Automated hybrid volumetric modulated arc therapy (HVMAT) for whole-breast irradiation with simultaneous integrated boost to lumpectomy area : A treatment planning study.

PURPOSE: To develop an automated treatment planning approach for whole breast irradiation with simultaneous integrated boost using an automated hybrid VMAT class solution (HVMAT). MATERIALS AND METHODS: Twenty-five consecutive patients with left breast cancer received 50 Gy (2 Gy/fraction) to the whole breast and an additional simultaneous 10 Gy (2.4 Gy/fraction) to the tumor cavity. Ipsilateral lung, heart, and contralateral breast were contoured as main organs-at-risk. HVMAT plans were inversely optimized by combining two open fields with a VMAT semi-arc beam. Open fields were setup to include the whole breast with a 2 cm flash region and to carry 80% of beams weight. HVMAT plans were compared with three tangential techniques: conventional wedged-field tangential plans (SWF), field-in-field forward planned tangential plans (FiF), and hybrid-IMRT plans (HMRT). Dosimetric differences among the plans were evaluated using Kruskal-Wallis one-way analysis of variance. Dose accuracy was validated using the PTW Octavius-4D phantom together with the 1500 2D-array. RESULTS: No significant differences were found among the four techniques for both targets coverage. HVMAT plans showed consistently better PTVs dose contrast, conformity, and homogeneity (p < 0.001 for all metrics) and statistically significant reduction of high-dose breast irradiation. V55 and V60 decreased by 30.4, 26.1, and 20.8% (p < 0.05) and 12.3, 9.9, and 6.0% (p < 0.05) for SWF, FIF, and HMRT, respectively. Pretreatment dose verification reported a gamma pass-rate greater than the acceptance threshold of 95% for all HVMAT plans. In addition, HVMAT reduced the time for full planning optimization to about 20 min. CONCLUSIONS: HVMAT plans resulted in superior target dose conformity and homogeneity compared to other tangential techniques. Due to fast planning time HVMAT can be applied for all patients, minimizing the impact on human or departmental resources.

Personalized Treatment Planning Automation in Prostate Cancer Radiation Oncology: A Comprehensive Dosimetric Study.

BACKGROUND: In radiation oncology, automation of treatment planning has reported the potential to improve plan quality and increase planning efficiency. We performed a comprehensive dosimetric evaluation of the new Personalized algorithm implemented in Pinnacle3 for full planning automation of VMAT prostate cancer treatments. MATERIAL AND METHODS: Thirteen low-risk prostate (without lymph-nodes irradiation) and 13 high-risk prostate (with lymph-nodes irradiation) treatments were retrospectively taken from our clinical database and re-optimized using two different automated engines implemented in the Pinnacle treatment system. These two automated engines, the currently used Autoplanning and the new Personalized are both template-based algorithms that use a wish-list to formulate the planning goals and an iterative approach able to mimic the planning procedure usually adopted by experienced planners. In addition, the new Personalized module integrates a new engine, the Feasibility module, able to generate an "a priori" DVH prediction of the achievability of planning goals. Comparison between clinically accepted manually generated (MP) and automated plans generated with both Autoplanning (AP) and Personalized engines (Pers) were performed using dose-volume histogram metrics and conformity indexes. Three different normal tissue complication probabilities (NTCPs) models were used for rectal toxicity evaluation. The planning efficiency and the accuracy of dose delivery were assessed for all plans. RESULTS: For similar targets coverage, Pers plans reported a significant increase of dose conformity and less irradiation of healthy tissue, with significant dose reduction for rectum, bladder, and femurs. On average, Pers plans decreased rectal mean dose by 11.3 and 8.3 Gy for low-risk and high-risk cohorts, respectively. Similarly, the Pers plans decreased the bladder mean doses by 7.3 and 7.6 Gy for low-risk and high-risk cohorts, respectively. The integral dose was reduced by 11-16% with respect to MP plans. Overall planning times were dramatically reduced to about 7 and 15 min for Pers plans. Despite the increased complexity, all plans passed the 3%/2 mm γ-analysis for dose verification. CONCLUSIONS: The Personalized engine provided an overall increase of plan quality, in terms of dose conformity and sparing of normal tissues for prostate cancer patients. The Feasibility "a priori" DVH prediction module provided OARs dose sparing well beyond the clinical objectives. The new Pinnacle Personalized algorithms outperformed the currently used Autoplanning ones as solution for treatment planning automation.

Campylobacter jejuni cell lysates differently target mitochondria and lysosomes on HeLa cells.

Campylobacter jejuni is the most common cause of bacterial gastroenteritis in humans. The synthesis of cytolethal distending toxin appears essential in the infection process. In this work we evaluated the sequence of lethal events in HeLa cells exposed to cell lysates of two distinct strains, C. jejuni ATCC 33291 and C. jejuni ISS3. C. jejuni cell lysates (CCLys) were added to HeLa cell monolayers which were analysed to detect DNA content, death features, bcl-2 and p53 status, mitochondria/lysosomes network and finally, CD54 and CD59 alterations, compared to cell lysates of C. jejuni 11168H cdtA mutant. We found mitochondria and lysosomes differently targeted by these bacterial lysates. Death, consistent with apoptosis for C. jejuni ATCC 33291 lysate, occurred in a slow way (>48 h); concomitantly HeLa cells increase their endolysosomal compartment, as a consequence of toxin internalization besides a simultaneous and partial lysosomal destabilization. C. jejuni CCLys induces death in HeLa cells mainly via a caspase-dependent mechanism although a p53 lysosomal pathway (also caspase-independent) seems to appear in addition. In C. jejuni ISS3-treated cells, the p53-mediated oxidative degradation of mitochondrial components seems to be lost, inducing the deepest lysosomal alterations. Furthermore, CD59 considerably decreases, suggesting both a degradation or internalisation pathway. CCLys-treated HeLa cells increase CD54 expression on their surface, because of the action of lysate as its double feature of toxin and bacterial peptide. In conclusion, we revealed that C. jejuni CCLys-treated HeLa cells displayed different features, depending on the particular strain.

Microbiological study of cosmetic products during their use by consumers: health risk and efficacy of preservative systems.

AIM: To evaluate the microbial contamination of 91 cosmetics (23 o/w emulsions, 47 tensiolytes, 21 aqueous pastes) in three different states of use (intact, in-use, ending product) and the protection efficacy of the preservative systems most frequently used in the analysed cosmetic formulations. METHODS AND RESULTS: Total bacterial count, isolation and identification of pathogenic isolates were performed on the collected cosmetics. About 10.6% of tensiolytes (13.5% bath foam, 6.7% shampoo, 10% liquid soaps) were contaminated by Staphylococcus warneri, Staphylococcus epidermidis and Pseudomonas putida. The efficacy of the preservative systems of two cosmetic products, tested against standard micro-organisms (Staphylococcus aureus ATCC 4338 and Pseudomonas aeruginosa ATCC 9027) and two isolates from cosmetics in this study (S. epidermidis and P. putida), satisfied the Cosmetics, Toiletries, and Fragrance Association and Official Italian Pharmacopeia criteria, while only one tested cosmetic respected the Rapid Challenge Test criterion. CONCLUSIONS: Contaminated cosmetic products are relatively uncommon, but some products, unable to suppress the growth of several micro-organisms, represent a potential health hazard. SIGNIFICANCE AND IMPACT OF THE STUDY: The challenge test may be performed not only during the preparation of the preservative system in the intact cosmetics, but also be used to evaluate the protection efficacy during their use.

'In vivo' studies on the pathophysiological mechanism of Vibrio parahaemolyticus TDH(+)-induced secretion.

The thermostable direct haemolysin (TDH) is considered to be the major virulence factors of Vibrio parahaemolyticus; however, poor information is available about its mechanism of action. In our study we examined the capacity of two V. parahaemolyticus TDH-producers (strains 2067 and 3305) to induce fluid secretion in rat ileal loop and to reveal the role of calcium ions (Ca(2+)), calmodulin (CaM), and protein kinase C (PKC) in V. parahaemolyticus TDH(+)-induced fluid secretion. The results show that V. parahaemolyticus TDH(+) strains were able to induce secretion in small intestine; on the contrary, this ability was not evidenced in the V. parahaemolyticus TDH(-) strain used as negative control. The data suggest an enterotoxic activity of haemolysin. Calcium ionophore A23187 and 1-verapamil (calcium channel blocker), when injected alone, induced fluid accumulation in the control loops. A further increase in fluid accumulation (P<0.001) was noted when calcium ionophore was injected along with bacterial suspension of both TDH(+) strains and a significant decrease (P<0.001) in experimental loops when 1-verapamil was inoculated along with bacterial suspension. The other modulating agents increased fluid accumulation in both control and experimental loops, without significant differences with respect to the positive control. Our findings suggest that Ca(2+) appears to be an important messenger involved in the stimulation of intestinal secretion, contrary to PKC and calmodulin which do not appear to have any role.

Recipient-induced transfer of the symbiotic plasmid pRL1JI in Rhizobium leguminosarum bv. viciae is regulated by a quorum-sensing relay.

Analysis of the regulation of plasmid transfer genes on the symbiotic plasmid pRL1JI in Rhizobium leguminosarum bv. viciae has revealed a novel regulatory relay that is specifically poised to detect an N-acyl-homoserine lactone (AHL) made by different cells (potential recipients of pRL1JI). Adjacent to the traI-trbBCDEJKLFGHI plasmid transfer operon on pRL1JI are two regulatory genes, bisR and traR, which encode LuxR-type quorum-sensing regulators required for conjugation. Potential recipients of pRL1JI induce the traI-trb operon and plasmid transfer via a quorum-sensing relay involving BisR, TraR and the traI-trb operon in donor cells. BisR induces expression of traR in response to N-(3-hydroxy-7-cis-tetradecenoyl)-l-homoserine lactone (3-OH-C14:1-HSL), which is produced by CinI in potential recipient strains. In donor strains (carrying pRL1JI), BisR represses the expression of the chromosomal gene cinI; this repression results in a very low level of formation of 3-OH-C14:1-HSL and hence relatively low levels of expression of traR and the traI-trb operon in strains carrying pRL1JI. However, if 3-OH-C14:1-HSL from potential recipients is present, then traR and plasmid transfer are induced. The induction of traR occurs at very low concentrations of 3-OH-C14:1-HSL (around 1 nm). TraR then induces the traI-trb operon in a quorum-sensing dependent manner in re-sponse to the TraI-made AHLs, N-(3-oxo-octanoyl)-l-homoserine lactone and N-(octanoyl)-l-homoserine lactone. The resulting autoinduction results in high levels of expression of the traI-trb operon. Premature expression of the traI-trb operon is reduced by TraM, which probably titres out TraR preventing expression of traI when there are low levels of traR expression. Expression of traR in stationary phase cells is limited by feedback inhibition mediated by TraI-made AHLs.

Retention of virulence in viable but non-culturable halophilic Vibrio spp.

The viable but non-culturable (VBNC) forms of two environmental strains of Vibrio alginolyticus 1 and Vibrio parahaemolyticus 66 and one strain of V. parahaemolyticus ATCC 43996 showing virulence characteristics (hemolysin production, adhesive and/or cytotoxic ability, in vivo enteropathogenicity) were obtained by culturing bacteria in a microcosm consisting of artificial sea water (ASW) and incubating at 5 degrees C with shaking. Every 2 days, culturability of the cells in the microcosm was monitored by spread plates on BHI agar and total count and the percentage of viable cells were determined by double staining with DAPI and CTC. When cell growth was not detectable (<0.1 CFU/ml), the population was considered non-culturable and, then, the VBNC forms were resuscitated in a murine model. For each strain, eight male Balb/C mice were intragastrically inoculated with 0.1 ml of concentrated ASW bacterial culture. Two mice from each group were sacrificed at 2, 4, 8, and 12 days after challenge for autopsy and re-isolation of the microorganisms from the intestinal tissue cultures. Isolation was obtained in 25% of the animals challenged with the VBNC V. alginolyticus strain, in 37.5% of those challenged with the VBNC V. parahaemolyticus strain of environmental origin and in 50% of the animals infected with VBNC V. parahaemolyticus ATCC 43996. The strains thus isolated were again subjected to biological assays to determine the retention of pathogenicity. The virulence characteristics that seemed to disappear after resuscitation in the mouse were subsequently reactivated by means of two consecutive passages of the strains in the rat ileal loop model. The results obtained indicate that VBNC forms of the strains examined can be resuscitated and retain their virulence properties.

On the outcome in stroke patients one year later: the role of atrial fibrillation.

The post-stroke aftermath of patients suffering from atrial fibrillation (AF) is investigated. A total of 104 consecutive patients (80 with AF and 24 without AF) were referred to a rehabilitation centre and enrolled. They underwent a rehabilitative programme. Disability was assessed by using the Functional Independence Measure (FIM), at the time of admission, discharge and after 1 year. Death was more frequent over the 1-year interval in AF group. Ten patients died (42%), whereas in the non-AF group death occurred in 15 cases (19%). By using chi-square test, the difference was significant (P < 0.02). The logistic regression analysis indicated that the unique variable affecting the FIM at follow-up was the FIM at discharge (P < 0.0001). AF was not a significant variable in the equation. The result can be attributed to subject attrition in the AF group, as the most severe patients died and survivors could benefit from a better functional status.

Importance and evaluation of comorbidity in rehabilitation.

PURPOSE: The aim of this study is to evaluate the relationship between comorbidity and the disability in patients referred to a rehabilitation centre. Patients often suffer from associated diseases which may negatively influence the outcome. METHOD: After evaluation of several scales, the Cumulative Illness Rating Scale (CIRS) was selected. For the study 413 consecutive patients were enrolled. RESULTS: In this series, neurological patients presented with higher severity and higher comorbidity than orthopaedic patients. Orthopaedic cases were older but neurological cases had higher severity and higher comorbidity. Indeed, a significant negative correlation between severity and dependence scores was found. Comorbidity, also, showed a significant negative correlation with FIM both in the orthopaedic and in the neurological patients. These data show that severe comorbid conditions influence functional autonomy. Severity and Comorbidity were correlated both in orthopaedic cases and in neurological cases as well. CONCLUSIONS: A review of geriatric literature demonstrates lower values in the patients in rehabilitation. The difference is due to our accurate selection of patients at admission, where general health conditions are considered. In conclusion the CIRS scale should be used as a method for selecting patients at admission or as a prognostic index for improvement at discharge. CIRS, however, has some inconveniences and amelioration is necessary, such as the inclusion of a double testing (admission-discharge), psychiatric disturbances and a new item for skin alone. CIRS should not be applied if a patient referred for rehabilitation has the worst score in even a single item.

Studies on the development and stability of resistance of Helicobacter pylori to metronidazole and clarithromycin.

Thirty-one strains of Helicobacter pylori, susceptible to metronidazole and clarithromycin, were isolated from 31 biopsy samples from patients with various gastric pathologies. We investigated the possibility of selecting resistant strains and the stability of resistance by exposing isolates to increasing subinhibitory concentrations of metronidazole and clarithromycin using a serial passages technique. Resistance to metronidazole was obtained in 100% of the isolates, while 32.2% displayed resistance to clarithromycin. Resistance to metronidazole was stable in 93.5% (29 of 31) of the isolates, whereas 100% (10 of 10) of the strains were stably resistant to clarithromycin. The stability of the resistance that occurred after three passages on medium containing the two drugs was statistically significant (P <0.001). Thus, the technique of serial passages in vitro could be useful as a first screening in selecting drugs for use in therapeutic protocols for clinical trials.

Very old people can have favorable outcome after hip fracture: 58 patients referred to rehabilitation.

We examined both impairment and disability of 58 patients who were referred to our rehabilitation center after surgery of hip fracture. The mean age was 86.7 years. The impairment was measured by the Motricity Index (MI) scale and disability by means of the Functional Independence Measure (FIM) scale. At admission, the MI median value was 64 and at discharge the value was 84 (range of scale, 0-100). The FIM median value was 57.5 at admission and 82 at discharge (range of scale, 18-126). Our data indicate that, on average, patients recover, even at very advanced age, but still require supervision at discharge. Twelve patients died after complications of previous risk factors. When general conditions were satisfactory, no complications arose.

Red blood cells as an antigen-delivery system.

The use of adjuvants is usually required to induce strong immunological responses to protein antigens. However, in many cases these adjuvants cannot be extensively applied in human and veterinary vaccinations because of associated inflammatory reactions or granuloma formation. We show here that protein antigens (bovine serum albumin, hog liver uricase, and yeast hexokinase), coupled to autologous red blood cells by way of a biotin-avidin-biotin bridge, elicit an immunological response in mice similar to or higher than that obtained by the use of Freund's adjuvant. Quantities as low as 0.5 micrograms/mouse are high enough to generate these immunological responses. Furthermore, splenocytes of mice immunized by red blood cell-coupled antigens can be used to generate hybridomas secreting monoclonal antibodies. Thus, the delivery of antigens by autologous red blood cells is an effective way to avoid the use of adjuvants for producing anti-peptide antibodies and possibly to generate peptide vaccines.

ATP modifications in hexokinase deficient fibroblasts exposed to nutrient shifts.

Human fibroblasts from a patient homozygous for hexokinase (HK) deficiency and normal controls were submitted to different nutrient shifts to investigate the role of glucose metabolism on ATP levels. HK-deficient cells maintained in the presence of both glucose and L-glutamine had 20 per cent less ATP than controls and an ATP/ADP ratio of 18 instead of 37-40. Glucose alone was not able to maintain normal ATP levels neither in the controls nor in the HK-deficient cells. However while in control cells the ATP/ADP ratio was not modified, it was reduced to 10 in HK deficiency. The effect of several hexoses on ATP levels was also investigated. Mannose, which is metabolized by HK phosphorylation, was the least efficient for ATP maintenance in HK-deficient cells. In contrast galactose, which is metabolized by a specific galactokinase, provided ATP values close to those observed in the presence of glutamine. These results suggest that glucose metabolism is an important determinant in the maintenance of ATP levels in cultured cells and that glutamine, although being a good metabolic substrate, is not sufficient to maintain normal ATP concentrations.