Targeting CD19-positive lymphomas with the antibody-drug conjugate loncastuximab tesirine: preclinical evidence as single agent and in combination therapy.

Antibody-drug conjugates (ADCs) represent one of the most successful therapeutic approaches introduced in clinical practice in the last few years. Loncastuximab tesirine (ADCT-402) is a CD19 targeting ADC, in which the antibody is conjugated through a protease cleavable dipeptide linker to a pyrrolobenzodiazepine (PBD) dimer warhead (SG3199). Based on the results of a phase 2 study, loncastuximab tesirine was recently approved for adult patients with relapsed/refractory large B-cell lymphoma. We assessed the activity of loncastuximab tesirine using in vitro and in vivo models of lymphomas, correlated its activity with CD19 expression levels, and identified combination partners providing synergy with loncastuximab tesirine. Loncastuximab tesirine was tested across 60 lymphoma cell lines. Loncastuximab tesirine had strong cytotoxic activity in B-cell lymphoma cell lines. The in vitro activity was correlated with CD19 expression level and intrinsic sensitivity of cell lines to the ADC's warhead. Loncastuximab tesirine was more potent than other anti-CD19 ADCs (coltuximab ravtansine, huB4-DGN462), albeit the pattern of activity across cell lines was correlated. Loncastuximab tesirine activity was also largely correlated with cell line sensitivity to R-CHOP. Combinatorial in vitro and in vivo experiments identified the benefit of adding loncastuximab tesirine to other agents, especially BCL2 and PI3K inhibitors. Our data support the further development of loncastuximab tesirine as a single agent and in combination for patients affected by mature B-cell neoplasms. The results also highlight the importance of CD19 expression and the existence of lymphoma populations characterized by resistance to multiple therapies.

Noninvasive prediction of axillary lymph node breast cancer metastasis using morphometric analysis of nodal tumor microvessels in a contrast-free ultrasound approach.

PURPOSE: Changes in microcirculation of axillary lymph nodes (ALNs) may indicate metastasis. Reliable noninvasive imaging technique to quantify such variations is lacking. We aim to develop and investigate a contrast-free ultrasound quantitative microvasculature imaging technique for detection of metastatic ALN in vivo. EXPERIMENTAL DESIGN: The proposed ultrasound-based technique, high-definition microvasculature imaging (HDMI) provides superb images of tumor microvasculature at sub-millimeter size scales and enables quantitative analysis of microvessels structures. We evaluated the new HDMI technique on 68 breast cancer patients with ultrasound-identified suspicious ipsilateral axillary lymph nodes recommended for fine needle aspiration biopsy (FNAB). HDMI was conducted before the FNAB and vessel morphological features were extracted, analyzed, and the results were correlated with the histopathology. RESULTS: Out of 15 evaluated quantitative HDMI biomarkers, 11 were significantly different in metastatic and reactive ALNs (10 with P << 0.01 and one with 0.01 < P < 0.05). We further showed that through analysis of these biomarkers, a predictive model trained on HDMI biomarkers combined with clinical information (i.e., age, node size, cortical thickness, and BI-RADS score) could identify metastatic lymph nodes with an area under the curve of 0.9 (95% CI [0.82,0.98]), sensitivity of 90%, and specificity of 88%. CONCLUSIONS: The promising results of our morphometric analysis of HDMI on ALNs offer a new means of detecting lymph node metastasis when used as a complementary imaging tool to conventional ultrasound. The fact that it does not require injection of contrast agents simplifies its use in routine clinical practice.

Heat Generation During Initial Osteotomy for Implant Site Preparation: An In Vitro Measurement Study.

Introduction: Controlling temperature generation during implant site preparation is important to prevent implant early failure. Aim: The present in vitro study aimed at measuring temperature variation generated during the initial osteotomy using both rotatory and piezo-surgical inserts. Methods: Nine groups were defined according to drill and insert type, cooling volume (mL/min) and cooling temperature. A total of 315 implant site preparations were performed in an artificial bone sample and the temperature was measured using an infrared camera. Drills' wear was assessed using scanning electron microscopy at baseline and after 10 and 35 utilizations. Results: Piezo-surgical insert groups determined a temperature increase that was significantly higher than the one generated by rotatory drills groups (p < 0.001). When considering rotatory drills groups a temperature ≥ 40 °C was never recorded. Conclusion: Lower saline temperature implied a significant temperature decrease (p < 0.001), while the increase in cooling volume did not imply a temperature decrease. The scanning electron microscopy analysis of the drills demonstrated that little drill wear occurred up to 35 utilization times.

Quantitative Biomarkers Derived from a Novel Contrast-Free Ultrasound High-Definition Microvessel Imaging for Distinguishing Thyroid Nodules.

Low specificity in current ultrasound modalities for thyroid cancer detection necessitates the development of new imaging modalities for optimal characterization of thyroid nodules. Herein, the quantitative biomarkers of a new high-definition microvessel imaging (HDMI) were evaluated for discrimination of benign from malignant thyroid nodules. Without the help of contrast agents, this new ultrasound-based quantitative technique utilizes processing methods including clutter filtering, denoising, vessel enhancement filtering, morphological filtering, and vessel segmentation to resolve tumor microvessels at size scales of a few hundred microns and enables the extraction of vessel morphological features as new tumor biomarkers. We evaluated quantitative HDMI on 92 patients with 92 thyroid nodules identified in ultrasound. A total of 12 biomarkers derived from vessel morphological parameters were associated with pathology results. Using the Wilcoxon rank-sum test, six of the twelve biomarkers were significantly different in distribution between the malignant and benign nodules (all p < 0.01). A support vector machine (SVM)-based classification model was trained on these six biomarkers, and the receiver operating characteristic curve (ROC) showed an area under the curve (AUC) of 0.9005 (95% CI: [0.8279,0.9732]) with sensitivity, specificity, and accuracy of 0.7778, 0.9474, and 0.8929, respectively. When additional clinical data, namely TI-RADS, age, and nodule size were added to the features, model performance reached an AUC of 0.9044 (95% CI: [0.8331,0.9757]) with sensitivity, specificity, and accuracy of 0.8750, 0.8235, and 0.8400, respectively. Our findings suggest that tumor vessel morphological features may improve the characterization of thyroid nodules.

Wearable Electrocardiography for Physical Activity Monitoring: Definition of Validation Protocol and Automatic Classification.

Wearable devices are rapidly spreading thanks to multiple advantages. Their use is expanding in several fields, from medicine to personal assessment and sport applications. At present, more and more wearable devices acquire an electrocardiographic (ECG) signal (in correspondence to the wrist), providing potentially useful information from a diagnostic point of view, particularly in sport medicine and in rehabilitation fields. They are remarkably relevant, being perceived as a common watch and, hence, considered neither intrusive nor a cause of the so-called "white coat effect". Their validation and metrological characterization are fundamental; hence, this work aims at defining a validation protocol tested on a commercial smartwatch (Samsung Galaxy Watch3, Samsung Electronics Italia S.p.A., Milan, Italy) with respect to a gold standard device (Zephyr BioHarness 3.0, Zephyr Technology Corporation, Annapolis, MD, USA, accuracy of ±1 bpm), reporting results on 30 subjects. The metrological performance is provided, supporting final users to properly interpret the results. Moreover, machine learning and deep learning models are used to discriminate between resting and activity-related ECG signals. The results confirm the possibility of using heart rate data from wearable sensors for activity identification (best results obtained by Random Forest, with accuracy of 0.81, recall of 0.80, and precision of 0.81, even using ECG signals of limited duration, i.e., 30 s). Moreover, the effectiveness of the proposed validation protocol to evaluate measurement accuracy and precision in a wide measurement range is verified. A bias of -1 bpm and an experimental standard deviation of 11 bpm (corresponding to an experimental standard deviation of the mean of ≈0 bpm) were found for the Samsung Galaxy Watch3, indicating a good performance from a metrological point of view.

Light-Controlled Rotational Speed of an Acoustically Levitating Photomobile Polymer Film.

In this work, we study the light-induced changes of the rotational speed of a thin photomobile film using a single-axis acoustic levitator operating at 40 kHz. In our experiments, a 50 µm thick photomobile polymer film (PMP) is placed in one of the nodes of a stationary acoustic field. Under the action of the field, the film remains suspended in air. By externally perturbing this stable equilibrium condition, the film begins to rotate with its natural frequency. The rotations are detected in real time by monitoring the light of a low power He-Ne laser impinging on and reflected by the film itself. During the rotational motion, an external laser source is used to illuminate the PMP film; as a consequence, the film bends and the rotational speed changes by about 20 Hz. This kind of contactless long-distance interaction is an ideal platform for the development and study of many electro-optics devices in microgravity and low-friction conditions. In particular, we believe that this technology could find applications in research fields such as 3D dynamic displays and aerospace applications.

Pharmacologic screen identifies active combinations with BET inhibitors and LRRK2 as a novel putative target in lymphoma.

Inhibitors of the Bromo- and Extra-Terminal domain (BET) family proteins have strong preclinical antitumor activity in multiple tumor models, including lymphomas. Limited single-agent activity has been reported in the clinical setting. Here, we have performed a pharmacological screening to identify compounds that can increase the antitumor activity of BET inhibitors in lymphomas. The germinal center B-cell like diffuse large B-cell lymphoma (DLBCL) cell lines OCI-LY-19 and WSU-DLCL2 were exposed to 348 compounds given as single agents at two different concentrations and in combination with the BET inhibitor birabresib. The combination partners included small molecules targeting important biologic pathways such as PI3K/AKT/MAPK signaling and apoptosis, approved anticancer agents, kinase inhibitors, epigenetic compounds. The screening identified a series of compounds leading to a stronger antiproliferative activity when given in combination than as single agents: the histone deacetylase (HDAC) inhibitors panobinostat and dacinostat, the mTOR (mechanistic target of rapamycin) inhibitor everolimus, the ABL/SRC (ABL proto-oncogene/SRC proto oncogene) inhibitor dasatinib, the AKT1/2/3 inhibitor MK-2206, the JAK2 inhibitor TG101209. The novel finding was the benefit given by the addition of the LRRK2 inhibitor LRRK2-IN-1, which was validated in vitro and in vivo. Genetic silencing demonstrated that LRRK2 sustains the proliferation of lymphoma cells, a finding paired with the association between high expression levels and inferior outcome in DLBCL patients. We identified combinations that can improve the response to BET inhibitors in lymphomas, and LRRK2 as a gene essential for lymphomas and as putative novel target for this type of tumors.

Accuracy and Precision of Wearable Devices for Real-Time Monitoring of Swimming Athletes.

Nowadays, the use of wearable devices is spreading in different fields of application, such as healthcare, digital health, and sports monitoring. In sport applications, the present trend is to continuously monitor the athletes' physiological parameters during training or competitions to maximize performance and support coaches. This paper aims to evaluate the performances in heart rate assessment, in terms of accuracy and precision, of both wrist-worn and chest-strap commercial devices used during swimming activity, considering a test population of 10 expert swimmers. Three devices were employed: Polar H10 cardiac belt, Polar Vantage V2, and Garmin Venu Sq smartwatches. The former was used as a reference device to validate the data measured by the two smartwatches. Tests were performed both in dry and wet conditions, considering walking/running on a treadmill and different swimming styles in water, respectively. The measurement accuracy and precision were evaluated through standard methods, i.e., Bland-Altman plot, analysis of deviations, and Pearson's correlation coefficient. Results show that both precision and accuracy worsen during swimming activity (with an absolute increase of the measurement deviation in the range of 13-56 bpm for mean value and 49-52 bpm for standard deviation), proving how water and arms movement act as relevant interference inputs. Moreover, it was found that wearable performance decreases when activity intensity increases, highlighting the need for specific research for wearable applications in water, with a particular focus on swimming-related sports activities.

A Pilot Cross-Sectional Study on Oral Health and Nutritional Status of Institutionalized Older Adults: A Focus on Sarcopenia.

The global population aged over 60 will double by 2050. This pilot cross-sectional study aims at evaluating nutritional and oral health status and the prevalence of sarcopenia in older adults living in an Italian residential aged care facility. Thirty-two adults aged ≥65 years were included. Individual sociodemographic data and nutritional and oral health data were collected. For sarcopenia diagnosis, muscle mass, physical performance, muscle strength and anthropometric parameters were recorded. Participants underwent a nutritional screening and a dental examination. Mini Nutritional Assessment and masticatory mixing ability test were performed. The results showed that men recorded a hand strength significantly higher than that of women, 25.5 ± 7.2 Kg vs. 12.8 ± 5.9 Kg (p < 0.01), respectively. Gait speed test showed that only 20.8% of the participants had a speed of more than 0.8 m/s. A strong negative correlation between masticatory performance and the number of missing teeth was detected (r = -0.84, 95% C.I. [-0.92; -0.69], p < 0.01). Overall, a high percentage of institutionalized older adults were diagnosed as being sarcopenic. Poor oral health in older adults is a major general health problem as it may restrict both food selection and nutrient intake, representing a risk factor for sarcopenia, although longitudinal studies are needed to confirm this relationship.

Wearable devices as a valid support for diagnostic excellence: lessons from a pandemic going forward.

Today, the use of wearable devices is continuously increasing with many different application fields. Their low-cost and wide availability make these devices proper instruments for long-term monitoring, potentially useful to detect physiological changes related to influenza or other viruses. The relevance of this aspect and the impact of such technology have become evident particularly in the last year, during COVID-19 emergency; (big) data from wearable devices (already worn by many citizens) together with artificial intelligence techniques gave birth to specific studies dedicated to quickly identify patterns discriminating between healthy and infected people. These evaluations are made on the basis of parameters measured by these devices, among which heart rate, physical activity, and sleep seem to play a dominant role. This could be extremely significant in terms of early detection and limit of contagion risk. However, there is still a lot of research to be conducted in terms of measurement accuracy, data management (privacy and security issues), and results exploitation, in order to reach an accurate and reliable solution helping the whole healthcare system particularly in epidemic events, such as the SARS-CoV-2 pandemic.

Heartbeat Detection by Laser Doppler Vibrometry and Machine Learning.

Background: Heartbeat detection is a crucial step in several clinical fields. Laser Doppler Vibrometer (LDV) is a promising non-contact measurement for heartbeat detection. The aim of this work is to assess whether machine learning can be used for detecting heartbeat from the carotid LDV signal. Methods: The performances of Support Vector Machine (SVM), Decision Tree (DT), Random Forest (RF) and K-Nearest Neighbor (KNN) were compared using the leave-one-subject-out cross-validation as the testing protocol in an LDV dataset collected from 28 subjects. The classification was conducted on LDV signal windows, which were labeled as beat, if containing a beat, or no-beat, otherwise. The labeling procedure was performed using electrocardiography as the gold standard. Results: For the beat class, the f1-score (f1) values were 0.93, 0.93, 0.95, 0.96 for RF, DT, KNN and SVM, respectively. No statistical differences were found between the classifiers. When testing the SVM on the full-length (10 min long) LDV signals, to simulate a real-world application, we achieved a median macro-f1 of 0.76. Conclusions: Using machine learning for heartbeat detection from carotid LDV signals showed encouraging results, representing a promising step in the field of contactless cardiovascular signal analysis.

Development of a Novel Medical Device for Mucositis and Peri-Implantitis Treatment.

In spite of all the developments in dental implantology techniques, peri-implant diseases are frequent (prevalence up to 80% and 56% of subjects for mucositis and peri-implantitis, respectively) and there is an urgency for an effective treatment strategy. This paper presents an innovative electromedical device for the electromagnetic treatment of mucositis and peri-implantitis diseases. This device is also equipped with a measurement part for bioimpedance, which reflects the health conditions of a tissue, thus allowing clinicians to objectively detect impaired areas and to monitor the severity of the disease, evaluate the treatment efficacy, and adjust it accordingly. The design of the device was realized considering literature data, clinical evidence, numerical simulation results, and electromagnetic compatibility (EMC) pre-compliance tests, involving both clinicians and engineers, to better understand all the needs and translate them into design requirements. The reported system is being tested in more than 50 dental offices since 2019, providing efficient treatments for mucositis and peri-implantitis, with success rates of approximately 98% and 80%, respectively.

Copanlisib synergizes with conventional and targeted agents including venetoclax in B- and T-cell lymphoma models.

Copanlisib is a pan-class I phosphoinositide 3-kinase (PI3K) inhibitor with preferred activity toward PI3Kα and PI3Kδ. Despite the clear overall clinical benefit, the number of patients achieving complete remissions with the single agent is relatively low, a problem shared by the vast majority of targeted agents. Here, we searched for novel copanlisib-based combinations. Copanlisib was tested as a single agent, in combination with an additional 17 drugs in 26 cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and T-cell lymphomas. In vivo experiments, transcriptome analyses, and immunoblotting experiments were also performed. Copanlisib as a single agent showed in vitro dose-dependent antitumor activity in the vast majority of the models. Combination screening identified several compounds that synergized with copanlisib. The strongest combination was with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax. The benefit of the combination over single agents was also validated in an MZL xenograft model and in MCL primary cells, and was due to increased induction of apoptosis, an effect likely sustained by the reduction of the antiapoptotic proteins myeloid cell leukemia 1 (MCL1) and BCL-XL, observed in MCL and MZL cell lines, respectively. These data supported the rationale for the design of the Swiss Group for Clinical Cancer Research (SAKK) 66/18 phase 1 study currently exploring the combination of copanlisib and venetoclax in relapsed/refractory lymphomas.

Experimental Assessment of Sleep-Related Parameters by Passive Infrared Sensors: Measurement Setup, Feature Extraction, and Uncertainty Analysis.

A simple sleep monitoring measurement method is presented in this paper, based on a simple, non-invasive motion sensor, the Passive InfraRed (PIR) motion sensor. The easy measurement set-up proposed is presented and its performances are compared with the ones provided by a commercial, ballistocardiographic bed sensor, used as reference tool. Testing was conducted on 25 nocturnal acquisitions with a voluntary, healthy subject, using the PIR-based proposed method and the reference sensor, simultaneously. A dedicated algorithm was developed to correlate the bed sensor outputs with the PIR signal to extract sleep-related features: sleep latency (SL), sleep interruptions (INT), and time to wake (TTW). Such sleep parameters were automatically identified by the algorithm, and then correlated to the ones computed by the reference bed sensor. The identification of these sleep parameters allowed the computation of an important, global sleep quality parameter: the sleep efficiency (SE). It was calculated for each nocturnal acquisition and then correlated to the SE values provided by the reference sensor. Results show the correlation between the SE values monitored with the PIR and the bed sensor with a robust statistic confidence of 4.7% for the measurement of SE (coverage parameter k = 2), indicating the validity of the proposed, unobstructive approach, based on a simple, small, and low-cost sensor, for the assessment of important sleep-related parameters.

Dataset from spirometer and sEMG wireless sensor for diaphragmatic respiratory activity monitoring.

We introduce a dataset to provide insights into the relationship between the diaphragm surface electromyographic (sEMG) signal and the respiratory air flow. The data presented had been originally collected for a research project jointly developed by the Department of Information Engineering and the Department of Industrial Enginering and Mathematical Sciences, Polytechnic University of Marche, Ancona, Italy. This article describes data recorded from 8 subjects, and includes 8 air flow and 8 surface electromyographic (sEMG) signals for diaphragmatic respiratory activity monitoring, measured with a sampling frequency of 2 kHz.

A Smart Sensing Architecture for Domestic Monitoring: Methodological Approach and Experimental Validation.

Smart homes play a strategic role for improving life quality of people, enabling to monitor people at home with numerous intelligent devices. Sensors can be installed to provide a continuous assistance without limiting the resident's daily routine, giving her/him greater comfort, well-being and safety. This paper is based on the development of domestic technological solutions to improve the life quality of citizens and monitor the users and the domestic environment, based on features extracted from the collected data. The proposed smart sensing architecture is based on an integrated sensor network to monitor the user and the environment to derive information about the user's behavior and her/his health status. The proposed platform includes biomedical, wearable, and unobtrusive sensors for monitoring user's physiological parameters and home automation sensors to obtain information about her/his environment. The sensor network stores the heterogeneous data both locally and remotely in Cloud, where machine learning algorithms and data mining strategies are used for user behavior identification, classification of user health conditions, classification of the smart home profile, and data analytics to implement services for the community. The proposed solution has been experimentally tested in a pilot study based on the development of both sensors and services for elderly users at home.

Measurement of the 100 MHz EMF radiation in vivo effects on zebrafish D. rerio embryonic development: A multidisciplinary study.

The augmented exposure of both environment and human being to electromagnetic waves and the concomitant lack of an unequivocal knowledge about biological consequences of these radiations, raised public interest on electromagnetic pollution. In this context, the present study aims to evaluate the biological effects on zebrafish (ZF) embryos of 100 MHz radiofrequency electromagnetic field (RF-EMF) exposure through a multidisciplinary protocol. Because of the shared synteny between human and ZF genomes that validated its use in biomedical research, toxicology and developmental biology studies, ZF was here selected as experimental model and a measurement protocol and biological analyses have been set up to clearly discriminate between RF-EMF biological and thermal effects. The results showed that a 100 MHz EMF was able to affect ZF embryonic development, from 24 to 72 h post fertilization (hpf) in all the analyzed pathways. Particularly, at the 48 hpf stage, a reduced growth, an increased transcription of oxidative stress genes, the onset of apoptotic/autophagic processes and a modification in cholesterol metabolism were detected. ZF embryos faced stress induced by EMF radiation by triggering detoxification mechanisms and at 72 hpf they partially recovered from stress reaching the hatching time in a comparable way respect to the control group. Data here obtained showed unequivocally the in vivo effects of RF-EMF on an animal model, excluding thermal outcomes and thus represents the starting point for more comprehensive studies on dose response effects of electromagnetic fields radiations consequences.

Bioimpedancemetry for the assessment of periodontal tissue inflammation: a numerical feasibility study.

In dentistry possible inflammatory episodes of oral cavity can be very frequent (periodontitis, mucositis, peri-implantitis) and they can have serious consequences. Indeed, peri-implantitis is still the principal cause of implant failure. Impedance values of biological tissues are related to the physiological/pathological state of the tissue itself. In fact, an inflamed site exhibits an impedance value lower than that of the corresponding healthy tissue. Based on these observations, the aim of this work is to determine if impedancemetric measurements are able to provide information about the inflammatory state of tissues. A numerical 3D model has been realized to simulate the measurement conditions present in the event of inflammation around a dental implant. The aim is to understand if it is possible to determine the presence of an inflamed tissue and to locate its site, so that the treatment could be specifically focused in that specific area. A simplified geometry reproducing the implant has been realized in order to validate the numerical model by means of experimental measurements. The obtained results are satisfactorily accurate, so the model can be considered reliable. Therefore, multiple simulations have been run on the original model to carry out a parametric study in terms of different conductivity values, different volumes of inflamed tissues and different measurement frequencies. The advantages and limits of such a method have been shown to properly define the main constraints for the system design.

An Electromagnetic Sensor for the Autonomous Running of Visually Impaired and Blind Athletes (Part I: The Fixed Infrastructure).

Sport is one of the best ways to promote the social integration of people affected by physical disability, because it helps them to increase their self-esteem by facing difficulties and overcoming their disabilities. Nowadays, a large number of sports can be easily played by visually impaired and blind athletes without any special supports, but, there are some disciplines that require the presence of a sighted guide. In this work, the attention will be focused on marathons, during which athletes with visual disorders have to be linked to the sighted guide by means of a non-stretchable elbow tether, with an evident reduction of their performance and autonomy. In this context, this paper presents a fixed electromagnetic infrastructure to equip a standard running racetrack in order to help a blind athlete to safely run without the presence of a sighted guide. The athlete runs inside an invisible hallway, just wearing a light and a comfortable sensor unit. The patented system has been homemade, designed, realized and finally tested by a blind Paralympic marathon champion with encouraging results and interesting suggestions for technical improvements. In this paper (Part I), the transmitting unit, whose main task is to generate the two magnetic fields that delimit the safe hallway, is presented and discussed.

An Electromagnetic Sensor for the Autonomous Running of Visually Impaired and Blind Athletes (Part II: The Wearable Device).

Currently, the availability of technology developed to increase the autonomy of visually impaired athletes during sports is limited. The research proposed in this paper (Part I and Part II) focuses on the realization of an electromagnetic system that can guide a blind runner along a race track without the need for a sighted guide. In general, the system is composed of a transmitting unit (widely described in Part I) and a receiving unit, whose components and main features are described in this paper. Special attention is paid to the definition of an electromagnetic model able to faithfully represent the physical mechanisms of interaction between the two units, as well as between the receiving magnetic sensor and the body of the user wearing the device. This theoretical approach allows for an estimation of the signals to be detected, and guides the design of a suitable signal processing board. This technology has been realized, patented, and tested with a blind volunteer with successful results and this paper presents interesting suggestions for further improvements.