Oxygen consumption rate of Caenorhabditis elegans as a high-throughput endpoint of toxicity testing using the Seahorse XFe96 Extracellular Flux Analyzer.

Caenorhabditis elegans presents functioning, biologically relevant phenotypes and is frequently used as a bioindicator of toxicity. However, most C. elegans in vivo effect-assessment methods are laborious and time consuming. Therefore, we developed a novel method to measure the oxygen consumption rate of C. elegans as a sublethal endpoint of toxicity. This protocol was tested by exposing 50 larval stage one C. elegans individuals for 48 h (at 20 °C) to different concentrations of two toxicants i.e. benzylcetyldimethylammonium chloride (BAC-C16) and cadmium (Cd). Following exposures, the oxygen consumption rate of the C. elegans individuals were measured using the high-throughput functionality of the Seahorse XFe96 Extracellular Flux Analyzer. Dose-response curves for BAC-C16 (R2 = 0.93; P = 0.001) and Cd (R2 = 0.98; P = 0.001) were created. Furthermore, a strong, positive correlation was evidenced between C. elegans oxygen consumption rate and a commonly used, ecologically relevant endpoint of toxicity (growth inhibition) for BAC-C16 (R2 = 0.93; P = 0.0001) and Cd (R2 = 0.91; P = 0.0001). The data presented in this study show that C. elegans oxygen consumption rate can be used as a promising functional measurement of toxicity.

[Five-year results of IMRT for prostate cancer - toxicity]. / Petileté výsledky IMRT karcinomu prostaty - toxicita.

BACKGROUND: Intensity modulated radiotherapy (IMRT) plays a crucial role in the treatment of prostate cancer thanks to its capacity for healthy tissue sparing. This work reports on the acute and late toxicity rates among 233 patients treated with high-dose IMRT. MATERIAL AND METHODS: From June 2003 to December 2007, 233 men with clinically localized prostate cancer underwent radical radiotherapy. One hundred sixty patients were treated with IMRT to the prostate and the base of seminal vesicles to 78 Gy in 39 fractions, 73 patients underwent simultaneous integrated boost. Prescribed doses were 82 Gy and 73,8 Gy in 41 fractions to the prostate and seminal vesicles, respectively. Late toxicity was evaluated prospectively using a RTOG/FC-LENT score. RESULTS: Thirty patients (12.8%) experienced acute Grade 2 gastrointestinal (GI) toxicity. No acute Grade 3 or 4 GI toxicity developed. Forty two patients (18.1%) experienced acute Grade 2 genitourinary toxicity and 23 patients (9.9%) had Grade 3 GU toxicity. Grade 4 Genitourinary toxicity was observed in nine (3.8%) patients, due to a need of short-term urinary catheterization. With a median follow-up of 49.2 months, the estimated 5-year cumulative incidence of Grade 2 gastrointestinal toxicity was 22.4%. The estimated 5-year cumulative incidence of Grade 2 genitourinary toxicity was 17.7%. CONCLUSION: Intensity modulated radiotherapy enables dose escalation to 78-82 Gy with an acceptable toxicity.

[Five-year results of IMRT for prostate cancer - tumor control]. / Petileté výsledky IMRT karcinomu prostaty - kontrola nádoru.

BACKGROUND: Intensity-modulated radiation therapy (IMRT) is the method of choice in external-beam radiotherapy tolocalized prostate cancer. This work analyses five year results of IMRT with a dose of 78/82 Gy. PATIENTS AND METHODS: From June 2003 to December 2007, the IMRT technique was employed to treat 233 patients with T1-3 N0 M0 prostate cancer. It was supplemented by hormone therapy especially in high-risk patients. Two IMRT techniques were applied - IMRT with a dose of 78 Gy in 39 fractions to prostate and seminal vesicles (SV) (IMRT 78) and IMRT with simultaneous integrated 82 Gy boost to prostate concurrently with 73,8 Gy in 41 fractions to SV (IMRT SIB 82). The IMRT 78 technique was used in 160 patients (69%). Seventy-three (31%) patients with intermediate (IR) or high-risk (HR) prostate cancer without SV involvement were treated with IMRT SIB 82 technique. The PSA relapse was defined as an increase in PSA of at least 2.0 ng/mL above the nadir or in comparison to the value at the initiation of hormone therapy. Clinical relapse was defined as an occurence of distant metastases and/or local recurrence. RESULTS: The median follow-up of our patients´ population was 4.3 years (range 0.6-8.9 years). The estimated 5-year PSA relapse-free survival in low-risk (LR), IR and HR patients was 86%, 89% and 83%, respectively (p = NS). In a multivariate analysis, Gleason score (GS) 8-10 was associated with significantly higher risk of PSA relapse (RR 2.76), while higher age at the time of diagnosis significantly decreased the PSA relapse risk (RR 0.94). The estimated 5-year clinical relapse-free survival in LR, IR and HR patients was 100%, 99% and 95%, respectively (p = NS). In a univariate analysis, both GS and PSA had a significant impact on the 5-year clinical relapse-free survival - GS 2-7 97 % vs GS 8-10 88 % (p = 0.03), PSA 20 98 % vs PSA > 20 85 % (p < 0.01). CONCLUSION: Treatment of localized prostate cancer using IMRT with a dose 78/82 Gy yielded an excellent 5-year tumour control with a risk of clinical relapse being less than 5%.

Identification and Characterization of Pale Potato Cyst Nematode (Globodera pallida) in Teplá, the Czech Republic.

Potato cyst nematode poses a significant threat to potato producers in the Czech Republic. Both species of potato cyst nematodes (Globodera rostochiensis and G. pallida) are listed as quarantine pests in the Czech Republic and also by the European Union, European and Mediterranean Plant Protection Organization, and North American Plant Protection Organization. To date, G. rostochiensis was responsible for all damage to potatoes caused by cyst nematodes in the Czech Republic, while G. pallida was recorded only once in the Czech Republic (2) 8 years ago. It is important to note that this occurrence of G. pallida was not located in the free-cultivation area. In July 2011, soil samples from a potato field located in the area of Teplá (Karlovy Vary Region) were collected, and the cysts extracted were identified as G. pallida according to microscopic observation of cyst fenestra and morphology of juveniles (1). Cyst morphometrics (means from 10 cysts) included: fenestra diameter 21.2 µm, distance fenestra to anus 56.8 µm, Granek's ratio 2.7, number of cuticular ridges between fenestra and anus 14; while second stage juvenile morphometrics (means of 13 specimens) were: L 466.7 µm, stylet 24.2 µm, tail 53.2 µm, body width at anus 13.2, h 28.6, c 8.8, c' 4.0. Terminus of juvenile tails was rounded, and stylet knobs possessed distinct forward projections. Total DNA was extracted from single cysts using the TriPure reagent (Roche), and the DNA samples were used to amplify cistron rDNA with the following primers: 18S, 5'-TTGATTAGGTCCCTGCCCTTT-3', and 21S, 5'-TTTCACTCGCCGTTACTAAGG-3'. The amplified region contains the 3' end of the 18S gene, ITS1, 5.8S, ITS2, and the 5' end of the 28S gene. Pfu DNA polymerase (Fermentas) was used for accurate amplification. A PCR product of approximately 1.0 kb was amplified from three individual cysts. The PCR amplicons were cloned into pJET1.2 using the CloneJET PCR Cloning Kit (Fermentas) and sequenced in both directions. The sequences of representative isolates were deposited in GenBank (Accession Nos. JQ692592, JQ692593, and JQ692594). The resultant 1.0 kb sequences showed 99% nucleotide identity to sequences of G. pallida from Canada (GenBank Accession Nos. GQ294522.1, GQ355975.1, and GQ294523.1), thus confirming the results of the morphological analyses. To our knowledge, this is the first detection of G. pallida in the free-cultivation area of Karlovy Vary Region, and only the second in the Czech Republic since the first report in 2003 (2). References: (1) M. W. Brzeski. Page 237 in: Nematodes of Tylenchina in Poland and Temperate Europe. Muzeum I Instytut Zoologii Polska Akademia Nauk, Warszawa 1998. (2) M. Zouhar et al. Plant Disease, 87:98, 2003.

First Report of Polymyxa graminis f. sp. temperata, a Vector of Soilborne Cereal Viruses in the Czech Republic.

Polymyxa graminis L. is an eukaryotic, obligate, biotrophic parasite of plant roots (1), which belongs to a poorly studied, discrete, taxonomic unit informally called the 'plasmodiophorids'. P. graminis has the ability to acquire and transmit a range of soilborne viruses that belong to at least three separate genera and can cause economically significant diseases in cereal crops. For example, the winter barley disease caused by Barley yellow mosaic virus (BaYMV) and/or Barley mild mosaic virus (BaMMV) is widespread in Europe, Japan, and China; yield losses of >50% may occur when susceptible barley cultivars are grown on severely infested soils (2). Monitoring for P. graminis was started in the Czech Republic in May 2008. Fifty-six soil samples were collected from different localities of cereal production (wheat and barley) in the Královéhradecký Region (eastern Czech Republic). Soil from each sample was placed in five replicate pots (12 × 12 cm) in a greenhouse at 22 to 25°C. Seeds of barley cv. Florian were sown into the soil (10 seeds per pot). Negative control soil (noninfested soils from the Czech Republic) and positive control soil (known P. graminis-infested soil from Germany) were also planted to barley in five replicate pots. After 90 days, plants were collected and the roots were washed thoroughly in sterilized water and examined with a light microscope without staining. The fungus was identified as P. graminis on the basis of morphology of resting spores (cystosori) and sporangia and the size of individual cystosori (4 to 5 µm in diameter) according to Thouvenel et al. (3). Cystosori of P. graminis were observed in the roots of plants grown in 20 of the 56 soil samples, especially the samples from Ceské Mezirici. The presence of P. graminis in the roots of plants grown in the soil samples and the positive control sample versus the absence of the vector in roots of plants in the negative control soil was verified by PCR assay with DNA extracts and the Psp1 and Psp2rev primers according to Legrève et al. (Page 40 in: Proceedings of the Fifth Symposium of the International Working Group on Plant Viruses with Fungal Vectors, 2003). The PCR assay included denaturation at 94°C for 2 min, then 35 cycles including denaturation of 30 s at 94°C, annealing at 60°C for 1 min, and elongation at 72°C for 35 s. A final elongation was completed at 72°C for 7 min. To characterize the P. graminis isolates, the amplified PCR product (a DNA fragment of 472 bp) was sequenced and blasted for each of the samples that tested positive. These sequences were aligned with a known sequence (GenBank Accession No. AM259276) for P. graminis. The sequences from P. graminis on barley were 100% homologous to the published sequence of P. graminis f. sp. temperata. To our knowledge, this is the first report of P. graminis f. sp. temperata in the Czech Republic. References: (1) G. A. Ledingham. Can. J. Res. 17:50, 1939. (2) R. T. Plumb et al. Plant Pathol. 35:314, 1986. (3) J. C. Thouvenel et al. Plant Dis. 64:957, 1980.

Time course of late rectal toxicity after radiation therapy for prostate cancer.

Rectum and bladder are the crucial organs at risk for curative radiation therapy of localized prostate cancer. We analyzed the incidence, profile and time course of late rectal radiation toxicity. A total of 320 patients with T1-3 prostate cancer were treated with three-dimensional conformal radiation therapy (3D-CRT). The prescription dose was 70 Gy for T1 and T2 patients (n=230) and 74 Gy for patients with locally advanced T3 tumors (n=90). Late rectal toxicity was graded according to the Fox Chase modification of the Radiation Therapy Oncology Group (RTOG) and Late Effects Normal Tissue Task Force (LENT) criteria. The median follow-up time was 6.2 years (range 0.2-10.7 years). At 5 years, the risk for the development of grade 2 and 3 rectal toxicities was 15.6 and 7.0%, respectively. All new cases of grade 2 and 3 rectal toxicities were observed within 5 years after treatment. Prevalence of grade 2 and 3 rectal symptoms showed fluctuation with maximum at 1.5 years and the minor peak at 4.5 years. Toxicity profile changed significantly over time. The proportion of rectal bleeding within grade 2 and 3 toxicity decreased from 85% at 1.5 years to 46% at 4.5 years. Conversely, the proportion of fecal incontinence among grade 2 and 3 rectal symptoms gradually increased (0% at 1.5 years vs 27% at 4.5 years). Late rectal radiation toxicity represents a dynamic process. Rectal bleeding decreases and fecal incontinence increases over time.

Intensive cyclic chemotherapy and transplantation of autologous peripheral blood progenitor cells (PBPC) or whole blood in high-risk breast cancer - follow up at 10 years.

AIM: The main aim of our paper is to contribute to objectification of currently widely discussed results of overall survival (OS), disease free survival (DFS) and time from relapse to tumor progression (TTP) in women with breast cancer. METHODS: Forty consecutive patients fulfilling the eligibility criteria were admitted to the study. Fifty-six women were included in the control group. All patients received 6 cycles of adjuvant intensive cyclic combined chemotherapy with epirubicin 150 mg/m(2) and cyclophosphamide 1250 mg/m(2) (EC) applied each 14 days. To overcome haematological toxicity transplantations of autologous peripheral blood progenitor cells (PBPCs) or whole blood enriched of PBPC were used. RESULTS: We found statistically significant difference in OS regardless of the stage of the disease to the benefit of women treated by intensive cyclic EC chemotherapy when compared with the control group. In evaluation of DFS no statistically significant difference was found in survival between the control group and the group with all stages of the disease. TTP in women without relation to the stage was statistically significantly longer than in the control group. CONCLUSION: In our study intensive cyclic EC chemotherapy did not show better curative effect when compared with conventional dosage chemotherapy.

Clinical results of intensity-modulated radiation therapy (IMRT) for tumors of the head and neck region.

Intensity-modulated radiation therapy (IMRT) is an advanced form of the three-dimensional conformal radiation therapy (3D-CRT). Highly conformal dose distribution is the basic feature of IMRT. The head and neck region is suitable for this new technology since the primary tumor is often surrounded by several critical structures. IMRT offers the ability of dose escalation due to steep dose gradient towards healthy tissues. In this review, clinical results of IMRT in several head and neck sites are presented, including intracranial tumors. Parotid-sparing strategies and patterns of local-regional failures are analyzed. The possibilities of irradiation of recurrent malignancies are mentioned. In perspective, the potential of IMRT should be explored in conjunction with altered fractionation regimens, including simultaneous integrated boost (SIB). Particularly, studies with dose escalation are desirable.

First Report of the Potato Cyst Nematode (Globodera pallida) in the Czech Republic.

Potato cyst nematode, Globodera pallida, was detected in several soil samples collected from various areas of the Czech Republic. Globodera rostochiensis pathotype Ro1 is known to be widespread in the Czech Republic. G. pallida was reported from the neighboring countries of Austria, Germany, and Poland, and also was suspected to be present in the Czech Republic, but it has never been unambiguously proved (1). Recently, nematode isolates have been recovered that multiply readily on Ro1 resistant potato cultivars. These isolates were identified on the basis of three tests: (i) microscopic observations of cyst vulval area morphology and stylets of second-stage larvae; (ii) enzyme-linked immunosorbent assay using a commercial kit; and (iii) a polymerase chain reaction method (2). A complete series of G. pallida and G. rostochiensis pathotypes from Scotland and Germany served as controls. Results were identical for all three methods used. One sample contained G. pallida only, five samples contained mixtures of G. rostochiensis and G. pallida, and one sample contained G. rostochiensis only. The origin of G. pallida contamination is unknown. Strict quarantine measures have been taken to prevent G. pallida from spreading into neighboring areas. To our knowledge, this is the first evidence of the occurrence of G. pallida in the Czech Republic. References: (1) J. Potocek et al. EPPO Bull. 21:81, 1991. (2) M. Zouhar et al. Plant Prot. Sci. 36:81, 2000.

Occurrence of the Root-Knot Nematode Meloidogyne hapla in the Czech Republic.

Meloidogyne hapla was detected in several commercial vegetable fields in the Province of Central Bohemia in the Czech Republic. Crops grown in the area of infestation include leek, onion, cauliflower, early potatoes, and carrot. During the past 2 to 3 years, growers have observed typical symptoms of Meloidogyne infection as stunting and galls on roots of carrot. Identification of the causal organism was based on microscopic observation of the perineal patterns of females obtained from root galls and polymerase chain reaction (1,2). Known isolates of M. chitwoodi, M. arenaria, M. javanica, M. incognita, and M. fallax from M. Phillips (SCRI, Dundee) and C. Zijlstra (PRI, Wageningen), and M. hapla from M. Liskova (PI, Kosice) were used as controls. M. hapla was identified in galled roots using both methods. Several fields with carrots were inspected in the area during vegetation in 2000 and 2001. In most fields, only isolated and limited outbreaks occurred, but recently, we observed a widespread and fairly homogeneous occurrence of root knot attributed to M. hapla in a 2-ha field. This nematode may become a more widespread and damaging pest, especially of carrot, in the Czech Republic. Other than carrot, vegetable crops grown in the area and tested in the greenhouse and field in soil highly infested by our isolates of M. hapla had no visible symptoms of infection. Therefore, exclusion of carrot cultivation or a longer crop rotation is recommended to minimize economic losses to growers. To our knowledge, this is the first report of the occurrence of M. hapla in the Czech Republic. References: (1) T. S. Harris et al. J. Nematol. 22:518, 1990. (2) C. Zijlstra et al. Genetics 85:1231, 1995.

Conformal radiotherapy for prostate cancer--longer duration of acute genitourinary toxicity in patients with prior history of invasive urological procedure.

The incidence and predictors of acute toxicity were evaluated in patients treated with three-dimensional conformal radiotherapy (3D-CRT) for localized prostate cancer. Between December 1997 and November 1999, 116 patients with T1-T3 prostatic carcinoma were enrolled in the study. Ninety patients were treated with 70 Gy and 26 patients with T3 tumors received 74 Gy. Of the 116 patients 42 (36.2%) had a prior history of invasive urological procedure (IUP) (transurethral resection of the prostate or transvesical prostatectomy for benign prostatic hyperplasia). Acute gastrointestinal (GI) and genitourinary (GU) symptoms were graded according to the EORTC/RTOG scoring system. Toxicity duration after the completion of 3D-CRT was recorded. The majority of patients experienced only mild or no (Grade 1) acute toxicities. Medications for GI and GU symptoms (Grade 2) were required by 28.4% and 12.9% of patients, respectively. Only one case of Grade 3 GI toxicity (0.9%) was observed. Seven patients (6.1%) experienced severe GU toxicity (Grade 3 or 4). No correlation was found between acute toxicity and age, stage, dose (70 Gy vs. 74 Gy), IUP and pelvic lymphadenectomy. A significant relationship was observed between the duration of acute GU toxicity and prior IUP. Symptoms persisted for more than 4 weeks in 51.9% and 26.0% of patients with and without a prior history of IUP, respectively (p = 0.02). The incidence of acute complications, associated with 3D-CRT for prostate cancer, was acceptable in our cohort of patients. A prior history of IUP resulted in a significantly longer duration of acute GU toxicity.

[Chronic progressive radiation myelopathy]. / Chronická progresívní radiacní myelopatie.

Radiation dose absorbed by spinal tissue in 4 cases of chronic progressive myelopathy was analysed so that the criteria could be established for choosing cases in which myelopathy was to be presumed. Nevertheless, neither regressive nor vascular lesion was found in 10 autopsies fitting the criteria. The fact was explained by individual variability or other unknown factors.