Marko Ušaj, Katja Trontelj, Rosana Hudej, Maša Kandušer and Damijan Miklavčič
Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization
Background. Various electrofusion parameters have to be adjusted to obtain the optimal electrofusion efficiency. Based on published data, good electrofusion conditions can be achieved with the hypotonic treatment. However, the duration of the hypotonic treatment before electroporation and buffer hypoosmolarity have to be adjusted in order to cause cell swelling, to avoid regulatory volume decrease and to preserve cell viability. The aims of our study were to determine cell size dynamics and viability of four different cell lines in hypotonic buffer and to study the influence of the electroporation on the selected cell line in hypotonic buffer.
Materials and methods. Cell size dynamics of different cell lines exposed to hypotonic buffer and electroporation were analyzed by time-resolved cell size measurements. The viability of hypotonically treated or/and electroporated cells was determined 24 h after the experiment by a modified crystal violet (CV) viability assay.
Results. In our experimental conditions the hypotonic treatment at 100 mOsm was efficient for CHO, V79 and B16-F1 cell lines. The optimal duration of the treatment was between two and five minutes. On the other hand the same hypotonic treatment did not cause cell swelling of NS1 cells. Cell swelling was also observed after electroporation of B16-F1 in isotonic buffer and it was amplified when hypotonic buffer was used. In addition, the regulatory volume decrease was successfully inhibited with electroporation.
Conclusions. Cell size dynamics in hypotonic conditions should be studied for each cell line since they differ in their sensitivity to the hypotonic treatment. The inhibition of cell regulatory volume decrease by electroporation may be beneficial in achieving higher electrofusion efficiency. The hypotonic treatment in itself did not significantly affect the cell viability; however, electric field parameters for electroporation should be carefully selected taking into account the hypotonically induced volume increase of cells.
Gonadal function in patients treated for Hodgkin's disease in childhood
Background. The long-term survival of patients treated for Hodgkin's disease (HD) in childhood is high and the chief concern is now being directed toward the late effects of the treatment, including the endocrine dysfunction.
Patients and methods. Testicular and ovarian functions were assessed in 64 long term survivors (24 females, 40 males) treated for HD in childhood in Slovenia between 1972 and 1994. At diagnosis they were 3-16 years old and had gonadal evaluation 4-27 years later at the age of 13-34. Fifty-four (84%) patients received chemotherapy (ChT), 49 in combination with radiation therapy (RT), 10 received RT alone. Gonadal function was assessed by the clinical examination and measurement of serum concentrations of estradiol and testosterone. Serum levels of LH and FSH were determined in the basal state and after the stimulation.
Results. Primary hypogonadism (PH) was found in 30 (47%) patients. Twenty-four of 40 (60%) males had PH with evidence of damage of germinal epithelium, 4 of them had evidence of damage of Leydig cells (LC) and 10 had evidence of dysfunction of LC as well. PH was found in 6 of 24 (25%) females.
Conclusions. After therapy for HD PH was more frequent in males than in females. Not only RT but also alkylating agents and procarbazine alone caused damage of LC. Age of patient at the time of treatment was not an important risk factor for gonadal toxicity. Pelvic RT in combination with ChT is the most important risk factor of the development PH both, in males and females.
Janez Podobnik, Igor Kocijancic, Viljem Kovac and Igor Sersa
3T MRI in evaluation of asbestos-related thoracic diseases - preliminary results
Disclosure No potential conflicts of interest were disclosed. Background. 3T high-field magnetic resonance imaging (MRI) scanners have recently become available for the clinical use and are being increasingly applied in the field of whole-body imaging and chest imaging as well. The aim of this study was to evaluate the diagnostic potential of 3 T MRI as a complementary imaging modality to CT in detecting the pathological changes of asbestos-related thoracic diseases.
Patients and methods. Fifteen patients with the asbestos-related thoracic disease were scheduled for 3T MRI. Five had a benign form of the disease and 10 had malignant pleural mesothelioma (MPM). From the patients with a benign form of the disease their last CT examination in digital form was acquired and patients with MPM were scheduled for CT examination with contrast media. The protocol of MR imaging consists of T2-weighted cardiac-gated breathhold turbo spin echo (TSE) sequences in coronal, sagittal and axial plane and T1-weighted cardiac-gated breath-hold TSE black blood in axial plane. In T2-weighted sequences in axial plane, fat saturation was also used. CT examinations were obtained with the administration of the contrast medium from lung apices to the lower end of the liver. Images of 5 mm (mediastinum window) and 3 mm (lung window) in axial plan were reconstructed. MRI signal intensity of lesions and adjacent muscles on Syngo MultiModality Work Place were measured.
Results. Compared to muscles pleural plaques appeared hypo-intense to iso-intense on T1 weighted images (in 100%) and also hypo-intense on T2 fs-weighted images (in 100%). MPM appeared inhomogeneous hypo-intense to iso-intense on T1-weighted and hyperintense on T2 fs-weighted images in all patients (100%).
Conclusions. These preliminary results pointed out that MRI was equal or even better compared with CT examination for detecting possible malignant potential of pleural changes in the asbestos-related pleural disease, using signal intensity measurements of T2 fs-weighted images. The 3T MRI enabled the accurate determination of chest pathology and it could be used for imaging of patients with the asbestos-related thoracic disease. MRI is particularly valuable because a patient is not exposed to the harmful radiation which is important if imaging methods are used repeatedly, like in screening programs or in monitoring of treatment results. This finding turned us to propose 3T MRI imaging technique as a non-ionizing imaging method for the follow-up of patients with the isolated pleural form of the asbestos-related disease.
Renata Rezonja Kukec, Iztok Grabnar, Tomaz Vovk, Ales Mrhar, Viljem Kovac and Tanja Cufer
Background. Chemotherapy with platinum agent and etoposide for small-cell lung cancer (SCLC) is supposed to be associated with intermediate risk (10-20%) of febrile neutropenia. Primary prophylaxis with granulocyte colonystimulating factors (G-CSFs) is not routinely recommended by the treatment guidelines. However, in clinical practice febrile neutropenia is often observed with standard etoposide/platinum regimen. The aim of this analysis was to evaluate the frequency of neutropenia and febrile neutropenia in advanced SCLC patients in the first cycle of standard chemotherapy. Furthermore, we explored the association between severe neutropenia and etoposide peak plasma levels in the same patients.
Methods. The case series based analysis of 17 patients with advanced SCLC treated with standard platinum/etoposide chemotherapy, already included in the pharmacokinetics study with etoposide, was performed. Grade 3/4 neutropenia and febrile neutropenia, observed after the first cycle are reported. The neutrophil counts were determined on day one of the second cycle unless symptoms potentially related to neutropenia occurred. Adverse events were classified according to Common Toxicity Criteria 4.0. Additionally, association between severe neutropenia and etoposide peak plasma concentrations, which were measured in the scope of pharmacokinetic study, was explored.
Results. Two out of 17 patients received primary GCS-F prophylaxis. In 15 patient who did not receive primary prophylaxis the rates of both grade 3/4 neutropenia and febrile neutropenia were high (8/15 (53.3%) and 2/15 (13.3%), respectively), already in the first cycle of chemotherapy. One patient died due to febrile neutropenia related pneumonia. Neutropenic events are assumed to be related to increased etoposide plasma concentrations after a standard etoposide and cisplatin dose. While the mean etoposide peak plasma concentration in the first cycle of chemotherapy was 17.6 mg/l, the highest levels of 27.07 and 27.49 mg/l were determined in two patients with febrile neutropenia.
Conclusions. Our study indicates that there is a need to reduce the risk of neutropenic events in chemotherapy treated advanced SCLC, starting in the first cycle. Mandatory use of primary G-CSF prophylaxis might be considered. Alternatively, use of improved risk models for identification of patients with increased risk for neutropenia and individualization of primary prophylaxis based on not only clinical characteristics but also on etoposide plasma concentration measurement, could be a new, promising options that deserves further evaluation.
Gabrijela Brzan Simenc, Jana Ambrozic, Katja Prokselj, Natasa Tul, Marta Cvijic, Tomislav Mirkovic, Helmut Karl Lackner and Miha Lucovnik
to adequately correct tissue hypoperfusion. 12 , 13 A quick, non-invasive, bedside test to assess fluid status of patients with preeclampsia would, therefore, be very helpful to clinicians working in obstetric units.
Ultrasound measurement of optic nerve sheath diameter (ONSD) has been described as a simple and reliable means of determining increased intracranial pressure due to cerebral edema in non-pregnant critically ill patients. 14 , 15 Signs of cerebral edema have been reported on magnetic resonance imaging in 71% to 100% of patients with preeclampsia
compression, is indicated in the lower right (B) The blue and green areas are shown mixed together in relatively equal percentages in the placenta (C) Most of the placenta is indicated in blue. In the left image, the strain ratio is calculated from the region of interest of the fat and placental tissue. In this case, the placental index is 19.83.
We took measurements by avoiding large blood vessels and thereby avoided bias caused by areas without echo. The ROI was selected within 10 cm from the body surface; areas without tissue elasticity, such as the deep part
Effect of response quality and line of treatment with rituximab on overall and disease-free survival of patients with B-cell lymphoma
Background. The introduction of rituximab into the treatment of patients with non-Hodgkin's lymphomas has improved the overall response rate, as well as the response duration and the overall survival of patients with B-cell lymphomas. But only a few studies have addressed the question whether the better response (complete response) and the early introduction of rituximab into the treatment translate into the better survival. The aim of this retrospective study was to assess the potential relationship between either the quality of the response or the line of the rituximab treatment and the overall survival (OS) as well as the disease-free survival (DFS) of patients with B-cell lymphomas.
Patients and methods. In the study, we analysed treatment outcomes in patients with different histological types of B-cell lymphomas who were treated at the Institute of Oncology between 2003 and 2007 with rituximab and chemotherapy. We included only patients who had the level of CD20 expression assessed prior to the introduction of the treatment with quantitative flow-cytometric measurements. The OS and DFS were evaluated by Kaplan-Meier survival curves.
Results. One hundred and fourteen patients were enrolled in the study. Patients who achieved a complete response after the rituximab containing treatment had a significantly longer OS than those reaching a partial response (hazard ratio [HR], 0.34; 95% CI, 0.05 to 0.91, P = 0.0375) and than patients with stable (hazard ratio [HR], 0.11; 95% CI, 0.0002 to 0.033, P < 0.0001) or progressive disease (hazard ratio [HR], 0.09; 95% CI, 0.003 to 0.03, P < 0.0001). Patients who achieved a complete response (CR; n = 70; 61.4%) had also a significantly longer DFS (hazard ratio [HR], 0.26; 95% CI, 0.021 to 0.538, P = 0.0068) than those reaching only a partial response (PR; n = 17; 14.9%). Patients treated with rituximab as the first-line treatment (n = 50; 43.9%) had a significantly longer OS than those treated with rituximab for the first (hazard ratio [HR], 0.27; 95% CI, 0.106 to 0.645, P = 0.0036) or second relapse (hazard ratio [HR], 0.22; 95% CI, 0.078 to 0.5, P = 0.0006). Also the DFS of patients treated with rituximab as the first-line treatment (n = 46; 52.9%) was significantly longer (hazard ratio [HR], 0.32; 95% CI, 0.088 to 0.9, P = 0.0325) than in patients treated with rituximab for their first relapse (n = 25; 28.7%).
Conclusions. These data indicate that a better response to rituximab therapy presumably translates into an improved OS and DFS for patients with B-cell lymphomas. The early introduction of rituximab into the treatment (i.e. first-line treatment) might improve OS. Therefore, the response adapted first-line therapy with rituximab should be considered when the treatment decision is taken in B-cell lymphoma patients.
Grzegorz Zwierzchowski, Grzegorz Bieleda and Janusz Skowronek
Radiochromic film dosimetry with gamma analysis is widely used in quality assurance procedures for external beam radiotherapy (EBRT). The main advantage of this method is the ability to collect dosimetric data with very high planar resolution in contrast to other methods that are based on point dose measurements.
Introducing fast and easily repeatable methods for commissioning procedures for brachytherapy (BT) treatment planning systems (TPS) is a complex undertaking in most cases. 1 , 2 The relatively low energy range of BT sources induce
Matej Podbregar, Ana Ursula Gavric, Eva Podbregar, Hugon Mozina and Sebastian Stefanovic
acquired from the Blood Transfusion Centre of Slovenia. The patients were transfused with two units of RBCs with maximal age difference of 3 days.
Near-infrared spectroscopy measurements and analysis
The thenar skeletal muscle StO 2 and THb concentrations were measured with tissue spectrometer (Equanox 7600; Nonin Medical, Minnesota, USA). The electrode (8004CA, Equanox Advance Sensor, Nonin Medical) was placed on the thenar eminence to measure the maximum resting StO 2 . During measurements, there were no additional treatment procedures in place, except for the