Extended nodal radiotherapy for prostate cancer relapse guided with [11C]-choline PET/CT: ten-year results in patients enrolled in a prospective trial.

AIMS: To report long-term outcomes of relapsed prostate cancer (PC) patients treated in a prospective single-arm study with extended-nodal radiotherapy (ENRT) and [11C]-choline positron emission tomography (PET)/computed tomography (CT)-guided simultaneous integrated boost (SIB) to positive lymph nodes (LNs). METHODS: From 12/2009 to 04/2015, 60 PC patients with biochemical relapse and positive LNs only were treated in this study. ENRT at a median total dose (TD) = 51.8 Gy/28 fr and PET/CT-guided SIB to positive LNs at a median TD = 65.5 Gy was prescribed. Median PSA at relapse was 2.3 (interquartile range, IQR:1.3-4.0) ng/ml. Median number of positive LNs: 2 (range: 1-18). Androgen deprivation therapy (ADT) was prescribed for 48 patients for a median of 30.7 (IQR: 18.5-43.1) months. RESULTS: Median follow-up from the end of salvage treatment was 121.8 (IQR: 116.1, 130.9) months; 3-, 5-, and 10-year BRFS were 45.0%, 36.0%, and 24.0%, respectively; DMFS: 67.9%, 57.2%, and 45.2%; CRFS: 62.9%, 53.9%, and 42.0%; and OS: 88.2%, 76.3%, and 47.9%, respectively. Castration resistance (p < 0.0001) and ≥ 6 positive LN (p = 0.0024) significantly influenced OS at multivariate analysis. Castration resistance (p < 0.0001 for both) influenced DMFS and CRFS in multivariate analysis. CONCLUSIONS: In PC relapsed patients treated with ENRT and [11C]-choline-PET/CT-guided SIB for positive LNs, with 10-year follow-up, a median Kaplan-Meier estimate CRFS of 67 months and OS of 110 months were obtained. These highly favorable results should be confirmed in a prospective, randomized trial.

A multicenter LArge retrospectIve daTabase on the personalization of stereotactic ABlative radiotherapy use in lung metastases from colon-rectal cancer: The LaIT-SABR study.

INTRODUCTION: Stereotactic ablative radiotherapy (SABR) has been shown to increase survival in oligometastatic disease, but local control of colorectal metastases remains poor. We aimed to identify potential predictive factors of SBRT response through a multicenter large retrospective database and to investigate the progression to the polymetastatic disease (PMD). MATERIAL AND METHODS: The study involved 23 centers, and was approved by the Ethical Committee (Prot. Negrar 2019-ZT). 1033 lung metastases were reported. Clinical and biological parameters were evaluated as predictive for freedom from local progression-free survival (FLP). Secondary end-point was the time to the polymetastatic conversion (tPMC). RESULTS: Two-year FLP was 75.4%. Two-year FLP for lesions treated with a BED < 00 Gy, 100-124 Gy, and ≥125 Gy was 76.1%, 70.6%, and 94% (p = 0.000). Two-year FLP for lesion measuring ≤10 mm, 10-20 mm, and >20 mm was 79.7%, 77.1%, and 66.6% (p = 0.027). At the multivariate analysis a BED ≥125 Gy significantly reduced the risk of local progression (HR 0.24, 95%CI 0.11-0.51; p = 0.000). Median tPMC was 26.8 months. Lesions treated with BED ≥125 Gy reported a significantly longer tPMC as compared to lower BED. The median tPMC for patients treated to 1, 2-3 or 4-5 simultaneous oligometastases was 28.5, 25.4, and 9.8 months (p = 0.035). CONCLUSION: The present is the largest series of lung colorectal metastases treated with SABR. The results support the use of SBRT in lung oligometastatic colorectal cancer patients as it might delay the transition to PMD or offer relatively long disease-free period in selected cases. Predictive factors were identified for treatment personalization.

Stereotactic ablative radiotherapy in castration-resistant prostate cancer patients with oligoprogression during androgen receptor-targeted therapy

Objectives To report outcomes of stereotactic body radiotherapy (SBRT) in metastatic castration-resistant prostate cancer (mCRPC) patients with oligoprogression (≤ 5 metastases) during first-line treatment with androgen receptor-targeted therapy (ARTT). Patients and methods Retrospective multi-institutional analysis of mCRPC patients treated with SBRT to oligoprogressive lesions during ARTT. End-points were time to next-line systemic treatment (NEST), radiological progression-free survival (r-PFS) and overall survival (OS). Toxicity was registered according to Common Terminology Criteria for Adverse Events (CTCAE) v4.0. Survival analysis was performed using the Kaplan–Meier method, univariate and multivariate analysis (MVA) were performed. Results Data from 34 patients were analyzed. Median NEST-free survival, r-PFS, and OS were 16.97, 13.47, and 38.3 months, respectively. At MVA, factors associated with worse NEST-free survival and r-PFS were polymetastatic burden at diagnosis of metastatic hormone-sensitive disease (hazard ratio [HR] 3.66, p = 0.009; HR 3.03, p = 0.034), PSA ≤ 7 ng/ml at mCRPC diagnosis (HR 0.23, p = 0.017; HR 0.19, p = 0.006) and PSADT ≤ 3 months at mCRPC diagnosis (HR 3.39, p = 0.026; HR 2.79, p = 0.037). Polymetastatic state at mHSPC diagnosis was associated with a decreased OS (HR 4.68, p = 0.029). No patient developed acute or late grade ≥ 2 toxicity. Conclusion Our results suggest that SBRT in oligoprogressive mCPRC is safe, effective and seems to prolong the efficacy of the ongoing systemic treatment positively affecting disease progression. Prospective trials are needed (AU)

Stereotactic ablative radiotherapy in castration-resistant prostate cancer patients with oligoprogression during androgen receptor-targeted therapy.

OBJECTIVES: To report outcomes of stereotactic body radiotherapy (SBRT) in metastatic castration-resistant prostate cancer (mCRPC) patients with oligoprogression (≤ 5 metastases) during first-line treatment with androgen receptor-targeted therapy (ARTT). PATIENTS AND METHODS: Retrospective multi-institutional analysis of mCRPC patients treated with SBRT to oligoprogressive lesions during ARTT. End-points were time to next-line systemic treatment (NEST), radiological progression-free survival (r-PFS) and overall survival (OS). Toxicity was registered according to Common Terminology Criteria for Adverse Events (CTCAE) v4.0. Survival analysis was performed using the Kaplan-Meier method, univariate and multivariate analysis (MVA) were performed. RESULTS: Data from 34 patients were analyzed. Median NEST-free survival, r-PFS, and OS were 16.97, 13.47, and 38.3 months, respectively. At MVA, factors associated with worse NEST-free survival and r-PFS were polymetastatic burden at diagnosis of metastatic hormone-sensitive disease (hazard ratio [HR] 3.66, p = 0.009; HR 3.03, p = 0.034), PSA ≤ 7 ng/ml at mCRPC diagnosis (HR 0.23, p = 0.017; HR 0.19, p = 0.006) and PSADT ≤ 3 months at mCRPC diagnosis (HR 3.39, p = 0.026; HR 2.79, p = 0.037). Polymetastatic state at mHSPC diagnosis was associated with a decreased OS (HR 4.68, p = 0.029). No patient developed acute or late grade ≥ 2 toxicity. CONCLUSION: Our results suggest that SBRT in oligoprogressive mCPRC is safe, effective and seems to prolong the efficacy of the ongoing systemic treatment positively affecting disease progression. Prospective trials are needed.

FDG PET-derived parameters as prognostic tool in progressive malignant pleural mesothelioma treated patients.

PURPOSE: The value of FDG PET-derived parameters in predicting overall survival (OS), local relapse-free survival (LRFS) and distant relapse-free survival (DRFS) in treated patients with malignant pleural mesothelioma (MPM) was evaluated. METHODS: This retrospective evaluation included 55 MPM patients treated between March 2006 and February 2015 with FDG PET/CT-guided salvage helical tomotherapy (HTT) after previous surgery plus chemotherapy. Univariate Cox regression analysis was performed to assess the impact of the following FDG PET-derived parameters: biological target volume (BTV), mean and maximum standardized uptake values (SUVmean/max), metabolic tumour volume (MTV) and total lesion glycolysis (TLG), measured using different uptake thresholds (40%, 50% and 60%). Logistic regression was then performed to identify the best FDG PET-derived parameters for selecting patients with poorer survival. RESULTS: The median OS was 9.1 months (range 0.0 - 69.6 months) after the end of HTT; 54/55 patients were dead at the last follow-up. BTV and TLG40, TLG50 and TLG60 were the most significant predictors of OS (p < 0.005). The median OS was 4.8 months in patients with MTV60 >5 cm3 and TLG40 >334.4, compared with 13.8 months and 16.1 months in patients with smaller values, respectively. The median LRFS and DRFS were 6.2 months (range 1.2 - 39.4 months) and 6.5 months (0.0 - 66.4 months), respectively. TLG40, TLG50 and TLG60 were significantly correlated with LRFS (p < 0.015). Median DRFS was 6.4 months in patients with MTV40 >39.6 cm3 and 6.2 months in patients with TLG40 >334.4, compared with 17 months and 18.8 months in patients with smaller values. BTV, TLG40 and MTV40 were also found to be good predictors in patients with poor OS/LRFS/DRFS (median survival times less than the median values). CONCLUSION: FDG PET-derived parameters effectively discriminated patients with a poor prognosis and may be helpful in the selection of MPM patients for salvage HTT.

Reduced Intestinal Motility, Mucosal Barrier Function, and Inflammation in Aged Monkeys.

OBJECTIVE: We aimed to examine the general health and intestinal physiology of young and old non-human primates with comparable life histories and dietary environments. DESIGN: Vervet monkeys (Chlorcebus aethiops sabaeus) in stable and comparable social and nutritional environments were selected for evaluation. Health phenotype, circulating cytokines and biomarkers of microbial translocation (MT) were measured (n=26-44). Subsets of monkeys additionally had their intestinal motility, intestinal permeability, and fecal microbiomes characterized. These outcomes document age-related intestinal changes present in the absence of nutritional stressors, which are all known to affect gastrointestinal motility, microbiome, and MT. RESULTS: We found that old monkeys have greater systemic inflammation and poor intestinal barrier function as compared to young monkeys. Old monkeys have dramatically reduced intestinal motility, and all changes in motility and MT are present without large differences in fecal microbiomes. CONCLUSION: We conclude that deteriorating intestinal function is a feature of normal aging and could represent the source of inflammatory burden yet to be explained by disease or diet in normal aging human primate populations. Intestinal changes were seen independent of dietary influences and aging within a consistent environment appears to avoid major microbiome shifts. Our data suggests interventions to promote intestinal motility and mucosal barrier function have the potential to support better health with aging.

The prepulse inhibition deficit appearance is largely independent on the circadian cycle, body weight, and the gender of vasopressin deficient Brattleboro rat.

OBJECTIVE: A disturbance of sensorimotor gating measured by prepulse inhibition of acoustic startle (PPI) is one of the best tests of the schizophrenia-like behavior. Vasopressin was implicated in the development of schizophrenia; therefore, the naturally occurring vasopressin-deficient Brattleboro rat has been suggested to be a reliable non-pharmacological animal model. However, previous studies focusing on PPI deficit did not use proper control and despite clear gender differences in the development of the disorder, the effect of gender has been mostly neglected. METHODS: First, we compared the "noise" and "tone" type prepulse at 73-77-81 dB intensity during the light or dark phase using small (~150 g) or big (~500 g) Wistar rats. The test parameters were validated by a pharmacological schizophrenia model (30 mg/kg ketamine i.p.). Than male, female, and lactating vasopressin-deficient animals were compared with +/+ ones. RESULTS: We established that the prepulse "noise" type is not optimal for PPI testing. The cycle of the day as well as the body weight had no effect on PPI. Even if we compared vasopressin-deficient animals with their closely related +/+ controls, the PPI deficiency was visible with more pronounced effect at 77 dB prepulse intensity similarly to pharmacological schizophrenia model. Despite our expectation, the gender as well as lactation had no effect on the vasopressin-deficiency induced PPI deficit. CONCLUSIONS: The present data confirmed and extended our previous studies that vasopressin-deficient rat is a good model of schizophrenia. It seems that female as well as lactating Brattleboro rats are useful tools for testing putative novel antipsychotics in line with special attention required for schizophrenic women.

Moderate Hypofractionation with Simultaneous Integrated Boost in Prostate Cancer: Long-term Results of a Phase I-II Study.

AIMS: To report 5 year outcome and late toxicity in prostate cancer patients treated with image-guided tomotherapy with a moderate hypofractionated simultaneous integrated boost approach. MATERIALS AND METHODS: In total, 211 prostate cancer patients, 78 low risk, 53 intermediate risk and 80 high risk were treated between 2005 and 2011. Intermediate- and high-risk patients received 51.8 Gy to pelvic lymph nodes and concomitant simultaneous integrated boost to prostate up to 74.2 Gy/28 fractions, whereas low-risk patients were treated to the prostate only with 71.4 Gy/28 fractions. Daily megavoltage computed tomography (MVCT) image guidance was applied. Androgen deprivation was prescribed for a median duration of 6 months for low-risk patients (for downsizing), 12 months for intermediate-risk and 36 months for high-risk patients. The 5 year biochemical relapse-free survival (bRFS), cancer-specific survival (CSS), overall survival and late gastrointestinal and genitourinary CTCAE.v3 toxicity were assessed. The effect of several clinical variables on both outcome and gastrointestinal/genitourinary toxicity was tested by uni- and multivariate Cox regression analyses. RESULTS: After a median follow-up of 5 years, the late toxicity actuarial incidence was: genitourinary ≥ grade 2: 20.2%; genitourinary ≥ grade 3: 5.9%; gastrointestinal ≥ grade 2: 17%; gastrointestinal ≥ grade 3: 6.3% with lower prevalence at the last follow-up visit (≥ grade 3: genitourinary: 1.9%; gastrointestinal: 1.9%). Major predictors of ≥ grade 3 genitourinary and gastrointestinal late toxicity were genitourinary acute toxicity ≥ grade 2 (hazard ratio: 4.9) and previous surgery (hazard ratio: 3.4). The overall 5 year bRFS was 93.7% (low risk: 94.6%; intermediate risk: 96.2%; high risk: 91.1%), overall survival and CSS were 88.6% (low risk: 90.5%; intermediate risk: 87.4%; high risk: 87%) and 97.5% (low risk: 98.7%; intermediate risk: 95%; high risk: 94.3%), respectively. Risk classes and androgen deprivation were not significantly correlated with either bRFS, overall survival or CSS. Twelve patients experienced a biochemical relapse but none experienced clinically proven local and/or pelvic recurrence. CONCLUSION: A satisfactory 5 year outcome with an acceptable toxicity profile was observed. The combination of image-guided radiotherapy-intensity-modulated radiotherapy, high equivalent 2 Gy dose (EQD2) with a moderate hypofractionated approach and extensive prophylactic lymph node irradiation also leads to very good outcome in high-risk patients.

(11)C-Choline PET/CT as a guide to radiation treatment planning of lymph-node relapses in prostate cancer patients.

PURPOSE: To evaluate, in prostate cancer (PCa) patients the potential of (11)C-choline PET/CT as a guide to helical tomotherapy (HTT) of lymph-node (LN) relapses with simultaneous integrated boost (SIB). The efficacy and feasibility of HTT in terms of acute toxicity were assessed. METHODS: We enrolled 83 PCa patients (mean age 68 years, range 51 - 82 years) with biochemical recurrence after radical primary treatment (mean serum PSA 7.61 ng/ml, range 0.37 - 187.00 ng/ml; PSA0) who showed pathological findings on (11)C-choline PET/CT only at the LN site. (11)C-Choline PET/CT was performed for restaging and then for radiation treatment planning (PET/CT0). Of the 83 patients, 8 experienced further LN relapse, of whom 5 were retreated once and 3 were retreated twice (total 94 radiotherapy treatments). All pelvic and/or abdominal LNs positive on PET/CT0 were treated with high doses using SIB. Doses were in the range 36 - 74 Gy administered in 28 fractions. After the end of HTT (mean 83 days, range 16 - 365 days), serum PSA was measured in all patients (PSA1) and compared with PSA0 to evaluate early biochemical response. In 47 patients PET/CT was repeated (PET/CT1) to assess metabolic responses at the treated areas. Toxicity criteria of the Radiation Therapy Oncology Group (RTOG) were used to assess acute toxicity. RESULTS: PET/CT0 revealed pathological LNs in the pelvis in 49 patients, pathological LNs in the abdomen in 15 patients pathological LNs in both the pelvis and abdomen in 18 patients, and pathological LNs in the pelvis or abdomen and other sites in 12 patients. All these sites were treated with HTT. With respect to PSA0, PSA1 (mean 6.28 ng/ml, range 0.00 - 220.46 ng/ml) showed a complete biochemical response after 66 of the 94 HTT treatments, a partial response after 12 treatments, stable disease after 1 treatment and progression of disease after 15 treatments. Of the 47 patients receiving PET/CT1, 20 showed a complete metabolic response at the treated area, 22 a partial metabolic response, 3 progression of disease and 2 stable disease. HTT with SIB was well tolerated in all patients. Grade 3 acute toxicity in the genitourinary tract was observed in two patients. CONCLUSION: (11)C-Choline PET/CT is a valuable tool for planning and monitoring HTT in LN relapse after primary treatment. High-dose hypofractionated (11)C-choline PET/CT-guided HTT with SIB is well tolerated and is associated with a high early biochemical response rate.

Lymph nodal metastases: diagnosis and treatment.

High risk prostate cancer patients have a significant risk to develop regional lymph node metastases, and this represent a major cause of biochemical failure. Although pelvic lymphadenectomy is the gold standard to assess the status of pelvic lymph nodes, a diagnostic imaging tool to non-invasively explore patients and to detect metastases, both in staging and in re-staging phase, would be of particular help in clinical management. In staging phase, while choline PET/CT specificity has been reported to be fairly high in lymph nodal detection, its sensitivity is not adequate due to its spatial resolution. Its role in the evaluation of patients with biochemical relapse or with suspected relapse has been successfully documented. In particular, choline PET/CT has great potential as a single step whole body diagnostic procedure to evaluate lymph nodal and bone metastatic involvement. Salvage lymph nodal dissection was recently listed as a possible experimental option for patients with nodal recurrent prostate cancer, even in the absence of solid prospective data. Radiation treatment for lymph-node recurrence is a therapeutic option evaluated in several studies, in particular by using stereotactic treatment or whole pelvic lymph-nodal irradiation plus a boost on choline PET/CT positive lymph nodes. In the present review an analysis of the specific role of choline PET/CT in guiding a specific treatment on lymph nodal site in prostate cancer patients is reported.

Use of culture and molecular analysis to determine the effect of antibiotic treatment on microbial community diversity and abundance during exacerbation in patients with cystic fibrosis.

BACKGROUND: Anaerobic bacteria are increasingly regarded as important in cystic fibrosis (CF) pulmonary infection. The aim of this study was to determine the effect of antibiotic treatment on aerobic and anaerobic microbial community diversity and abundance during exacerbations in patients with CF. METHODS: Sputum was collected at the start and completion of antibiotic treatment of exacerbations and when clinically stable. Bacteria were quantified and identified following culture, and community composition was also examined using culture-independent methods. RESULTS: Pseudomonas aeruginosa or Burkholderia cepacia complex were detected by culture in 24/26 samples at the start of treatment, 22/26 samples at completion of treatment and 11/13 stable samples. Anaerobic bacteria were detected in all start of treatment and stable samples and in 23/26 completion of treatment samples. Molecular analysis showed greater bacterial diversity within sputum samples than was detected by culture; there was reasonably good agreement between the methods for the presence or absence of aerobic bacteria such as P aeruginosa (κ=0.74) and B cepacia complex (κ=0.92), but agreement was poorer for anaerobes. Both methods showed that the composition of the bacterial community varied between patients but remained relatively stable in most individuals despite treatment. Bacterial abundance decreased transiently following treatment, with this effect more evident for aerobes (median decrease in total viable count 2.3×10(7) cfu/g, p=0.005) than for anaerobes (median decrease in total viable count 3×10(6) cfu/g, p=0.046). CONCLUSION: Antibiotic treatment targeted against aerobes had a minimal effect on abundance of anaerobes and community composition, with both culture and molecular detection methods required for comprehensive characterisation of the microbial community in the CF lung. Further studies are required to determine the clinical significance of and optimal treatment for these newly identified bacteria.

Isolation and activity of Xenorhabdus antimicrobial compounds against the plant pathogens Erwinia amylovora and Phytophthora nicotianae.

AIMS: Broad-spectrum antibiotics produced by symbiotic bacteria [entomopathogenic bacterium (EPB)] of entomopathogenic nematodes keep monoxenic conditions in insect cadavers in soil. This study evaluated antibiotics produced by EPB for their potential to control plant pathogenic bacteria and oomycetes. METHODS AND RESULTS: Entomopathogenic bacterium produce antibiotics effective against the fire blight bacterium Erwinia amylovora, including streptomycin resistant strains, and were as effective in phytotron experiments as kasugamycin or streptomycin. Xenorhabdus budapestensis and X. szentirmaii antibiotics inhibited colony formation and mycelial growth of Phytophthora nicotianae. From X. budapestensis, an arginine-rich fraction (bicornutin) was adsorbed by Amberlite((R)) XAD 1180, and eluted with methanol : 1 n HCI (99 : 1). Bicornutin inactivated zoospores, and inhibited germination and colony formation of cystospores at <<25 ppm. An UV-active molecule (bicornutin-A, MW = 826), separated by HPLC and thin-layer chromatography, was identified as a novel hexa-peptide : RLRRRX. CONCLUSIONS: Xenorhabdus budapestensis produces metabolites with strong antibacterial and cytotoxic activity. Individual compounds can be isolated, identified and patented, but their full antimicrobial potential may be multiplied by synergic interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: Active compounds of two new Xenorhabdus species might control plant diseases caused by pathogens of great importance to agriculture such as Erw. amylovora and P. nicotianae.

Xenorhabdus antibiotics: a comparative analysis and potential utility for controlling mastitis caused by bacteria.

AIMS: The role of antibiotics produced by bacterial symbionts of entomopathogenic nematodes is to suppress growth of microbes in the soil environment. These antibiotics are active against Gram-positive and Gram-negative bacteria, and were tested against mastitis isolates from dairy cows. METHODS AND RESULTS: Two bioassays were adapted for Xenorhabdus antibiotics; an overlay method on agar plates, and serially diluted, cell-free, Xenorhabdus cultures. The antimicrobial activities of the liquid cultures of 13 strains from five Xenorhabdus species were further evaluated. Antimicrobial activities of the type strains of X. nematophila, X. budapestensis and X. szentirmaii were tested on mastitis isolates of Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae with both bioassays. A previously reported antibiotic from X. nematophila, nematophin, was synthesized in three steps from tryptamine and 4-methyl-2-oxovaleric acid sodium salt. CONCLUSIONS: The antibiotics of all three Xenorhabdus strains were powerful in either bioassay, but the sensitivity of the isolates differed from each other. While Kl. pneumoniae was the least susceptible, Staph. aureus had the highest sensitivity to each Xenorhabdus strain. Xenorhabdus szentirmaii and X. budapestensis were more potent antibiotic producers than X. nematophila, and raceme nematophin was ineffective against all mastitis isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that Xenorhabdus antibiotics are effective against mastitis isolates and should be further evaluated for their potential in mastitis control or prevention.

Adult rat liver cells transdifferentiated with lentiviral IPF1 vectors reverse diabetes in mice: an ex vivo gene therapy approach.

AIMS/HYPOTHESIS: We examined a clinical model of ex vivo transdifferentiation of primary adult hepatocytes to insulin-secreting cells for the treatment of type 1 diabetes. MATERIALS AND METHODS: Isolated rat hepatocytes were transduced in primary culture with a human lentivirus containing pancreatic duodenal homeobox 1 (PDX1, now known as insulin promoter factor 1, homeodomain transcription factor [IPF1]). Insulin expression and secretion of the newly engineered cells were assessed in vitro by RT-PCR, in situ hybridisation, immunostaining and radioimmunoassay. PDX1-transduced hepatocytes were further studied in vivo by injecting them under the renal capsule of diabetic SCID mice. RESULTS: Isolated rat hepatocytes were efficiently transduced with the lentiviral vector, as assessed by green fluorescent reporter gene expression. The transduced cells exhibited insulin at both mRNA (RT-PCR, in situ hybridisation) and protein levels (immunostaining and radioimmunoassay). Moreover, insulin secretion by the engineered cells was dependent on glucose and sulfonylurea. Other beta cell genes, including those encoding solute carrier family 2 (facilitated glucose transporter), member 2 (Slc2a2), glucokinase (Gck), ATP-binding cassette, sub-family C (CFTR/MRP), member 8 (Abcc8), the potassium inwardly-rectifying channel, subfamily J, member 11 (Kcnj11) and proprotein convertase subtilisin/kexin type 1 (Pcsk1) were also expressed. The PDX1-transduced hepatocytes expressed several pancreatic transcription factors related to early pancreatic endocrine development (endogenous Pdx1, neurogenic differentiation factor 1 [Neurod1], and NK6 transcription factor related, locus 1 [Nkx6-1]) as well as the late-stage pancreatic transcription factors (paired box gene 4 [Pax4], paired box gene 6 [Pax6], and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A [Mafa]). Transplantation of 3 x 10(6) transdifferentiated liver cells under the renal capsule of seven streptozotocin-induced diabetic SCID mice resulted in significant reduction of non-fasting blood glucose levels from 30.7 +/- 1.3 to 8.7 +/- 3.7 mmol/l (mean +/- SEM, p = 0.01), in 6 to 8 weeks. Removal of the graft resulted in severe hyperglycaemia. CONCLUSIONS/INTERPRETATION: Ex vivo lentiviral-mediated PDX1 expression in isolated adult liver cells represents a potential model for type 1 diabetes mellitus therapy.

Evaluation of efficacy of entomopathogenic nematodes against larvae of Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae).

The blowfly Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) is the primary agent of cutaneous myiasis of sheep in northern Europe, southern Africa, Australia and New Zealand. As the application of chemicals has several disadvantages, alternative control measures of traumatic myiasis of livestock must be developed. In this study, the use of entomopathogenic nematodes (EPNs) as potential biocontrol agents against second instar larvae of Lucilia sericata was considered. The following nematode species were tested: Heterorhabditis bacteriophora (IS 5, HHU 1, Hmol, HNC 1, HAZ 36, Hbrecon, HHU 2, HAZ 29, HHP 88, HHU 3, HHU 4 and HGua), Steinernema intermedia, NC513 strain of S. glaserii, S. anomali, S. riobrave, Steinernema sp. and 5 strains of S. feltiae (22, Vija Norway, HU 1, scp, and IS 6). None of the examined EPN species or strains showed larvicidal efficacy at 37 degrees C (no killing effect was observed in the case of the two heat-tolerant strains--H. bacteriophora and S.feltiae) against L. sericata larvae. At lower temperatures (20 degrees C and 25 degrees C) only strains of S. feltiae were found to be active. The overall odds ratios calculated for L. sericata maggots to contract S. feltiae nematode infection show significant (p < 0.05) effect only in the case of strains HU 1, 22 and IS 6. In the case of strains HU 1 and 22 parasitic forms of S. feltiae could be detected in the dead larvae of L. sericata. Strain IS 6 (and also Vija Norway at 20 degrees C) penetrated and killed fly larvae, but only adult forms of the nematode occurred in the cadavers.

Inactivation of a novel gene produces a phenotypic variant cell and affects the symbiotic behavior of Xenorhabdus nematophilus.

Xenorhabdus nematophilus is an insect pathogen that lives in a symbiotic association with a specific entomopathogenic nematode. During prolonged culturing, variant cells arise that are deficient in numerous properties. To understand the genetic mechanism underlying variant cell formation, a transposon mutagenesis approach was taken. Three phenotypically similar variant strains of X. nematophilus, each of which contained a single transposon insertion, were isolated. The insertions occurred at different locations in the chromosome. The variant strain, ANV2, was further characterized. It was deficient in several properties, including the ability to produce antibiotics and the stationary-phase-induced outer membrane protein, OpnB. Unlike wild-type cells, ANV2 produced lecithinase. The emergence of ANV2 from the nematode host was delayed relative to the emergence of the parental strain. The transposon in ANV2 had inserted in a gene designated var1, which encodes a novel protein composed of 121 amino acid residues. Complementation analysis confirmed that the pleiotropic phenotype of the ANV2 strain was produced by inactivation of var1. Other variant strains were not complemented by var1. These results indicate that inactivation of a single gene was sufficient to promote variant cell formation in X. nematophilus and that disruption of genetic loci other than var1 can result in the same pleiotropic phenotype.

Novel application of PhastSystem polyacrylamide gel electrophoresis using restriction fragment length polymorphism--internal transcribed spacer patterns of individuals for molecular identification of entomopathogenic nematodes.

différences! [editorial] [editorial]onomic way of identifying and assigning nematodes to taxons, which had already been determined either by comparative sequence analysis of nuclear rDNA internal transcribed spacer (ITS) region or by other methods of molecular or conventional taxonomy, is provided. Molecular identification of entomopathogenic nematodes (EPN) can be upgraded by basing it on PhastSystem polyacrylamide gel electrophoresis (PAGE) analysis of restriction fragment length polymorphism (RFLP) patterns of polymerase chain reaction (PCR)-amplified DNA derived from single nematodes of Steinernema or Heterorhabditis spp. Although analysis from single worms has previously been made on agarose gel, the resolution on PhastSystem PAGE gel is much higher. The DNA sequences selected for analysis were those constituting the internal transcribed spacer region between the 18S and 26S rDNA genes within the rRNA operon. RFLP analysis was carried out by gel electrophoresis on the PhastSystem (Pharmacia) as detailed elsewhere (Triga et al., Electrophoresis 1999, 20, 1272-1277. The downscaling from conventional agarose to PhastSystem gels resulted in pattern of DNA fragments differing from those obtained with agarose gel electrophoresis under conventional conditions by increasing the number of detected fragments. The approach supported previous species identifications and was able to identify several unclassified isolates, such as those from Hungary and Ireland, and provides a method for identification of previously unclassified strains. We confirmed that Heterorhabditis "Irish Type", represented by two strains of different geographical origin, comprise a species different from H. megidis. We also confirmed that strain IS5 belongs to the species H. indicus rather than to H. bacteriophora, as had been suggested previously.

Gel electrophoretic restriction fragment length polymorphism analysis of DNA derived from individual nematodes, using the PhastSystem.

The DNA sequences constituting the internal transcribed spacer region, located between 18S and 26S rDNA genes within the rRNA operon, derived from single nematodes of two genera (Steinernema and Heterorhabditis) were amplified by polymerase chain reaction (PCR) and subjected to digestion by four restriction enzymes. The digests were analyzed by restriction fragment length polymorphism (RFLP) gel electrophoresis on the PhastSystem, using 7.5%T, 5%C(Bis) polyacrylamide. The downscaling from conventional agarose to PhastSystem gels permitted the analysis to be done on individual nematodes, rather than on mixed samples with average properties. The analysis time was reduced so as to allow for the electrophoretic separation on 200 samples/workday. The resulting patterns of DNA fragments differed from those obtained by agarose gel electrophoresis under conventional conditions by an increased number of detected fragments. The PhastSystem gel analysis provides the basis for taxonomical revisions.

Analytical characterisation of a capacitively coupled plasma torch with a central tube electrode.

A new type of radiofrequency capacitively coupled plasma torch is presented. The torch electrode geometry is coaxial with a tubular central electrode and one or two outer ring electrodes. The argon plasma is generated at 275 W radiofrequency power and 27.12 MHz and it has a very good stability and a low gas consumption of 0.4 l min(-1). The nebulized sample is introduced through the tubular electrode into the core of the annular shaped plasma thus achieving a better atomisation and a lower background. The limits of detection for 20 elements are in the range of ng ml(-1) and the dynamic range between 2.5 and 3.5. The best results are obtained with the torch with two outer ring electrodes.

Phase Variation in Xenorhabdus nematophilus.

Xenorhabdus nematophilus is a symbiotic bacterium that inhabits the intestine of entomopathogenic nematodes. The bacterium-nematode symbiotic pair is pathogenic for larval-stage insects. The phase I cell type is the form of the bacterium normally associated with the nematode. A variant cell type, referred to as phase II, can form spontaneously under stationary-phase conditions. Phase II cells do not elaborate products normally associated with the phase I cell type. To better define phase variation in X. nematophilus, several strains (19061, AN6, F1, N2-4) of this bacterium were analyzed for new phenotypic traits. An analysis of pathogenicity in Manduca sexta larvae revealed that the phase II form of AN6 (AN6/II) was significantly less virulent than the phase I form (AN6/I). The variant form of N2-4 was also avirulent. On the other hand, F1/II and 19061/II were as virulent as the respective phase I cells. Strain 19061/II was found to be motile, and AN6/II regained motility when the bacteria were grown in low-osmolarity medium. In contrast, F1/II remained nonmotile. The phase II cells did not produce the outer membrane protein, OpnB, that is normally induced during the stationary phase. Both phase I and phase II cells were able to support nematode growth and development. These findings indicate that while certain phenotypic traits are common to all phase II cells, other characteristics, such as virulence and motility, are variable and can be influenced by environmental conditions.