Deep in situ microscopy for real-time analysis of mammalian cell populations in bioreactors.

An in situ microscope based on pulsed transmitted light illumination via optical fiber was combined to artificial-intelligence to enable for the first time an online cell classification according to well-known cellular morphological features. A 848 192-image database generated during a lab-scale production process of antibodies was processed using a convolutional neural network approach chosen for its accurate real-time object detection capabilities. In order to induce different cell death routes, hybridomas were grown in normal or suboptimal conditions in a stirred tank reactor, in the presence of substrate limitation, medium addition, pH regulation problem or oxygen depletion. Using such an optical system made it possible to monitor real-time the evolution of different classes of animal cells, among which viable, necrotic and apoptotic cells. A class of viable cells displaying bulges in feast or famine conditions was also revealed. Considered as a breakthrough in the catalogue of process analytical tools, in situ microscopy powered by artificial-intelligence is also of great interest for research.

High contamination in the areas surrounding abandoned mines and mining activities: An impact assessment of the Dilala, Luilu and Mpingiri Rivers, Democratic Republic of the Congo.

Abandoned mines and mining activities constitute important sources of toxic metals and Rare Earth Elements (REEs) affecting surrounding environmental compartments and biota. This study investigates the contamination degree and distribution of toxic metals and REEs in contrasting sediment, soil and plant samples surrounding rivers in the African copperbelt area characterized by the presence of numerous abandoned mines, artisanal and industrial mining activities. ICP-MS results highlighted the highest concentration of Cu, Co and Pb in sediments reaching values of 146,801, 18,434 and 899 mg kg-1, respectively. In soil, the values of 175,859, 21,134 and 1164 mg kg-1 were found for Cu, Co and Pb, respectively. These values are much higher than the sediment guidelines for the protection of aquatic life and international soil clean-up standards. Enrichment factor and geoaccumulation index results indicated important contribution of mining activities to the study sites pollution in addition to natural background. Highest metal accumulation in leaves of Phalaris arundinacea L., was observed, reaching values of 34,061, 5050 and 230 mg kg-1 for Cu, Co, and Pb, respectively. The ∑REE concentration reached values of 2306, 733, 2796 mg kg-1 in sediment, soil and plant samples, respectively. The above results were combined with geographical information including satellite imagery, hydrography and mining concessions. Maps were produced to present the results in a comprehensive and compelling visual format. The results will be disseminated through an innovative mapping online platform to simplify access to data and to facilitate dialogue between stakeholders.

A web platform for landuse, climate, demography, hydrology and beach erosion in the Black Sea catchment.

The Black Sea catchment (BSC) is facing important demographic, climatic and landuse changes that may increase pollution, vulnerability and scarcity of water resources, as well as beach erosion through sea level rise. Limited access to reliable time-series monitoring data from environmental, statistical, and socio-economical sources is a major barrier to policy development and decision-making. To address these issues, a web-based platform was developed to enable discovery and access to key environmental information for the region. This platform covers: landuse, climate, and demographic scenarios; hydrology and related water vulnerability and scarcity; as well as beach erosion. Each data set has been obtained with state-of-the-art modelling tools from available monitoring data using appropriate validation methods. These analyses were conducted using global and regional data sets. The data sets are intended for national to regional assessments, for instance for prioritizing environmental protection projects and investments. Together they form a unique set of information, which lay out future plausible change scenarios for the BSC, both for scientific and policy purposes.

Cardiovascular safety pharmacology profile of etamicastat, a novel peripheral selective dopamine-ß-hydroxylase inhibitor.

Etamicastat, a peripheral reversible dopamine-ß-hydroxylase inhibitor, blocked the hERG current amplitude with an IC50 value of 44.0µg/ml in HEK 293 cells. At 0.3 and 3µg/ml, etamicastat had no effects on the action potential (AP) in male dog Purkinje fibers. At 30µg/ml, etamicastat significantly affected resting membrane potential (+4%), AP amplitude (-4%), AP duration at 60% (-14%) and AP duration at 90% (+5%) repolarization, and AP triangulation (+79%). In the telemetered conscious male dog, etamicastat (up to 20mg/kg) had no effects on arterial blood pressure, heart rate and the PR interval. At 10 and 20mg/kg, the QTc interval was slightly prolonged (8-9% max, P<0.05). No arrhythmia or other changes in the morphology of the ECG were observed. The maximum observed plasma concentrations (Cmax) of etamicastat (i.e. 3h post-administration) were 1.4 and 3.7µg/ml at 10 and 20mg/kg, respectively. No deleterious effects, including ECG disturbance were observed in male and female dogs dosed by gavage with etamicastat (up to 20mg/kg/day) for 28 days. Mean plasma Cmax etamicastat levels ranged between 2.4 and 6.3µg/ml on Day 1 and Day 28 of treatment, respectively. It is concluded that the blockade of the delayed rectifier potassium channels by etamicastat together with the QTc interval prolongation observed in conscious dogs can be considered as modest with respect to the measured plasmatic concentrations. These findings suggest that etamicastat is not likely to prolong the QT interval at therapeutic doses (~0.2µg/ml).

Cultured autologous keratinocytes in the treatment of large and deep burns: a retrospective study over 15 years.

AIM: The aim was to review the use and indications of cultured autologous epidermis (CAE) in extensive burns and to evaluate the efficiency of our strategy of burn treatment. MATERIALS AND METHODS: This retrospective study comprised 15 years (1997-2012). INCLUSION CRITERIA: all patients who received CAE. EXCLUSION CRITERIA: patients who died before complete healing and patients who received exclusively cultured allogeneic keratinocytes. Evaluation criteria were clinical. Time and success of wound healing after CAE graft were evaluated. RESULTS: A total of 63 patients were included with severity Baux score of 107 (from 70 to 140) and mean percentage of TBSA of 71% (from 40% to 97%). The CAE were used as Cuono method, in STSG donor sites and deep 2nd degree burns and in combination with large-meshed STSG (1:6-1:12) in extensively burned patients. Cuono method was used in 6 patients. The final take was 16% (0-30) because of the great fragility of the obtained epidermis. Nine patients with deep 2nd degree burns (mean TBSA 81%, from 60 to 97%) were successfully treated with only CAE without skin grafting. Combined technique (STSG meshed at 1:6-1:12 covered with CAE) was used in 27 patients (mean TBSA 69%, from 49% to 96%) with 85% success rate. Finally, donor sites treated with CAE in 49 patients could be harvested several times thanks to rapid epithelialization (time of wound healing was 7 days (from 5 to 10 days)). CONCLUSION: The CAE allow rapid healing of STSG donor sites and deep 2nd second degree burns in extensively burned patients.

Cultured allogenic keratinocytes for extensive burns: a retrospective study over 15 years.

UNLABELLED: The aim was to review the use and indications of cultured allogenic keratinocytes (CAlloK) in extensive burns and their efficiency. MATERIALS AND METHODS: This retrospective study comprised 15 years (1997-2012). INCLUSION CRITERIA: all patients who received CAlloK. EXCLUSION CRITERIA: patients who died before complete healing. Evaluation criteria were clinical. Time and success of wound healing after CAlloK use were evaluated. RESULTS: The CAlloK were used for 2 indications - STSG donor sites and deep 2nd degree burns in extensively burned patients. A total of 70 patients were included with severity Baux score of 99.2 (from 51 to 144) and mean percentage of TBSA of 63.49% (from 21 to 96%). Fifty nine patients received CAlloK for STSG donor sites with a mean number of applications of 4 and mean surface of 3800 cm(2) per patient. Treated donor sites were re-harvested 2.5 times. The mean time of complete epithelialization was 7 days. In 11 patients, CAlloK were used for deep 2nd degree burns. The mean percentage of burned surface was 73.7%. The mean surface of CAlloK per patient was 2545 cm(2). Complete healing was achieved in 6.4 days. CONCLUSION: The CAlloK allow rapid healing of STSG donor-sites and deep 2nd second degree burns in extensively burned patients.

No altered blood pressure and serum markers of oxidative stress after a long time dietary fish oil in the genetically 9 month-old type-2 diabetes Zucker rat.

In this study, we investigated the effect of a high n-3 fatty acid diet (eicosapentaenoic and docosahexaenoic acids) in Zucker obese and lean rats on blood pressure in association with physiological parameters, serum biochemistry and oxidative stress analysis. After 150 days of treatment, dietary fish oil supplementation in Zucker obese rats (9 months of age) reduces bodyweight gain and serum triglyceridemia and nitrite levels, increases serum glucose and angiotensin converting enzyme activity, but does not alter blood pressure, cholesterol levels and serum markers of oxidative stress (malondialdehyde, glutathione), compared to the Zucker rats fed control diet. According to these results, we can consider that after 150 days of treatment, fish oil is not enough to regulate parameters involved in the metabolic syndrome, such as cholesterolemia and blood pressure, in a 9 month-old genetically type-2 diabetes rat.

Models of cardiovascular disease: measurement of arrhythmogenic/antiarrhythmic activity in the guinea pig.

The protocols described in this unit are used to assess the effects of new chemical entities on arrhythmias in the guinea pig. In the anesthetized guinea pig, arrhythmias are induced by a slow intravenous infusion of digoxin, which provokes extrasystoles, ventricular tachyarrhythmias, and ultimately cardiac arrest. Imipramine precipitates the occurrence of arrhythmias, whereas propranolol shows protection against them.

Models of cardiovascular disease: measurement of antihypertensive activity in the conscious rat (SHR, DOCA-salt, and Goldblatt hypertension models).

The protocols described in this unit are used to assess the effects of new chemical entities on hypertension in conscious rats. In the spontaneously hypertensive rat (SHR) model, the results obtained with the reference compounds clonidine, prazosin, propranolol, and captopril are provided for illustration. All compounds demonstrate antihypertensive activity, with captopril and prazosin being the least and the most active, respectively. In the deoxycorticosterone acetate (DOCA)-salt model in the rat, the test substance shown as an example (a potential endothelin ET(A)-receptor antagonist) prevents the development of hypertension in the first phase. However, the effects of treatment disappear in the very last phase of the study, suggesting the development of a malignant hypertension resistant to treatment in this model. In the Goldblatt hypertension rat model (renal artery stenosis), losartan prevents the development of hypertension. It does not modify the weight of the right and left kidneys but slightly reduces the degree of cardiac hypertrophy.

Orthostatic hypotension induced by postural change in the rat (tilt test).

Postural-change-induced (orthostatic) hypotension is defined as an excessive drop in arterial blood pressure occurring when moving toward an upright position. This side effect, which may limit the therapeutic use of some agents, can occur with drugs, such as adrenoceptor blockers and vasodilators, that dampen sympathetic reflex activity. Described in this unit is a procedure for evaluating the effects of test substances on the changes in blood pressure and heart rate that occur in an anesthetized, normotensive rat during a tilting challenge (head-up position). In addition to being a relatively simple technique, this assay yields reproducible orthostatic hypotensive responses and allows for the investigation, in the same preparation, of several ascending doses of a test substance. Examples of results obtained with prazosin, an α1-adrenoceptor antagonist that is notorious for causing orthostatic hypotension, are provided for illustrative purposes.

Gastrointestinal models: intestinal transit, gastric emptying, and ulcerogenic activity in the rat.

The protocols described in this unit are designed to assess the effects of substances on intestinal transit and gastric emptying and to evaluate their ulcerogenic potential on the stomach and duodenum of the rat. Examples of results obtained with atropine or morphine (intestinal transit), loperamide (gastric emptying), or indomethacin (ulcerogenic activity) used as reference substances are provided for illustrative purposes. Atropine and morphine clearly reduce intestinal transit. Atropine, morphine, and loperamide clearly reduce gastric emptying. Indomethacin shows a marked ulcerogenic potential.

Cardiomyopathic Syrian hamster as a model of congestive heart failure.

Cardiomyopathic Syrian hamsters (Bio TO-2 dilated strain) constitute an animal model of congestive heart failure, which progressively develops an alteration of cardiac function leading to decreased arterial blood pressure and musculo-cutaneous blood flow associated with a complex process of cardiac remodeling including left ventricle dilation, wall thinning, and greater collagen density. The protocols described in this unit are designed to assess the pharmacological effects of new therapeutic strategies on cardiac and systemic hemodynamics, morphometry (body and target organs weight), cardiac remodeling (left ventricle dilation and collagen density), and survival in this model of dilated cardiomyopathy. Examples of results obtained with enalapril, an angiotensin I converting enzyme inhibitor, are provided for illustrative purposes.

The use of the dog electroencephalogram (EEG) in safety pharmacology to evaluate proconvulsant risk.

INTRODUCTION: The dog is frequently used for cardiovascular safety pharmacology and for toxicology studies, but is not often used for central nervous system (CNS) safety pharmacology purposes. We have therefore examined the electroencephalogram (EEG) in conscious dogs by means of radio-telemetry methods using the proconvulsant agent pentylenetetrazole (PTZ) as reference substance. Assessment of proconvulsant risk is an important aspect of CNS safety evaluation and the EEG is a sensitive technique for identifying pathologic brain activity, most importantly paroxysmal activity. METHODS: Dogs were implanted with epidural electrodes wired to subcutaneously placed radiotransmitters. Following baseline recording, the test substance was administered and the EEG and electromyogram (EMG) activities were recorded from dogs placed in slings. The EEG was assessed visually for abnormal activity and dogs were also continuously observed for the appearance of overt convulsive activity. The PTZ infusion was stopped and diazepam was administered as soon as clear and sustained EEG effects and/or behavioural symptoms occurred. RESULTS: Slow i.v. infusion of PTZ (1.5 mg/kg/min) induced clear paroxysmal effects on the EEG trace in the form of spike and wave trains of 4-5 Hz. Paroxysmal activity associated with clonic convulsions occurred between 17 and 36 min after the start of infusion (a mean of 24 min) but in most cases paroxysmal activity was observed approximately 60 s prior to any overt convulsive activity. DISCUSSION: These data show the usefulness of the telemetered dog EEG in safety pharmacology. The dog EEG is appropriate in situations where results from cardiovascular and CNS safety tests in the same species are required, or where the use of other species is contraindicated because of metabolic or pharmacokinetic particularities.

An overview of QT interval assessment in safety pharmacology.

Medicinal products that prolong cardiac repolarization unintentionally, as assessed in terms of prolongation of the QT interval of the electrocardiogram, may trigger a potentially fatal arrhythmia called torsade de pointe (TDP). This lethal risk necessitates a detailed preclinical evaluation before initiating clinical trials. There are two different and complementary approaches to assess the potential of drugs to cause QT interval prolongation. The in vivo approach provides information on the potential of the compound to prolong the QT interval under near-physiological conditions. It is mostly descriptive and not explanatory in terms of mechanisms of action. The in vitro approach provides much more mechanistic information, but is far removed from the clinical situation. While both approaches appear to possess reasonable predictive value, the results may depend largely on the experimental conditions employed. This unit reviews these issues and discusses a strategy aimed at understanding the problems associated with this cardiovascular risk.

Prulifloxacin: in vitro (HERG current) and in vivo (conscious dog) assessment of cardiac risk.

Prulifloxacin, a new thiazeto-quinoline derivative with antibiotic properties, was evaluated for cardiac risk both in vitro on the ether-à-go-go-related gene (HERG) K+ channel, and in vivo in the conscious dog monitored by telemetry. HERG current was measured from stably transfected human embryonic kidney (HEK) 293 cells by means of the patch-clamp technique. Application of AF 3013, the active metabolite of prulifloxacin, produced only minor reduction of HERG current amplitude (tail current=-40 mV), producing a maximum blockade of 12.3 +/- 3.3% at the highest concentration tested (335 microM). In comparison, ciprofloxacin also failed to produce a 50% inhibition of HERG current amplitude, although the maximum blockade was greater than that observed with prulifloxacin (47.6 +/- 1.9% at the highest concentration tested (335 microM). In contrast, moxifloxacin blocked HERG current amplitude with an IC50 value of 74.7 microM. Prulifloxacin had no effect on the QTc interval (Fridericia's) following 5 days of repeated oral administration (150 mg/kg/day) in the conscious dog monitored by telemetry. These findings suggest that prulifloxacin is not likely to prolong the QT interval.

QT interval prolongation and cardiac risk assessment for novel drugs.

Fatal cardiac arrhythmias, known as torsades de pointes, can occur with a wide variety of medicinal drugs and are associated with prolongation of the QT interval. This review critically evaluates the major strategies for assessing QT prolongation risk: ion channel studies, in vitro cardiac electrophysiology, and in vivo cardiac electrophysiology and hemodynamics. Disease- or drug-induced QT prolongation is mainly associated with reduced amplitude of the repolarizing outward K+ current in myocardial cells, particularly those carried by the human ether-a-go-go-related gene (HERG) channel. Thus, measuring HERG currents using patch-clamp technology and cloned HERG channels represents a first approach for evaluating adverse effects of drugs on ion channel function, under physiological conditions. Evaluation of changes in transmembrane action potential in isolated rabbit or dog Purkinje fibers reflects mixed ion channel blocking properties of the test substance and therefore permits a greater understanding of the mechanisms underlying the genesis of arrhythmias. Both HERG channel and Purkinje fiber procedures are clinically predictive, however, no in vitro technique can fully reproduce the in vivo situation. Therefore, both in vitro and in vivo approaches should be employed to maximize the chances of an accurate assessment of risk in an area where prolonged QT can result in death.

Electrocardiographic toxicity in the guinea pig.

Abnormalities of cardiac rhythm are one of the most common clinical problems in cardiology and arise as the result of either disorders of cardiac impulse formation or conduction, or a combination of both. It has been established that some classes of drugs, such as tricyclic antidepressants (e.g., imipramine), cardiac glycosides (e.g., digoxin), and Class I or Class III antiarrhythmic drugs (e.g., quinidine or amiodarone) can produce electrocardiographic toxicity in humans. It is therefore highly advisable to assess the effect of any new compound in this respect, during the early phases of drug development. This unit presents a protocol to detect the electrocardiographic toxicity of compounds in the anesthetized guinea pig.