Adjuvant accelerated partial-breast irradiation of early-stage breast cancer using stereotactic approach - methodology, technical challenges and early results of prospective randomized trial.

BACKGROUND: The adjuvant radiotherapy (RT) of the early-stage breast cancer patients as local treatment aims to eliminate potential microscopic residual disease in the surgery bed or satellites in its neighborhood. Based on published studies, accelerated partial breast irradiation (APBI) is recommended for strictly selected patients. The aim of this single-institution prospective randomized study was to compare the targeted APBI delivered by stereotactic approach with the currently more commonly used accelerated whole breast irradiation with the boost to the tumor bed in terms of feasibility, safety, tolerance, and cosmetic effects. MATERIALS AND METHODS: Early-stage breast cancer patients after partial mastectomy were screened for eligibility. The inclusion criteria were age > 50 years, non-lobular carcinoma histology, size 2cm, negative margins 2mm, L0, ER-positive, BRCA negative. Enrolled patients were equally randomized into two arms according to radiotherapeutic regiment - external APBI (5× 6 Gy) and accelerated whole breast irradiation with the boost (15× 2,67 Gy + 5× 2 Gy). These preliminary results of the ongoing study evaluated the first 57 from 84 planned patients. RESULTS: The median age was 65 years. The tumors were of grade 1 in 60 % of patients, the median size of 9mm and 70 % were classified as invasive ductal carcinoma. Statistical significant differences between the groups in baseline characteristics were not observed. A total of 29 patients was enrolled in the APBI group by the end of 2020. All enrolled patients were evaluated one month after RT. A total of 40 (70,2 %) a 33 (58 %) had examinations 3 and 6 months after RT, respectively. Toxicity evaluation showed statistically significantly fewer acute adverse events in the APBI group in terms of skin erythema, desquamation, skin tenderness, dryness, edema, pigmentation, breast pain and fatigue. Late toxicity evaluated in 3 and 6 months after RT was significantly higher in the control group. The cosmetic effect (independently evaluated by a physician, nurse and patient) was more favorable to the APBI group. CONCLUSION: The technique using the principles of targeted radiotherapy turned out to be a less toxic and easier feasible approach for adjuvant radiation of early-stage breast cancer patients. Consequently, the presented study increases the level of evidence for RT-indicated patients to the establishment of external APBI into daily clinical practice.

Investigation of discordant sibling pairs from hereditary breast cancer families and analysis of a rare PMS1 variant.

BACKGROUND: It is likely that additional genes for hereditary breast cancer can be identified using a discordant sib pair design. Using this design we identified individuals harboring a rare PMS1 c.605G>A variant previously predicted to result in loss of function. OBJECTIVES: A family-based design and predictive algorithms were used to prioritize candidate variants possibly associated with an increased risk of hereditary breast cancer. Functional analyses were performed for one of the candidate variants, PMS1 c.605G>A. METHODS: 1) 14 discordant sister-pairs from hereditary breast cancer families were identified. 2) Whole exome sequencing was performed and candidate risk variants identified. 3) A rare PMS variant was identified in 2 unrelated affected sisters but no unaffected siblings. 4) Functional analysis of this variant was carried out using targeted mRNA sequencing. RESULTS: Genotype-phenotype correlation did not demonstrate tracking of the variant with cancer in the family. Functional analysis revealed no difference in exon 6 incorporation, which was validated by analyzing PMS1 allele specific expression. CONCLUSIONS: The PMS1 c.605G>A variant did not segregate with disease, and there was no variant-dependent impact on PMS1 exon 6 splicing, supporting this variant is likely benign. Functional analyses are imperative to understanding the clinical significance of predictive algorithms.

Prognosis of HPV-Positive and -Negative Oropharyngeal Cancers Depends on the Treatment Modality.

BACKGROUND: The association between human papilloma virus (HPV) and oropharyngeal carcinoma is a topical issue due mainly to the rapid increase in incidence over recent years. These tumors are etiopathogenetically, epidemiologically, and clinically different from other carcinomas at this location. They have a better prognosis in that they are more chemo-and radiosensitive. Indeed, this has been shown by many extensive retrospective and prospective studies. HPV status is considered an integral part of a standard histopathological examination and is included as a new biological parameter in TNM classification. MATERIALS AND METHODS: The results of 77 patients who were treated non-surgically for locally advanced oropharyngeal carcinoma at a single university ear, nose, and throat clinic were analyzed retrospectively. RESULTS: Overall and specific survival of those with HPV-positive (HPV+) tumors was better that those for HPV negative (HPV-) tumors. With the exception of TNM classification, HPV positivity appeared to be the strongest predictor of local control, and of overall and specific survival, regardless of the type of treatment. However, smoking and p53 positivity were significant negative predictors of overall survival. Patients with HPV-associated tumors had a significantly better prognosis, regardless of treatment type. The difference between treatment modalities was confirmed for the whole group of patients, but not for the HPV+ and HPV-patients specifically, most probably due to the small number of patients enrolled. CONCLUSION: The results obtained herein may constitute the first step toward the concept of treatment de-escalation in those with HPV-associated oropharyngeal carcinoma; however, this decision can be based only on the results of current extensive randomized trials. Specification of the optimal de-escalation scheme, or the choice of treatment modality, for which the difference in treatment results is most pronounced, has yet to be identified. This work was supported by grants of the Ministry of Health of the Czech Republic IGA NT12483-4/2011 and AZV 15-31627A. he authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 21. 9. 2018 Accepted: 14. 5. 2019.

Stereotactic Body Radiotherapy - Current Indications.

BACKGROUND: Stereotactic body radiotherapy (SBRT) is used to treat localized tumor lesions and consists of applying high doses of radiation to a small number of fractions using specially equipped linear accelerators, modern immobilization devices, and imaging methods, which are considered special, advanced techniques in modern day radiotherapy. SBRT is a very well tolerated, non-invasive, short-term treatment that does not require hospitalization or any complicated preparation. Compared to standard radiotherapy techniques, SBRT allows, due to its precision, significantly higher doses to be applied to the target with less damage to surrounding healthy tissues. If dose constraints are not exceeded, the risk of damage to tissues and organs around the irradiated volume is reduced to minimum. The principle of SBRT is the application of ablative doses of radiation that cause necrosis of the irradiated tissue. PURPOSE: The aim of this review is to provide a basic overview of SBRT indications, radiation doses used, and potential side effects. It is not intended to be a detailed description of treatment itself (such as discussion of patient fixation systems, management of respiratory movements, or image guided strategies of treatment). This review also discusses rarer indications for SBRT, such as pancreatic carcinoma or hepatocellular carcinoma. CONCLUSION: Advances in image navigation, radiation planning, and dose application have enabled successful introduction of SBRT as a treatment regimen for many primary tumors and oligometastatic disease. If surgery is not possible or the patient refuses surgery, it is always reasonable to consider SBRT. SBRT has curative potential for the treatment of primary lung or prostate tumors. High-dose irradiation of oligometastases of various primary tumors can lead to long-term survival without disease symptoms, delay administration of toxic systemic therapies, and improve the quality of life of oncological patients. Key words radiotherapy - stereotactic body radiotherapy - review - ablative radiotherapy - lung cancer - prostate cancer - oligometastatic disease This work was supported in part by the Ministry of Health, Czech Republic - Conceptual Development of Research Organization (MMCI 00209805). The results of this research have been acquired within CEITEC 2020 (LQ1601) project with the financial contribution made by the Minis-try of Education, Youths and Sports of the Czech Republic within special support paid from the National Programme for Sustainability II funds. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 30. 10. 2018 Accepted: 4. 11. 2018.

Effect of Tumor Size and p16 Status on Treatment Outcomes - Achievement of Complete Remission in Prospectively Followed Patients with Oropharyngeal Tumors.

BACKGROUND: Oropharyngeal squamous cell tumors associated with human papillomavirus infection (p16 positive tumors) have better prognosis than p16 negative tumors regardless of the more advanced stage of the disease. Tumor volume (GTVt+n) is generally an important factor affecting treatment results of ionizing radiation. The aim of this prospective non-randomized study is to evaluate the effect of tumor volume on the (chemo)radiation treatment results in a group of patients with p16 negative and p16 positive oropharyngeal tumors. PATIENTS AND METHODS: Patients with confirmed squamous cell tumor of the oropharynx of stages III and IV, according to the 7th version of the TNM (tumor-nodes-metastases) classification, were eligible for this study. The main exclusion criteria were palliative treatment, neoadjuvant chemotherapy or planned concomitant therapy with cetuximab. Patients were treated according to standardized protocols with curative intent. Primary tumor volume (GTVt) and involved nodes volume (GTVn) were obtained from radiotherapy planning system for further statistical analysis. The differences in tumor volumes between the groups according to p16 expression were assessed with subsequent testing of probability to achieve complete remission (CR) of the disease in both groups. RESULTS: In total, 49 patients - 84% men, median age 60.5 years, 25 (51%) patients p16 positive, 40 (82%) underwent concomitant chemoradiotherapy. Median of GTVt in the whole patients group is 40.2 ccm, GTVn 11.78 ccm and median volume of the whole tumor burden (GTVt+n) 70.21 ccm (range 11.05-249). Median of GTVn was greater in the p16 positive cohort (p = 0.041). In the entire group, the median time to reach CR was 91 days (95% CI 86-107 days) from the end of radiotherapy. In the group of p16 negative patients, 14 achieved CR (61%) out of 23 patients, in p16 positive group 20 (80%) out of 25 patients (p = 0.111). P16 negative patients had a longer time to CR (p = 0.196, HR 1.58, 95% CI 0.79-3.18). None of the independently assessed volumetric parameters of the tumor (GTVt, GTVn, GTVt+n) affected CR in the p16 positive patients group, while there was a significant impact of the whole tumor burden (GTVt+n) in the p16 negative cohort (median 58.1 ccm in CR patients vs. 101.9 ccm, p = 0.018). CONCLUSION: We have showed less GTVt+n dependence to achieve CR in p16 positive tumors in comparison with p16 negative tumors. Thus, p16 positive oropharyngeal squamous cell cancers should not be withdrawn from the curative treatment intent based on the greater GTVt+n. Key words oropharyngeal neoplasms - p16 status - treatment outcome - tumor burden - complete remission This work was supported by grant of the Ministry of Health of the Czech Republic AZV 15-31627A and by grant of the Ministry of Health of the Czech Republic - Conceptual development of a research organization (MMCI 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 2. 11. 2018 Accepted: 11. 11. 2018.

Ablative dose stereotactic body radiation therapy for oligometastatic disease: a prospective single institution study.

Localized, metastasis-directed stereotactic body radiation therapy (SBRT) of oligometastatic disease (OD) is currently rapidly evolving standard of care in many institutions. Further reports of outcomes are required to strengthen the level of evidence in the absence of comparative trials evaluating different practical procedures. The aim of this prospective single institutional study is to analyse, in unselected cohort of patients from real-world clinical practice, the long-term survival, tumor control outcomes and safety of SBRT in OD (radical ablative radiotherapy with biological equivalent dose BED10>100 Gy). In addition to standard toxicity and survival parameters, we report unique outcomes as FFWD - Freedom from widespread dissemination, FFNT - Freedom from the need of subsequent treatment and functional survival with Karnofsky performance status higher than 70%. A total of 110 patients were prospectively evaluated, 60% and 40% were treated for lung and liver oligometastatic disease, respectively. No grade 3 or 4 acute toxicities (CTCAE) were reported. With median follow up of 22.2 months and 2-year overall survival of 88.3%, four patients (6.1%) experienced local progression in the lung SBRT cohort. In the liver SBRT cohort, median follow up was 33 months, 2-year overall survival was 68.5% and 11 patients (25%) experienced local and 36 (81.8%) distal progression. Higher BED10 of 150-170 Gy compared to 100-150 Gy was an independent positive prognostic factor for local progression-free survival for all patients with hazard ratio 0.25. This confirms SBRT ablative radiobiology effects to be independent of OD primary histology and location. The best outcomes in terms of FFNT were observed in the multivariable analysis of patients with 1-2 lung OD compared to both the liver OD cohort and patients with more than 2 lung metastases. Better FFNT in the liver SBRT cohort was observed in patients with 1-2 liver metastases and in patients whose liver OD was irradiated by higher BED10. In conclusion, SBRT is a suitable option for patients who are not surgical candidates; with approximately 30% of patients not requiring subsequent treatment 2 years after SBRT. We believe that this treatment represents a safe and effective option for oligometastatic involvement in patients with various primary tumors.

[MicroRNAs in Prediction of Response to Radiotherapy in Head and Neck Cancer Patients - Pilot Study]. / MikroRNA v predikci odpovedi na radioterapii u pacientu s nádory hlavy a krku - pilotní studie.

BACKGROUND: Radiotherapy plays a key role in the treatment of squamous cell head and neck cancers (HNSCC). The effectivity of radiation therapy is often limited by radioresistance of these tumours. microRNAs (miRNAs) are endogenous, evolutionary conserved, small non-coding RNAs involved in regulation of cellular processes associated with radioresistance. The objective of this study was to identify miRNA profile enabling to predict the radiation treatment outcomes in HNSCC patients. MATERIAL AND METHODS: The retrospective study included HNSCC patients who underwent a definitive radiotherapy. Patients were divided into two groups according to loco-regional control (LRC) as follows - short LRC (n = 22; median 5.1 months (min. 1.3, max, 18.6)) vs. long LRC (n = 21; 60.4 (min. 46.8, max. 98.8)) group. Global miRNA expression profiles were obtained by use of Affymetrix microarray technology (GeneChip miRNA 4.0 Array). RESULTS: We identified 24 miRNAs to be significantly associated with LRC (p < 0.05), all of these miRNAs were upregulated in patients with short LRC. Out of these miRNAs, 12 miRNAs with p < 0.025 and 4 miRNAs with p < 0.01 have been identified. CONCLUSION: miRNAs seems to be promising as potential biomarkers predicting radiotherapy treatment outcomes in patients with HNSCC.Key words: microRNAs - radiotherapy - head and neck cancer The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Supported by Ministry of Health of the Czech Republic, grant No. 15-31627A. All rights reserved.Submitted: 19. 3. 2018Accepted: 20. 3. 2018.

Patterns of failure after brain metastases radiotherapy: reflections on the importance for treatment and clinical trials reporting.

In many ongoing clinical trials, new strategies for radiotherapy of brain metastases are currently being investigated. A post surgical focal cavity stereotactic radiosurgery and the developing role of a hippocampal-sparing whole brain radiotherapy are of the highest importance. The evaluation of spatial patterns of metastases failure after radiotherapy is a powerful tool for assessing the potential benefit of new different radiotherapy approaches, which enables to identify possible directions leading to better radiotherapy techniques and to modify general management for newly diagnosed brain metastases. The purpose of this article is to present a mix between trial data and philosophical point of view for discussion about the importance of systematic evaluation of spatial patterns of failure in all ongoing trials investigating new approaches in local brain metastases treatment.

Utilization of Prognostic Indexes for Patients with Brain Metastases in Daily Radiotherapy Routine -  is the Complexity and Intricacy Still an Issue?

BACKGROUND: Many prognostic indexes are available for patients with brain metastases in order to estimate remaining lifetime before selection of appropriate treatment including palliative radiotherapy. Their routine utilization is often deprecated for their complexity. We developed a practical tool based on widely available spreadsheet editors for facilitation of daily clinical use of selected indexes (RPA, GPA and WBRT 30) and evaluated its usage for retrospective single institutional survival analysis of patients irradiated for brain metastases. PATIENTS AND METHODS: Spreadsheet platform was prepared and adjusted for automatic calculation of selected prognostic indexes after input of the relevant parameters. The consecutive series of newly diagnosed patients referred during 2011 to the palliative brain radiotherapy were analyzed, and real calculated survival parameters of individual subgroups of RPA, GPA and WBRT 30 were compared with estimated ones. Correlation of radiotherapy technique and estimated survival at the time of treatment indication was evaluated. RESULTS: Total of 121 patients (61% with multiple metastases) were irradiated with the majority undergoing whole brain radiotherapy. Median overall survival from the time of radiotherapy indication was 3.13 months. Non balanced distribution into individual scoring systems subgroups was observed with 8 (7%), 89 (73%) and 24 (20%) patients assigned to RPA 1, 2 and 3 subgroup, 3 (3%), 9 (7%), 57 (47%) and 52 (43%) patients assigned to GPA 3.5- 4, GPA 3.0, GPA 1.5- 2.5 and GPA 0- 1.0 subgroup and 10 (8%), 88 (73%) and 23 (19%) patients assigned to WBRT 30 subgroup D, B and A. Entire differences in overall survival between subgroups are significant among all three scoring systems. CONCLUSION: Routine calculation of available prognostic indexes is useful in decision making regarding the best radiotherapy of brain metastases, and their calculation is greatly facilitated by properly prepared widely available spreadsheet tools.

Toxicity and survival outcomes of adjuvant chemoradiation for gastric and gastroesophageal junction cancer patients treated in period 2006-2009: an institutional experience.

Surgical resection is the mainstay of gastric or gastroesophageal junction cancer treatment and has curative potential for patients with early-stage disease. In order to improve the poor survival rates, there are two complementary treatment strategies used at most - perioperative chemotherapy based on UK Magic trial or adjuvant chemoradiation based on INT-0116 trial. Daily treatment decision making should be led also by institutional experiences with toxicity evaluation. We evaluated survival and toxicity outcomes of 47 consecutive patients who underwent adjuvant chemoradiation in our institution in the years 2006-2009. 45Gy in 5 weeks with concurrent two cycles of FUFA Mayo regimen chemotherapy were administrated as part of combined treatment. The acute toxicity was relatively mild (CTCAE scale): grade 2 nausea in 26%, vomiting in 13%, and diarrhoea grade 1 in 15% and general abdominal discomfort in 57% of patients. Grade 3 haematological and infectious complications in 6% and 2% respectively. Late adverse events were as follows: grade 1 esophageal toxicity in 17%, signs of mild chronic esophageal ulceration and esophageal stenosis in 9% of patients (50% of them had tracheoesophageal fistula). The Kaplan- Meier estimate of the median overall survival was 30.5 months with median 25.7 months disease free survival. The overall survival was statistically significantly affected by the amount of removed positive lymph nodes. For the proper evaluation of radiotherapy role in multimodal treatment approach, results of other clinical trials investigating role of concurrent radiotherapy in administration of perioperative chemotherapy will be necessary. Meanwhile, two equally approaches are possible, all having their pros and cons. Institutional toxicity evaluation is recommended in order to provide the best care possible.

Toxicity and survival outcomes of adjuvant chemoradiation for gastric and gastroesophageal junction cancer patients treated in period 2006-2009: an institutional experience.

Surgical resection is the mainstay of gastric or gastroesophageal junction cancer treatment and has curative potential for patients with early-stage disease. In order to improve the poor survival rates, there are two complementary treatment strategies used at most - perioperative chemotherapy based on UK Magic trial or adjuvant chemoradiation based on INT-0116 trial. Daily treatment decision making should be led also by institutional experiences with toxicity evaluation. We evaluated survival and toxicity outcomes of 47 consecutive patients who underwent adjuvant chemoradiation in our institution in the years 2006-2009. 45Gy in 5 weeks with concurrent two cycles of FUFA Mayo regimen chemotherapy were administrated as part of combined treatment. The acute toxicity was relatively mild (CTCAE scale): grade 2 nausea in 26%, vomiting in 13%, and diarrhoea grade 1 in 15% and general abdominal discomfort in 57% of patients. Grade 3 haematological and infectious complications in 6% and 2% respectively. Late adverse events were as follows: grade 1 esophageal toxicity in 17%, signs of mild chronic esophageal ulceration and esophageal stenosis in 9% of patients (50% of them had tracheoesophageal fistula). The Kaplan- Meier estimate of the median overall survival was 30.5 months with median 25.7 months disease free survival. The overall survival was statistically significantly affected by the amount of removed positive lymph nodes. For the proper evaluation of radiotherapy role in multimodal treatment approach, results of other clinical trials investigating role of concurrent radiotherapy in administration of perioperative chemotherapy will be necessary. Meanwhile, two equally approaches are possible, all having their pros and cons. Institutional toxicity evaluation is recommended in order to provide the best care possible. Keywords: adjuvant chemoradiation, gastric cancer, early toxicity, late toxicity, survival outcomes.

Stereotactic body radiation therapy for colorectal cancer liver metastases; early results.

BACKGROUND: Stereotactic body radiation therapy (SBRT) is well feasible and effective method for treatment of colorectal cancer liver metastases. MATERIALS AND METHODS: From September 2009 to December 2011, 11 patients with 15 inoperable liver metastases of colorectal cancer were treated by SBRT using Varian Clinac iX linear accelerator. We treated 6 men and 5 women of age from 51 to 81 years (median 68). SBRT doses ranged from 40 to 56 Gy (median 54 Gy) and were administered in 3 to 8 fractions. RESULTS: Local control rates at 2, 4, 6, 9 and 12 months after completion of SBRT were 100%, 91%, 91%, 67% and 50%, respectively. Disease progression-free survival rates at 2, 4, 6, 9 and 12 months were 82%, 82%, 64%, 50% and 50%, respectively. Median follow-up was 15 months. No severe side effects were attributed to the therapy. CONCLUSION: Our study assessed the feasibility of SBRT in selected group of patients with 1 to 3 colorectal cancer liver metastases with no other treatment option. We achieved excellent local control and very moderate acute and late side effects. Distant metastases were the most common recurrence form after SBRT. SBRT demonstrated excellent local control and resulted in occasional long-term survivors without any serious side effects of therapy.

Campylobacter biofilm phenotype exhibits reduced colonization potential in young chickens and altered in vitro virulence.

In this study, we evaluated the ability of different Campylobacter phenotypes (biofilm versus planktonic) to colonize young poultry. It has been suggested that a persistent Campylobacter biofilm reservoir may be involved in the initial contamination of poultry flocks. Campylobacter jejuni cultured adherent to agar was utilized as the biofilm model and C. jejuni cultured in broth was evaluated as the planktonic model. In 2 independent trials, 1-d-old broiler chicks were given 1 of 3 treatments: 1) 10(5) cfu.mL(-1) of C. jejuni cultured in broth, 2) 10(5) cfu.mL(-1) of C. jejuni cultured adherent to agar, or 3) no C. jejuni (negative control). Cecal contents of all birds were evaluated by culturing 12 d after the initial challenge with C. jejuni. In both trials, birds challenged with C. jejuni cultured in broth had approximately 3 to 4 log higher cecal Campylobacter concentration than birds challenged with C. jejuni cultured adherent to agar. Using 2 cell lines (INT 407 and DF1), virulence of C. jejuni cultured in broth versus adherent to agar also was evaluated by challenging monolayers of eukaryotic cells with 1 of 3 treatments: 1) 10(5) cfu.mL(-1) of C. jejuni cultured in broth, 2) 10(5) cfu.mL(-1) of C. jejuni cultured adherent to agar, or 3) no C. jejuni (negative control). The virulence study also showed differences of C. jejuni cultured in broth or agar in attachment and invasion abilities to tissue culture cells, but differences were not as consistent as with the chick colonization study. This study indicates that phenotype may play a role in colonization of chickens and virulence by C. jejuni.

Can automated blood film analysis replace the manual differential? An evaluation of the CellaVision DM96 automated image analysis system.

Automation of differentials is desirable for economic and time-saving reasons. Over the last 20 years, automated imaging processes have started to be introduced where stained blood films are scanned by a computer-driven microscope and leucocytes classified; however, early methods were slow and had difficulty in classifying abnormal cells. More recently the CellaVision DM96 (CellaVision AB, Lund, Sweden) has been introduced with added features such as continuous loading of slides and a faster throughput than previous instruments. The accuracy of CellaVision DM96 has been evaluated by comparing results to reference manual differentials. Results from different operators using the DM96 were compared with their own manual differential and to a 400-cell reference manual differential. Precision of the instrument was compared to the manual differential. The preclassification accuracy of the DM96 was 89.2%. Precision was similar to that of the 100-cell manual differential. The DM96 was faster than the manual method, even after reclassification by a laboratory scientist of any cells wrongly categorized by the instrument. The DM96 accuracy in morphological classification of leucocytes and red blood cells; depends upon both blood pathology and experience of the laboratory scientist using the instrument. For some cell types and operators, DM96 accuracy was better than the individual's 100 cell manual differential.

Campylobacter jejuni as a secondary colonizer of poultry biofilms.

AIMS: The objective of this study was to determine if survival of culturable Campylobacter jejuni outside the host was increased by entrapment in pre-established biofilms. METHODS AND RESULTS: Campylobacter jejuni was inoculated into four biofilm populations isolated from poultry environments and cultured at three temperatures. Survival of culturable Camp. jejuni in some pre-established biofilms was extended vs survival of culturable Camp. jejuni in broth. But some biofilms were detrimental to survival of culturable Camp. jejuni. Denaturing gradient gel electrophoresis analysis indicated differences in bacterial profiles depending on initial source and temperature of culturing, which may have had impacts on survival of culturable Camp. jejuni. Further investigation showed no evidence of interspecies cell signalling indicating that secondary colonization was only physical. CONCLUSIONS: The results of this study show Camp. jejuni's attachment to surfaces is facilitated by pre-established biofilms and survival of culturable Camp. jejuni may be extended in some pre-established biofilms, but these biofilms do not fully explain long-term survival of culturable Camp. jejuni outside hosts. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information concerning survival of culturable Camp. jejuni outside the host and shows biofilms may be important in transmission and prevalence of Camp. jejuni.

Determination of toxicity of Campylobacter jejuni isolated from humans and from poultry carcasses acquired at various stages of production.

AIM: The research focused on the determination of the toxicity variation associated with Campylobacter jejuni isolated from humans and chickens. METHODS AND RESULTS: Campylobacter jejuni isolates were obtained from chicken carcasses and from humans exhibiting symptoms of campylobacteriosis. Using HeLa cells as the in vitro model, toxicity was determined for each isolate. The mean toxicity level of the chicken isolates was significantly lower than that of the human isolates (P < 0.001). There was a wide range of toxicity in C. jejuni isolated from chickens and the percentage of isolates exhibiting low toxicity remaining relatively constant. All C. jejuni isolates from humans possessed either medium or high levels of toxicity. CONCLUSIONS: All wildtype C. jejuni isolates obtained from poultry carcasses may not be equally important as a human foodborne pathogen. SIGNIFICANCE AND IMPACT OF STUDY: Campylobacter jejuni remains a primary foodborne pathogen and increased efforts are needed to determine the impact of wildtype isolates in causing human illness. The present research indicates that all isolates may not be equally important in regards to disease potential. The information found should be included in efforts to develop C. jejuni detection, control and infection modelling.

Detection of Campylobacter jejuni in poultry samples using an enzyme-linked immunoassay coupled with an enzyme electrode.

An enzyme-linked immunoassay coupled with a tyrosinase modified enzyme electrode was used for rapid detection of Campylobacter jejuni. The immunomagnetic separation (IMS) method was investigated to achieve optimal isolation of C. jejuni cells. Eight types of beads with three different sizes and function groups were coated with anti-C. jejuni to isolate C. jejuni from the sample solution. Bead size and coating methods were found to be major factors that influenced the capture efficacy. Streptavidin-labeled beads (2.8 microm) provided the greatest capture ability. Three blocking reagents were tested to minimize non-specific binding. Bovine serum albumin (BSA) showed the best blocking capability. Two IMS formats were tested. Competitive immunoassay cut the detection time to 1.5 h, but the detection limit was relatively high (10(6) CFU/ml). This system was evaluated using C. jejuni pure culture and poultry samples inoculated with C. jejuni. This detection method for C. jejuni could be completed within 2.5 h and had a detection limit of 2.1 x 10(4) CFU/ml. No significant difference was found between pure culture samples and poultry samples (P>0.01). A linear relationship was found between C. jejuni cell numbers and the peak current ratio in a range of 10(2)-10(7) CFU/ml (R(2)=0.94).

Cloning and function of the rat colonic epithelial K+ channel KVLQT1.

KVLQT1 (KCNQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of KVLQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to KVLQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of KVLQT1 in crypt cells and surface epithelium. Expression of rKVLQT1 in Xenopus oocytes induced a typical delayed activated K+ current, that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that KVLQT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.

Efficacy of cetylpyridinium chloride in immersion treatment for reducing populations of pathogenic bacteria on fresh-cut vegetables.

The efficacy of cetylpyridinium chloride (CPC) immersion to reduce the numbers of three pathogenic bacteria (Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli O157:H7) on three different fresh-cut vegetables (broccoli, cauliflower, and radishes) was studied. The fresh-cut vegetables were inoculated with one of the three pathogenic bacteria at a concentration of 10(5) CFU/ml for 1 h at room temperature and then treated with 0.1 or 0.5% CPC immersion for 1 min. Both Salmonella Typhimurium and E. coli O157:H7 plates were incubated from 48 to 72 h at 37 degrees C, and L. monocytogenes plates were incubated from 72 to 96 h before being counted. The results of three experiments showed that for the average of the three vegetables treated with 0.1 and 0.5% CPC, L. monocytogenes was reduced by 2.85 and 3.70 log CFU/g, Salmonella Typhimurium by 2.37 and 3.15 log CFU/g, and E. coli O157:H7 by 1.01 and 1.56 log CFU/g, respectively, in comparison with the vegetables treated with water only. The 0.5% CPC treatment was significantly different (P < 0.05) from the 0.1% CPC treatment on reduction of L. monocytogenes, Salmonella Typhimurium, and E. coli O157:H7. The CPC residual on the treated vegetables and their washing solutions were evaluated by using high-performance liquid chromatography.

Rapid detection of Salmonella typhimurium in chicken carcass wash water using an immunoelectrochemical method.

An immunoelectrochemical method coupled with immunomagnetic separation was developed for rapid detection of Salmonella Typhimurium in chicken carcass wash water. Samples of chicken carcass wash water were inoculated with Salmonella Typhimurium at different cell numbers. Possible nonspecified inhibitors in the wash water were minimized by filtration and centrifugation. An approximately 9.4% loss of Salmonella cells was found after filtration (P < 0.01). The samples were mixed with anti-Salmonella-coated magnetic beads (ASCMB) and alkaline phosphatase-labeled anti-Salmonella (APLAS) to form ASCMB-Salmonella-APLAS conjugates. The conjugates were separated from the solution using a magnetic separator and then incubated with phenylphosphate substrate to produce phenol. The number of Salmonella was determined by measuring the phenol concentration using an amperometric tyrosinase carbon paste electrode in a flow injection analysis system. Under optimized parameters (1 mM MgCl2, 0.2 microg/ml APLAS, and 1 mM phenylphosphate in pH 7.0 Tris buffer solution), Salmonella Typhimurium in chicken carcass wash water could be identified and enumerated within 2.5 h with a detection limit of 5 x 10(3) CFU/ml. A linear relationship on a log-log scale was found between Salmonella cell number and the peak current ratio for Salmonella concentrations ranging from 10(3) to 10(7) CFU/ml (R2 = 0.963). The peak currents of multibacteria samples, containing Salmonella Typhimurium, Listeria monocytogenes, and Campylobacter jejuni, were not significantly different from Salmonella-only samples (P > 0.01).