Design and optimization of a high-energy optical parametric amplifier for broadband, spectrally incoherent pulses.

Spectrally incoherent laser pulses with sufficiently large fractional bandwidth are in demand for the mitigation of laser-plasma instabilities occurring in high-energy laser-target interactions. Here, we modeled, implemented, and optimized a dual-stage high-energy optical parametric amplifier for broadband, spectrally incoherent pulses in the near-infrared. The amplifier delivers close to 400 mJ of signal energy through noncollinear parametric interaction of 100-nJ-scale broadband, spectrally incoherent seed pulses near 1053 nm with a narrowband high-energy pump operating at 526.5 nm. Mitigation strategies for high-frequency spatial modulations in the amplified signal caused by index inhomogeneities in the Nd:YLF rods of the pump laser are explored and discussed in detail.

Evaluation of second cancer induction risk by CT follow-up in oncological long-surviving patients.

The goal of establishing prompt localization of the malignant spread or recurrence of a tumor has found a powerful solution in the definition of follow-up protocols, which include the indication for CT scans on an annual or semiannual basis. In the case of long-surviving patients, however, this approach will lead to a considerable integrated dose level over a period of several years after recovery from the illness. Pathologies treated primarily by surgery and/or chemotherapy have been considered, not taking into account cancers treated with adjuvant or radical radiotherapy. Given that the most likely protocols for these cancers often call for total body scans, an estimation of the consequent effective and organ doses can be performed with acceptable accuracy. The data acquired from five centers have been collected and the related effective and organ doses calculated by means of IMPACT software. Use of the effective dose concept, however, has lately become the subject of criticism, and the recently proposed Effective Risk Model has therefore also been applied. The evaluated absolute additional risk of second tumor induction ranges between 0.1% and 10%, depending primarily on age and pathology. These results depict this additional risk as an issue of significant importance for clinical practice. A revision of follow-up and scan parameter protocols, as well as the introduction of new algorithms for dose reduction, could significantly improve the risk-benefit ratio for all the pathologies studied.

Endocytosis and intracellular localisation of type 1 ribosome-inactivating protein saporin-s6.

Saporin-S6 is a single-chain ribosome-inactivating protein (RIP) that has low toxicity in cells and animals. When the protein is bound to a carrier that facilitates cellular uptake, the protein becomes highly and selectively toxic to the cellular target of the carrier. Thus, saporin-S6 is one of the most widely used RIPs in the preparation of immunoconjugates for anti-cancer therapy. The endocytosis of saporin-S6 by the neoplastic HeLa cells and the subsequent intracellular trafficking were investigated by confocal microscopy that utilises indirect immunofluorescence analysis and transmission electron microscopy that utilises a direct assay with gold-conjugated saporin-S6 and an indirect immunoelectron microscopy assay. Our results indicate that saporin-S6 was taken up by cells mainly through receptor-independent endocytosis. Confocal microscopy analysis showed around 30% co-localisation of saporin-S6 with the endosomal compartment and less than 10% co-localisation with the Golgi apparatus. The pathway identified by the immunofluorescence assay and transmission electron microscopy displayed a progressive accumulation of saporin-S6 in perinuclear vesicular structures. The main findings of this work are the following: i) the nuclear localisation of saporin-S6 and ii) the presence of DNA gaps resulting from abasic sites in HeLa nuclei after intoxication with saporin-S6.

Combined treatment of non-small cell lung cancer with synchronous brain metastases: a single center experience.

AIM: The aim of this study was to analyze our experience with combined treatment of non-small cell lung cancer with synchronous brain metastases. METHODS: Between 1992 and 2008, 31 patients were treated by performing neurosurgery (or stereotactic radiosurgery) and lung surgery. Patients were divided into two groups according to their preoperative mediastinal work-up: group A (CT scan) and group B (FDG-PET scan). RESULTS: Twenty-six patients had one brain metastasis and five had two. Neurosurgery was performed in 10 patients, stereotactic radiosurgery in 20 and both approaches in 1. Seven patients underwent chemotherapy after cerebral procedure. Pulmonary resection was complete in 27 cases and incomplete in 4. Histological findings showed: adenocarcinoma in 19 cases, squamous cell carcinoma in 8 and large cell carcinoma in 4. All patients underwent adjuvant chemotherapy. Overall 1, 2 and 5-year survival rates were 83%, 47% and 21%, respectively. The median survival was 22 months. Univariate analysis showed a better prognosis for complete resection (P=0.008), adenocarcinomas (P=0.015), N0 disease (P=0.038), and Group B (P=0.045). Multivariate analysis showed that only the radicality of the resection (P=0.027) and Group B (P=0.047) were independent prognostic factors. CONCLUSION: Our experience confirms that selected patients with non-small cell lung cancer and synchronous brain metastases may be effectively treated by combined therapy. Complete resection, adenocarcinoma histology and N0 disease were prognostic factors. The incorporation of FDG-PET scan into the preoperative work-up may translate into a survival benefit.

Increased serum level of xanthine oxidoreductase in liver transplanted patients.

Xanthine oxidoreductase (XOR) leakage into serum has been observed in various types of liver pathology as well as after liver transplantation (LT). We determined the amount of XOR associated with LT to investigate the changes in serum enzyme level during the LT procedure and the post-operative period. Additionally, we examined whether there was any correlation between XOR levels and the surgical technique. XOR levels were measured by a competitive ELISA. In a first group of patients, the portal vein was flushed before the liver and systemic reperfusions, which occurred simultaneously. In the second group, the graft was flushed with blood from the portal vein before the systemic reperfusion. XOR showed a marked elevation in the caval effluent collected during LT and was higher compared to control serum levels at all time points that were examined after LT. The XOR levels during LT were also higher than samples taken pre-LT or from the portal blood flush before reperfusion. The XOR level was higher in Group 2 than in Group 1. Enhancement of the XOR serum level during LT was not derived from enterocytes, and it should be attributed to enzyme leakage from graft liver cells. We report the elevation of serum XOR during the three weeks following LT for the first time, as well as the influence of the graft reperfusion technique on XOR serum levels.

Toxicity and pollutant impact analysis in an urban river due to combined sewer overflows loads.

The Navile Channel (Bologna, Italy) is an ancient artificial water course derived from the Reno river. It is the main receiving water body for the urban catchment of Bologna sewer systems and also for the Waste Water Treatment Plant (WWTP) main outlet. The aim of this work is to evaluate the Combined Sewer Overflows (CSOs) impact on Navile Channel's water quality. In order to collect Navile flow and water quality data in both dry and wet weather conditions, two measuring and sampling stations were installed, right upstream and downstream the WWTP outflow. The study shows that even in case of low intensity rain events, CSOs have a significant effect on both water quantity and quality, spilling a considerable amount of pollutants into the Navile Channel and presenting also acute toxicity effects. The collected data shown a good correlations between the concentrations of TSS and of chemical compounds analyzed, suggesting that the most part of such substances is attached to suspended solids. Resulting toxicity values are fairly high in both measuring points and seem to confirm synergistic interactions between heavy metals.

The subjective feeling of burden in caregivers of elderly with dementia: how to intervene?

The aim of this study was to know how caregiver burden was influenced by caregivers' and patients' characteristics and by social support; the intention was to use these elements in order to suggest how to optimize the interventions which may reduce caregivers' subjective feeling of burden. A sample of 99 caregivers was studied using a descriptive-correlational design. Caregiver burden was assessed by using the caregiver burden inventory (CBI). The burden was due to restrictions on their personal time and to the sense of failure regarding their hopes and expectations. These results suggest that it could be useful to intervene in two ways. On one hand, improving all those interventions targeted to reduce restrictions on the personal caregivers' time, making respite care and day care units more accessible to elderly with dementia. On the other hand, giving caregivers the opportunity to benefit from interventions oriented to cope the sense of failure and the physical stress, to say, individual counseling or continued informal support.

Occupational sensitization to ribosome-inactivating proteins in researchers.

BACKGROUND: Ribosome-inactivating proteins (RIPs) are expressed in many plants. Because of their anti-infectious and anti-proliferative effects, intensive research is going on for applying these toxins in therapy against viral infections or malignancies. Recently, we demonstrated that type I allergy against RIPs from elderberry can occur. OBJECTIVE: Stimulated by our study, a group of RIP researchers reported that some of the employees had suspected allergy to RIPs. METHODS AND RESULTS: We tested their sera in ELISA on natural RIPs. Specific IgE in four subjects were found against dianthin30, gelonin, momordin, PAP-S, saporin, ricin and volkensin. In contrast, asparin and lychnin did not show any IgE binding. When separating extracts of plants containing the toxins in SDS-PAGE, RIPs appeared to be the predominant constituents. Interestingly, among the other plant proteins, they were exclusively recognized by IgE in immunoblot. RIPs derived from close botanical families share high sequence homologies. Nevertheless, in IgE inhibition experiments with human sera, cross-reactivity between RIPs also derived from non-related plants could be demonstrated. CONCLUSION: We conclude that sensitization and IgE induction to RIPs may occur upon exposure. This has to be considered when applying them in therapy against malignancies or viral infections.

Self-organization of polystyrenes into ordered microstructured films and their replication by soft lithography.

We report on the formation of ordered arrays of micrometric holes on the surface of polystyrene (PS) films cast from volatile solvents in the presence of humidity at different temperatures. The formation mechanism is investigated for PS having different molecular weights, polydispersities, and carboxylic terminations. Among the chosen materials, a highly regular honeycomb microstructured morphology is obtained on the surface of films prepared with dicarboxy-terminated PS with = 100,000. Experiments and observations on film formation indicate that polar groups are playing a fundamental role in this process. Tuning the surface tension by means of polar terminations allows the film morphology to be modified and in particular the preparation of two- or three-dimensional microstructured films. Finally, we show how these structures can be replicated by soft lithography and then used in the fields of photonic crystals and organic electronics.

Toxicity of xanthine oxidoreductase to malignant B lymphocytes.

Reactive oxygen species (ROS) generated by xanthine oxidoreductase (XOR) were toxic to B lymphoma-derived Raji cells (positive for 8A monoclonal antibody, mAb). The sensitivity of these malignant cells to the hypoxanthine/XOR system was higher than that observed in peripheral human lymphocytes. The understanding of the mechanisms of cytotoxicity induced by XOR-produced ROS is essential in view of a possible clinical application. Cell death mostly had the feature of apoptosis and post-apoptotic necrosis and depended on the activity of XOR. Catalase, but not superoxide dismutase, protected cells from the toxicity of XOR, thus indicating that cell damage depended on the production of hydrogen peroxide. The toxicity of ROS was selectively targeted to malignant Raji cells by antibody-XOR conjugation, either directly, with an 8A-XOR conjugate, or indirectly, with an 8A mAb plus an anti-mouse IgG-XOR. Both direct and indirect immunotoxins induced apoptotic death to target cells in a dose-dependent manner. These conjugates showed no aspecific cytotoxicity in conditions very similar to the ex vivo treatment of cell suspension for bone marrow transplantation. Moreover, the prevalence of apoptotic death over necrosis may reduce the in vivo inflammatory response and its local and systemic consequences, thus becoming relevant in the construction of immunotoxins with therapeutic potential.

CD38 as a target of IB4 mAb carrying saporin-S6: design of an immunotoxin for ex vivo depletion of hematological CD38+ neoplasia.

An anti-CD38 mAb (IB4) coupled to saporin-S6, a type 1 ribosome-inactivating protein (RIP), was designed for ex vivo or loco-regional therapeutical applications in myeloma and lymphoma. The ability of this immunotoxin to eliminate CD38+ cells was studied in vitro on selected CD38+ human cell lines (Raji, HBL6, L540 and CEM) and on CD38+ neoplastic cells from a Non Hodgkin Lymphoma (NHL) patient. HBL6, Raji and L540 cells resulted very sensitive to the IB4/saporin-S6 conjugate, concentrations as low as 100 pM of the immunotoxin completely inhibited protein synthesis. CD38+ neoplastic cells from the NHL patient were completely eliminated after treatment with immunotoxin at 10 nM concentration. CFU-c rescue by bone marrow precursors was maintained after exposure to the immunotoxin. These results indicate that IB4/saporin-S6 is endowed with strong and specific cytotoxic effects on selected CD38+ tumor cells lineages. Consequently, it is reasonable to propose a clinical use of the IB4/saporin-S6 for ex vivo purging of unwanted cells (e.g. depletion of contaminating neoplastic cells in aphereses obtained from G-CSF-treated patients) or for loco-regional therapies of CD38+ tumors.

Immunotoxins and other conjugates: pre-clinical studies.

A wide variety of conjugates containing RIPs, of either chemical or recombinant type, have been made and tested against dangerous cells in vitro and in animal models. Many of these pre-clinical studies will be reviewed here dividing them on the basis of the target cell and the surface molecule specifically recognized.

Multicenter randomized phase III trial of epirubicin plus paclitaxel vs epirubicin followed by paclitaxel in metastatic breast cancer patients: focus on cardiac safety.

The aim of the study was to evaluate cardiac safety of two different schedules of Epirubicin and Paclitaxel in advanced breast cancer patients enrolled into a multicenter randomized phase III trial. Patients received Epirubicin 90 mg m(-2) plus Paclitaxel 200 mg m(-2) (3-h infusion) on day 1 every 3 weeks for eight courses (arm A), or Epirubicin 120 mg m(-2) on day 1 every 3 weeks for four courses followed by four courses of Paclitaxel 250 mg m(-2) on day 1 every 3 weeks (arm B). Left ventricular ejection fraction was evaluated by bidimesional echocardiography at baseline, after four and eight courses of chemotherapy and every 4 months during follow-up. Baseline median left ventricular ejection fraction was 60% in arm A and 65% in arm B; after four courses, figures were 57 and 60%, respectively. After eight courses, the median left ventricular ejection fraction in arm A declined to 50% while no further reduction was detected in arm B by adding four courses of high-dose Paclitaxel. Seven episodes of congestive heart failure were observed during treatment in arm A. Present monitoring demonstrated that the risk of congestive heart failure or impairment in the cardiac function correlated only with the cumulative dose of Epirubicin; no impact on cardiotoxicity can be attributed to high-dose Paclitaxel.

The conjugate Rituximab/saporin-S6 completely inhibits clonogenic growth of CD20-expressing cells and produces a synergistic toxic effect with Fludarabine.

Immunotoxins are chimeric proteins consisting of a toxin coupled to an antibody. To date, several clinical trials have been conducted, and some are still ongoing, to evaluate their anti-tumor efficacy. In this view, we chemically constructed an anti-CD20 immunotoxin with the mAb Rituximab and the type 1 ribosome-inactivating protein (RIP) saporin-S6, designed for B cells non-Hodgkin's lymphoma (NHL) therapy. This immunotoxin showed a specific cytotoxicity for the CD20+ cell lines Raji and D430B, evidenced by inhibition of protein synthesis, evaluation of apoptosis and clonogenic assay. Upon conjugation, saporin-S6 increased its toxicity on target cells by at least 2 logs, with IC(50) values of 0.1-0.3 nM. The percentage of AnnexinV+ cells was over 95% in both cell lines treated with 10 nM immunotoxin. A complete elimination of Raji clones was reached with the 10 nM immunotoxin, whereas a mixture of free RIP and mAb gave about 90% of clonogenic growth. Rituximab/saporin-S6, at 10 nM concentration, also induced apoptosis in 80% of lymphoma cells from NHL patients. Moreover, sensitivity of Raji to Rituximab/saporin-S6 was augmented when cells were coincubated with Fludarabine. The synergistic toxic effect of the two drugs led to a total elimination of the neoplastic population.

Expression of CTLA-4 in nonhuman primate lymphocytes and its use as a potential target for specific immunotoxin-mediated apoptosis: results of in vitro studies.

T-cell-mediated immunoregulation is one of the main mechanisms implicated in induction and maintenance of transplantation tolerance. In this regard, deletion or modulation of xeno/alloantigen-specific T cells, as well as blocking of their interactions with other cell populations, are currently being pursued for tolerance induction in humans as well as nonhuman primates. In order to investigate whether cytotoxic T-lymphocyte antigen-4 (CTLA-4) may represent a suitable target for a T cell depletion approach in nonhuman primate models, we analysed CTLA-4 expression in peripheral blood mononuclear cells (PBMCs) from nonhuman primates and the potential role of two anti-CTLA-4 saporin-conjugated immunotoxins. The analysis was performed in PBMCs from 8 cynomolgus monkeys from Philippines and from Mauritius both at protein level by flow cytometry and at transcriptional level by RT-PCR. In addition, the apoptotic role of the immunotoxins was investigated. The results showed that CTLA-4 was expressed at variable levels depending on the origin of the cynomolgus monkeys and the resting or activated cell condition. CTLA-4 was not expressed on resting Mauritius PBMCs and showed a lower up-regulation upon PMA/PHA activation compared to the Philippines PBMCs that expressed CTLA-4 also before activation. Two CTLA-4 RNA transcripts (672 and 550 bp) were detected with levels variations after cell stimulation. Two anti-CTLA-4 immunotoxins induced in vitro apoptosis of activated PBMCs from both sources of cynomolgus monkeys. This is the first report that documents CTLA-4 expression both at protein and transcriptional level by nonhuman primate PBMCs and provides novel perspectives of xeno/allograft rejection immunotherapy based on CTLA-4 targeting.

Multimodal treatment with primary single-agent epirubicin in operable breast cancer: 5-year experience of the Michelangelo Cooperative Group.

BACKGROUND: To assess the efficacy of primary single-agent epirubicin (120 mg/m(2) every 3 weeks for three cycles) in reducing tumor burden in operable breast cancer >or=2.5 cm in largest diameter at diagnosis and its effect on the rate of conservative surgery. PATIENTS AND METHODS: A total of 319 eligible patients, who were all candidates for mastectomy, were enrolled on to a multicenter prospective non-randomized study. Tumor response was assessed clinically and pathologically. Relapse-free and overall survival were assessed on major prognostic variables. RESULTS: After primary epirubicin, complete disappearance of invasive neoplastic cells accounted for only 2.6% of patients, but 40% of patients had their primary tumor downstaged to

Transferrin toxin but not transferrin receptor immunotoxin is influenced by free transferrin and iron saturation.

BACKGROUND: Cytotoxic agents can be targeted successfully to cancer cells. The efficacy of such novel and potent anticancer strategies may be influenced by variables of iron metabolism. METHODS: The in vitro cytotoxicity against glioma cells of transferrin (Tf)-based targeted toxins was compared with that of alpha-transferrin receptor (TfR)-immunotoxin. RESULTS: Of four Tf-based targeted toxins, Tf-gelonin, Tf-pokeweed antiviral protein, Tf-momordin and Tf-saporin, inhibitory concentration 50% values against glioma-derived cell lines HS683 and U251, ranged from [4.8 +/- 1.5] x 10(-10) m for Tf-saporin to [26.9 +/- 15.3] x 10(-10) m for Tf-gelonin in [(3)H]-leucine incorporation assays. Tf-saporin and alpha-TfR-saporin-immunotoxin had similar efficacy, even in the more quantitative clonogenic assay (4-5 log kill with 1 x 10(-9) m) using the myeloma cell line RPMI 8226 and glioma cell line U251. However, on RPMI 8226, the efficacy of Tf-saporin 1 x 10(-9) m was reduced by 90% in the presence of 150 microg mL(-1)(=20% of normal plasma value) competing diferric transferrin, whereas the efficacy of the corresponding immunotoxin was affected only marginally. In addition, the efficacy of Tf-based conjugates will depend on their iron saturation state. Iron desaturation of Tf-saporin was demonstrated by [(59)Fe]-labelling, subsequent CM-Sepharose chromatography and SDS-PAGE. Desaturation led to virtually complete loss of affinity for the transferrin receptor, as determined by flow cytometry, which could be largely restored upon resaturation. CONCLUSION: Transferrin-based toxin conjugates are strongly influenced by the presence of free transferrin and the iron saturation state. The corresponding alpha-transferrin receptor-immunotoxin does not show these disadvantages, has similar efficacy and should be preferred for further experiments.

Immunotoxins containing recombinant anti-CTLA-4 single-chain fragment variable antibodies and saporin: in vitro results and in vivo effects in an acute rejection model.

Immunotoxins containing recombinant human-derived single-chain fragment variable (scFv) reagents (83 and 40) against CTLA-4 (CD152) linked to saporin, a ribosome-inactivating protein, were prepared and tested on CD3/CD28-activated T lymphocytes, MLRs, CTLA-4-positive cell lines, and hemopoietic precursors. Immunotoxins induced apoptosis in activated T lymphocytes and were able to specifically inhibit MLR between T lymphocytes and dendritic cells. The 83-saporin immunotoxin also inhibited the T cell activation in an MLR between T lymphocytes and an EBV-positive lymphoblastoid B cell line. Toxicity tests on hemopoietic precursors showed little or no effects in inhibiting colonies' growth. As the 83 scFv Ab was reactive also with activated mouse T lymphocytes, 83-saporin was tested in a model of tumor rejection consisting of C57BL/6 mice bearing a murine H.end endothelioma cell line, derived from DBA/2 mice. The lymphoid infiltration due to the presence of the tumor was reduced to a high extent, demonstrating that the immunotoxin was actually available and active in vivo. Thus, taking the results altogether, this study might represent a new breakthrough for immunotherapy, showing the possibility of targeting CTLA-4 to kill activated T cells, using conjugates containing scFv Abs and type 1 ribosome-inactivating protein.

Therapeutic outcome according to histologic subtype in 121 patients with malignant pleural mesothelioma.

One-hundred and twenty-one cases of malignant pleural mesothelioma (MPM) seen between 1986 and 1999 at the authors' Institution were reviewed. Histotype was epithelial in 88 patients (73%), sarcomatous in 21 (17%) and mixed in 12 (10%). Ninety-one patients received a treatment (38 palliative pleurectomy and no further therapy, 16 palliative pleurectomy followed by chemotherapy, 37 chemotherapy alone), while 30 were referred to supportive care only. Median survival of the whole population was 10.5 months. The 1-, 2- and 3-year survival were 40, 17 and 8%, respectively. Univariate analysis of subgroups showed that poor performance status (PS), non-epithelial histotype, Butchart stage>I and International Mesothelioma Interest Group (IMIG) stage>I were individually associated with lower survival. Patients receiving any therapy survived longer than patients treated with supportive care only (P=0.0004). Treatment modality had an independent prognostic value (P=0.00005), with a survival advantage for patients receiving surgery and adjuvant chemotherapy. Multivariate analysis confirmed the independent prognostic value of PS (P=0.001; HR=2.48) and treatment modality (P=0.003; HR=1.38). The prognostic role of PS (P=0.02) and treatment modality (P=0.01) was confirmed in the subset of patients with epithelial histology. On the contrary, therapy had no impact on survival in patients with sarcomatoid MPM (P=0.74). Despite the predicted bias of a retrospective non-randomized evaluation of treatment-related factors, patients with good PS and epithelial histology seemed to have a survival benefit from surgery or multimodality therapy, as opposite to patients with poor PS or non-epithelial histotype. However, these results must be confirmed in a larger prospective trial with uniform treatment.