cent pour cent (21/21) des patients ont eu une durée appropriée d'antibiothérapie avec des taux plasmatiques supérieures à la CMI du céfépime (T>CMI>50%) pour les agents pathogènes retrouvés dans cette étude (CMI<4 mg/I), mais seulement 45-65% d'entre eux ont eu une couverture appropriée pour les agents pathogènes potentiels présentant une CMI> 8 mg/I pour le céfépime. Deux patients avec une insuffisance rénale (Clcr<30 ml/min) ont présenté des symptômes compatibles avec une épilepsie non-convulsive (état confusionnel et myoclonies) attribuée dans un 2ème temps à une toxicité du céfépime après que les taux plasmatiques aient été communiqués aux soignants qui ont suspendu l'antibiothérapie avec disparition des symptômes. Les résultats de cette étude empirique confirment l'existence d'effets secondaires cachés et de paramètres PK/PD inappropriés (pour les agents pathogènes ayant des CMI de limite supérieure) dans notre population de SIAD. En outre, ils mettent en évidence une fenêtre thérapeutique efficace pour une posologie de céfépime de 2 g toutes les 12 heures chez les patients ayant une Clcr>50 ml/min infectés par des pathogènes avec des CMI pour le céfépime <4 mg/I. Les échecs thérapeutiques constatés dans cette étude sont probablement liés à des taux sériques inadaptés, résultant de la difficulté de prescription dans les situations cliniques complexes. Dans ce contexte, un prompt dosage plasmatique du céfépime doit être considéré en cas de diminution de la Clcr ou en présence de CMI élevées.

Fractal temporal organisation of motricity is altered in major depression.

OBJECTIVE: Motor changes in major depression (MD) may represent potential markers of treatment response. Physiological rhythms (heart rate/gait cycle/hand movements) have been recently shown to be neither random nor regular but to display a fractal temporal organisation, possibly reflecting a unique central "internal clock" control. Sleep and mood circadian rhythm modifications observed in MD also suggest a role for this "internal clock". We set out to examine the fractal pattern of motor activity in MD. METHODS: Ten depressed patients (46±20 years) and ten age- and gender-matched healthy controls (48±21 years) underwent a 6-h ambulatory monitoring of spontaneous hand activity with a validated wireless device. Fractal scaling exponent (α) was analysed. An α value close to 1 means the pattern is fractal. RESULTS: Healthy controls displayed a fractal pattern of spontaneous motor hand activity (α: 1.0±0.1), whereas depressed patients showed an alteration of that pattern (α:1.2±0.15, p<0.01), towards a smoother organisation. CONCLUSION: The alteration of fractal pattern of hand activity by depression further supports the role of a central internal clock in the temporal organisation of movements. This novel way of studying motor changes in depression might have an important role in the detection of endophenotypes and potential predictors of treatment response.

Numerical analysis of wave-induced fluid flow effects on seismic data: Application to monitoring of CO2 storage at the Sleipner Field

In this work we analyze how patchy distributions of CO2 and brine within sand reservoirs may lead to significant attenuation and velocity dispersion effects, which in turn may have a profound impact on surface seismic data. The ultimate goal of this paper is to contribute to the understanding of these processes within the framework of the seismic monitoring of CO2 sequestration, a key strategy to mitigate global warming. We first carry out a Monte Carlo analysis to study the statistical behavior of attenuation and velocity dispersion of compressional waves traveling through rocks with properties similar to those at the Utsira Sand, Sleipner field, containing quasi-fractal patchy distributions of CO2 and brine. These results show that the mean patch size and CO2 saturation play key roles in the observed wave-induced fluid flow effects. The latter can be remarkably important when CO2 concentrations are low and mean patch sizes are relatively large. To analyze these effects on the corresponding surface seismic data, we perform numerical simulations of wave propagation considering reservoir models and CO2 accumulation patterns similar to the CO2 injection site in the Sleipner field. These numerical experiments suggest that wave-induced fluid flow effects may produce changes in the reservoir's seismic response, modifying significantly the main seismic attributes usually employed in the characterization of these environments. Consequently, the determination of the nature of the fluid distributions as well as the proper modeling of the seismic data constitute important aspects that should not be ignored in the seismic monitoring of CO2 sequestration problems.

Objective evaluation of shoulder function using body-fixed sensors: a new way to detect early treatment failures?

Background: Variable definitions of outcome (Constant score, Simple Shoulder Test [SST]) have been used to assess outcome after shoulder treatment, although none has been accepted as the universal standard. Physicians lack an objective method to reliably assess the activity of their patients in dynamic conditions. Our purpose was to clinically validate the shoulder kinematic scores given by a portable movement analysis device, using the activities of daily living described in the SST as a reference. The secondary objective was to determine whether this device could be used to document the effectiveness of shoulder treatments (for glenohumeral osteoarthritis and rotator cuff disease) and detect early failures.Methods: A clinical trial including 34 patients and a control group of 31 subjects over an observation period of 1 year was set up. Evaluations were made at baseline and 3, 6, and 12 months after surgery by 2 independent observers. Miniature sensors (3-dimensional gyroscopes and accelerometers) allowed kinematic scores to be computed. They were compared with the regular outcome scores: SST; Disabilities of the Arm, Shoulder and Hand; American Shoulder and Elbow Surgeons; and Constant.Results: Good to excellent correlations (0.61-0.80) were found between kinematics and clinical scores. Significant differences were found at each follow-up in comparison with the baseline status for all the kinematic scores (P < .015). The kinematic scores were able to point out abnormal patient outcomes at the first postoperative follow-up.Conclusion: Kinematic scores add information to the regular outcome tools. They offer an effective way to measure the functional performance of patients with shoulder pathology and have the potential to detect early treatment failures.Level of evidence: Level II, Development of Diagnostic Criteria, Diagnostic Study. (C) 2011 Journal of Shoulder and Elbow Surgery Board of Trustees.