Generation of three-octave-spanning transient Raman comb in hydrogen-filled hollow-core PCF.

A noise-seeded transient comb of Raman sidebands spanning three octaves from 180 to 2400 nm, is generated by pumping a hydrogen-filled hollow-core photonic crystal fiber with 26-µJ, 300-fs pulses at 800 nm. The pump pulses are spectrally broadened by both Kerr and Raman-related self-phase modulation (SPM), and the broadening is then transferred to the Raman lines. In spite of the high intensity, and in contrast to bulk gas-cell based experiments, neither SPM broadening nor ionization are detrimental to comb formation.

Preliminary results of a program for the implementation of laparoscopic colorectal surgery in an Italian comprehensive cancer center during the COVID-19 pandemic.

Despite operative benefit and oncological non-inferiority, videolaparoscopic (VLS) colorectal surgery is still relatively underutilized. This study analyzes the results of a program for the implementation of VLS colorectal surgery started in an Italian comprehensive cancer center shortly before COVID-19 outbreak. A prospective database was reviewed. The study period was divided in four phases: Phase-1 (Open surgery), Phase-2 (Discretional phase), Phase-3 (VLS implementation phase), and Phase-4 (VLS consolidation phase). Formal surgical and perioperative protocols were adopted from Phase-3. Postoperative complications were scored by the Clavien-Dindo classification. 414 surgical procedures were performed during Phase-1, 348 during Phase-2, 360 during Phase-3, and 325 during Phase-4. In the four phases, VLS primary colorectal resections increased from 11/214 (5.1%), to 55/163 (33.7%), 85/151 (57.0%), and 109/147 (74.1%), respectively. The difference was statistically significant (P < 0.001). All-type VLS procedures were 16 (3.5%), 61 (16.2%), 103 (27.0%), and 126 (38.6%) (P < 0.001). Conversions to open surgery of attempted laparoscopic colorectal resections were 17/278 in the overall series (6.1%), and 12/207 during Phase-3 and Phase-4 (4.3%). Severe (grades IIIb-to-V) postoperative complications of VLS colorectal resections were 9.1% in Phase-1, 12.7% in Phase-2, 12.8% in Phase-3, and 5.3% in Phase-4 (P = 0.677), with no significant differences with open resections in each of the four phases: 9.4% (P = 0.976), 11.1% (P = 0.799), 13.8% (P = 1.000), and 8.3% (P = 0.729). Despite the difficulties deriving from the COVID-19 outbreak, our experience suggests that volume of laparoscopic colorectal surgery can be significantly and safely increased in a specialized surgical unit by means of strict operative protocols.

Consensus conference on laparoscopic appendectomy: development of guidelines.

AIM: Laparoscopic appendectomy (LA) is not yet unanimously considered the gold standard treatment for appendicitis, despite the increasing use of advanced laparoscopic operations and the high incidence of the disease. METHOD: Due to the results of an audit which classified LA as widespread in Italy, a Consensus Conference was organized, in order to give evidence-based answers to the most debated problems regarding the operation. After researching the literature, a panel of 20 experts were selected and interviewed on hot topics; a subsequent discussion using the Delphi methodology was utilized in the course of the consensus conference and submitted to the evaluation of an audience of surgeons. RESULTS: Checkpoint statements were formulated whenever an agreement was reached. A level of evidence was then assigned to single statements and the process revised by two external reviewers. CONCLUSION: Consensus development guidelines are herein reported and regard diagnostic pathway, diagnostic laparoscopy, indications, behaviour in case of innocent appendix, technical aspects, learning curve; however, some questions remain unsolved due to the lack of evidence.

Forward modeling of pile-up events in liquid scintillator detectors for neutron emission spectroscopy.

When using liquid scintillator detectors to measure the neutron emission spectrum from fusion plasmas, the problem of pile-up distortion can be significant. Because of the large neutron rates encountered in many fusion experiments, some pile-up distortion can remain even after applying traditional pile-up elimination methods, which alters the shape of the measured light-yield spectrum and influences the spectroscopic analysis. Particularly, pile-up events appear as a high-energy tail in the measured light-yield spectrum, which obfuscates the contribution that supra-thermal ions make to the energy spectrum. It is important to understand the behavior of such "fast ions" in fusion plasmas, and it is hence desirable to be able to measure their contribution to the neutron spectrum as accurately as possible. This paper presents a technique for incorporating distortion from undetected pile-up events into the analysis of the light-yield spectrum, hence compensating for pile-up distortion. The spectral contribution from undetected pile-up events is determined using Monte Carlo methods and is included in the spectroscopic study as a pile-up component. The method is applied to data from an NE213 scintillator detector at JET and validated by comparing with results from the time-of-flight spectrometer TOFOR, which is not susceptible to pile-up distortion. Based on the results, we conclude that the suggested analysis method helps counteract the problem of pile-up effects and improves the possibilities for extracting accurate fast-ion information from the light-yield spectrum.

Neutron emission spectroscopy of D plasmas at JET with a compact liquid scintillating neutron spectrometer.

Neutron emission spectroscopy is a diagnostic technique that allows for energy measurements of neutrons born in nuclear reactions. The JET tokamak fusion experiment (Culham, UK) has a special role in this respect as advanced spectrometers for 2.5 MeV and 14 MeV neutrons have been developed here for the first time for measurements of the neutron emission spectrum from D and DT plasmas with unprecedented accuracy. Twin liquid scintillating neutron spectrometers were built and calibrated at the Physikalisch-Technische Bundesanstalt (PTB) (Braunschweig, Germany) and installed on JET in the recent years with tangential-equatorial (KM12) and vertical-radial (KM13) view lines, with the latter only recently operational. This article reports on the performance of KM12 and on the development of the data analysis methods in order to extract physics information upon D ions kinematics in JET auxiliary-heated D plasmas from 2.5 MeV neutron measurements. The comparison of these results with the correspondents from other JET neutron spectrometers is also presented: their agreement allows for JET unique capability of multi-lines of sight neutron spectroscopy and for benchmarking other 14 MeV neutron spectrometers installed on the same lines of sight in preparation for the DT experimental campaign at JET.

JET diagnostic enhancements testing and commissioning in preparation for DT scientific campaigns.

In order to optimize the scientific exploitation of JET (Joint European Torus) during the upcoming deuterium-tritium experiments, a set of diagnostic systems is being enhanced. These upgrades focus mainly on the experimental and operational conditions expected during tritium campaigns. It should be stressed that measurements relevant for burning plasmas are specifically targeted. Previously non-available capabilities, such as a current measurement system fully covering all poloidal field circuits, are described in detail. Instrument descriptions, performance prediction, testing, and initial commissioning results of these systems are presented.

Polyketone polymer: a new support for direct enzyme immobilization.

Polyketone polymer -[-CO-CH(2)-CH(2)-](n)-, obtained by copolymerization of ethene and carbon monoxide, is utilized for immobilization of three different enzymes, one peroxidase from horseradish (HRP) and two amine oxidases, from bovine serum (BSAO) and lentil seedlings (LSAO). The easy immobilization procedure is carried out in diluted buffer, at pH 7.0 and 3 degrees C, gently mixing the proteins with the polymer. No bifunctional reagents and spacer arms are required for the immobilization, which occurs exclusively via a large number of hydrogen bonds between the carbonyl groups of the polymer and the -NH groups of the polypeptidic chain. Experiments demonstrate a high linking capacity of polymer for BSAO and an extraordinary strong linkage for LSAO. Moreover, activity measurements demonstrate that immobilized LSAO totally retains the catalytic characteristics of the free enzyme, where only a limited increase of K(M) value is observed. Finally, the HRP-activated polymer is successfully used as active packed bed of an enzymatic reactor for continuous flow conversion and flow injection analysis of hydrogen peroxide containing solutions.

Extremely broadband single-shot cross-correlation frequency-resolved optical gating using a transient grating as gate and dispersive element.

A cross-correlation frequency-resolved optical gating (FROG) concept, potentially suitable for characterizing few or sub-cycle pulses in a single shot, is described in which a counter-propagating transient grating is used as both the gate and the dispersive element in a FROG spectrometer. An all-reflective setup, which can operate over the whole transmission range of the nonlinear medium, within the sensitivity range of the matrix sensor, is also proposed, and proof-of-principle experiments for the ultraviolet and visible-to-near-infrared spectral ranges are reported.

Autologous dendritic cells derived from CD34+ progenitors and from monocytes are not functionally equivalent antigen-presenting cells in the induction of melan-A/Mart-1(27-35)-specific CTLs from peripheral blood lymphocytes of melanoma patients with low frequency of CTL precursors.

Peptide presentation by autologous dendritic cells (DCs) is a new tool to activate tumor antigen-specific T cells in melanoma patients. However, it is not known whether autologous DCs, differentiated by two of the most efficient protocols (from CD34+ progenitors or from monocytes), are equally effective as professional antigen-presenting cells (APCs) when the patients have a low frequency of peptide-specific precursors. To this end, a limiting dilution assay was applied to evaluate the frequency of antigen-specific CTL precursors (CTLps) in peripheral blood of HLA-A*0201+ melanoma patients. Then, from two melanoma patients showing low frequency of CTLps to melanoma antigen-A/melanoma antigen recognized by T cell (Melan-A/Mart-1)(27-35) peptide, autologous DCs were differentiated from granulocyte colony-stimulating factor-mobilized CD34+ progenitors or from monocytes. CD34+- and monocyte-derived DCs were characterized by a similar proportion of CD1a+ cells expressing HLA class II antigens and CD54, CD80, and CD86 molecules. Both types of DC presented Melan-A/Mart-1(27-35) and tyrosinase(369-377) peptides to melanoma-specific CTL clones and were equally effective as peptide-pulsed APCs in the activation of influenza A matrix(58-66)-specific CTLs from high-frequency precursors (1294/10(6) and 1789/10(6) lymphocytes in the two patients). However, efficient activation of Melan-A/Mart-1(27-35)-specific CTLs from low-frequency precursors (158/10(6) and 77/10(6) lymphocytes) of the two patients was markedly dependent on the use of peptide-loaded CD34+-derived DCs. These results suggest that CD34+- and monocyte-derived DCs are not functionally equivalent APCs for the activation of low-frequency peptide-specific CTLps.

Differences between in vivo and in vitro activation of cancer patient lymphocytes by recombinant interleukin 2: possible role for lymphokine-activated killer cell infusion in the in vivo-induced activation.

In this study 15 consecutive melanoma patients were treated with two courses of bolus recombinant interleukin 2 (rIL2) and rIL2 plus in vitro-generated lymphokine-activated killers (LAK), respectively. The immunological monitoring performed after 4 days of rIL2 or rIL2 plus LAK, indicate that the in vivo peripheral blood lymphocyte (PBL), activation (spontaneous proliferation, tumor cytotoxicity, number of DR+ PBL, obtained after the second cycle of rIL2 plus LAK is significantly higher than after the first cycle of rIL2 alone. During the 5-day interval between the two courses, PBL activation returns to baseline levels and no evidence for increased sensitivity of PBL to rIL2 is present. To further confirm this, two additional patients were studied, in whom rIL2 was administered by continuous i.v. infusion. In these two patients the in vitro versus in vivo PBL activation could be directly and simultaneously compared by using in vitro the same concentration of rIL2 reached and maintained in the patients' sera. The PBL activation induced in vivo by a cycle of rIL2 alone was significantly less (about 10 times) than that obtained in vitro with a comparable rIL2 concentration. Thus, the infusion of in vitro highly activated PBL could explain the increased in vivo lymphocyte activation of the second cycle of rIL2 plus LAK over the first cycle of rIL2 alone.

A superagonist variant of peptide MART1/Melan A27-35 elicits anti-melanoma CD8+ T cells with enhanced functional characteristics: implication for more effective immunotherapy.

In the present study, we show that a singly substituted peptide derived from the epitope MART1(27-35) and containing a Leu in position 1 (LAGIGILTV; 1L) behaves as a superagonist by in vitro inducing specific T cells with enhanced immunological functions. 1L-specific CTLs can be raised from peripheral blood of HLA-A2+ melanoma patients more efficiently than T cells specific for the cognate peptide. These T cells show a greater sensitivity to native MART1(27-35) when compared with CTL variable raised to parental peptide from the same patients. More importantly, anti-1L but not anti-native T cells display high levels of interferon gamma production at early time points, and readily secreted interleukin-2 in response to native epitope endogenously presented by melanoma cells. Additionally, anti-1L T cells are insensitive to the inhibitory effects of MART1(27-35) natural analogues that antagonize the lytic response of CTLs raised to the cognate peptide. Analysis of T-cell receptor variable beta usage suggests that the native and 1L peptides stimulate different components of the MART1(27-35)-reactive T cell population. These data provide rationale to the use of superagonist analogues of tumor antigens for inducing in vivo immunization potentially able to overcome tumor immune escape and mediate a more significant control of tumor growth.

First neutron spectroscopy measurements with a pixelated diamond detector at JET.

A prototype Single crystal Diamond Detector (SDD) was installed at the Joint European Torus (JET) in 2013 along an oblique line of sight and demonstrated the possibility to carry out neutron spectroscopy measurements with good energy resolution and detector stability in discharges heated by neutral beam injection and radio-frequency waves. Starting from these positive results, within the Vertical Neutron Spectrometer project of the Joint European Torus, we have developed a pixelated instrument consisting of a matrix of 12 independent SDDs, called the Diamond Vertical Neutron Spectrometer (DVNS), which boosts the detection efficiency of a single SDD by an order of magnitude. In this paper we describe the main features of the DVNS, including the detector design, energy resolution, and data acquisition system for on-line processing. Preliminary spectroscopy measurements of 2.5 MeV neutrons from the present deuterium plasma at JET are finally presented.

JET diagnostic enhancements in preparation for DT operations.

In order to complete the exploitation of the JET ITER-like Wall and to take full benefit from deuterium-tritium experiments on JET, a set of diagnostic system refurbishments or upgrades is in progress. These diagnostic enhancements focus mainly on neutron, gamma, fast ions, instabilities, and operations support. These efforts intend to provide better spatial, temporal, and energy resolution while increasing measurement coverage. Also previously non-existing capabilities, such as Doppler reflectometry is now available for scientific exploitation. Guaranteeing diagnostic reliability and consistency during the expected DT conditions is also a critical objective of the work and systems being implemented. An overview of status and scope of the ongoing projects is presented.

Recognition of melanoma-derived antigens by CTL: possible mechanisms involved in down-regulating anti-tumor T-cell reactivity.

Several T cell-recognized epitopes presented by melanoma cells have been identified recently. Despite the large array of epitopes potentially available for clinical use, it is still unclear which of these antigens could be effective in mediating anti-tumor responses when used as a vaccine. Preliminary studies showed that immunization of melanoma patients with epitopes derived from proteins of the MAGE family may result in significant clinical regressions. However, no sign of systemic immunization could be observed in peripheral blood of treated patients. Conversely, significant immunization (detected as increased antigen-specific CTL activity in peripheral blood) was obtained by vaccinating HLA-A2.1+ melanoma patients with the immunodominant epitope (residues 27-35) of the differentiation antigen MART-1, but this immunization was not accompanied by a significant clinical response. To implement immunotherapeuties capable of significantly impacting disease outcome, it is necessary to identify the potential mechanisms responsible for the failure of some antigens to mediate significant anti-tumor responses in vivo. In the case of the MART-1(27-35) epitope, we hypothesize that one of these mechanisms may be related to the existence of natural analogs of this peptide in other human normal proteins.

Limited antitumor T cell response in melanoma patients vaccinated with interleukin-2 gene-transduced allogeneic melanoma cells.

We have immunized advanced melanoma patients with a HLA-A2-compatible human melanoma line genetically modified to release interleukin-2 (IL-2), to elicit or increase a T cell-mediated anti-melanoma response that may affect distant lesions. Twelve stage-IV patients were injected subcutaneously at days 1, 13, 26, and 55 with IL-2 gene-transduced and irradiated melanoma cells at doses of 5 or 15 x 10(7) cells. Both local and systemic toxicities were mild, consisting of transient erythema at the vaccination site; fever occurred in a minority of patients. Three mixed responses were recorded. Seven patients were evaluable for immunological studies. Mixed tumor-lymphocyte cultures carried out with different allogeneic HLA-A2-matched melanoma lines as stimulators and targets revealed an increase in the MHC-unrestricted, but no changes in the MHC-restricted, cytotoxicity in peripheral blood lymphocytes (PBL) obtained after vaccination as compared with those obtained before vaccination. Increased recognition of the tyrosinase 368-376 peptide occurred in post-vaccination PBL of one patient, whereas a weak increase in recognition of the gp100 280-288 peptide was detectable in another patient; these 2 patients also recognized the gp100 457-466 peptide. After in vitro, stimulation with the only available autologous melanoma line, CD4+ cells with autologous tumor-specific cytotoxicity and ability to release interferon-gamma (IFN-gamma) were found in post- but not in pre-vaccination PBL. In the same patient, as well as in another patient, limiting dilution analysis showed that vaccination resulted in an increased frequency of melanoma-specific cytotoxic T lymphocyte (CTL) precursors. These results indicate that vaccination with cells releasing IL-2 locally can expand a T cell response against antigen(s) of autologous, untransduced tumor, although this response occurred in a minority of the melanoma patients studied.

Interleukin-2 gene-transduced human melanoma cells efficiently stimulate MHC-unrestricted and MHC-restricted autologous lymphocytes.

Two human melanoma lines were transduced by a retroviral vector with the gene of the human interleukin-2 (IL-2) and characterized for their immunological properties in comparison with the parental lines. Transduction resulted in the production of biologically active IL-2 in the average amounts of 2,282 and 2,336 pg/ml per 10(5) cells per 24 hr over 3 and 2 months by the Me14932/IL-2 and the Me1B6/IL-2 lines, respectively. Melanoma-transduced cells lost their tumorigenicity in nude mice. No major changes in the phenotype were observed in IL-2 gene-transduced lines. In fact, more than 90% of cells expressed class I and II(DR) HLA, adhesion molecules, integrins, and melanoma-associated antigens. Irradiation with 100-400 Gy, while inhibiting tumor cell growth in vitro, allowed the release of IL-2 by the transduced cells for at least 5 weeks. The two melanoma lines also maintained susceptibility to lysis by lymphokine-activated killer (LAK) cells and by a HLA-A2-restricted melanoma-specific cytotoxic T lymphocyte (CTL) clone recognizing the melanoma antigen (Melan-A). In a limiting dilution assay, transduced, but not parental melanoma lines unless added with an amount of IL-2 comparable to that released by the transduced cells, were able to expand both nonspecific and melanoma-specific CTL precursors from autologous peripheral blood lymphocytes (PBL). In mixed lymphocytes-tumor cultures, IL-2 gene-transduced melanoma cells stimulated the expansion of major histocompatibility complex (MHC)-unrestricted effectors from autologous PBL, and of CD3+ CD8+ MHC-restricted CTL from tumor-invaded lymph nodes. These results indicate that IL-2 gene transduction does not alter significantly the expression of the immunologically relevant molecules of human melanoma lines while increasing their ability to stimulate both specific and nonspecific lymphocyte responses. These lines will be of value in the vaccination of melanoma patients.

Vaccination of melanoma patients with interleukin 4 gene-transduced allogeneic melanoma cells.

A human melanoma line genetically modified to release interleukin 4 (IL-4) was utilized to immunize advanced melanoma patients in order to elicit or increase a specific anti-melanoma immune response, which may affect distant lesions. Twelve metastatic melanoma patients were injected subcutaneously at least three times with 5 x 10(7) IL-4 gene-transduced and irradiated allogeneic melanoma cells per dose. Both systemic and local toxicities were mild, consisting of transient fever and erythema, swelling, and induration at the vaccination site. Two mixed but not complete or partial clinical responses were recorded. To assess the immune response of vaccinated patients, both serological and cell-mediated activities were evaluated. Antibodies to alloantigens could be detected in 2 of 11 patients tested. Mixed tumor-lymphocyte cultures were performed, utilizing autologous and allogeneic HLA-A2-matched melanoma lines as simulators and targets. A significant increase in IFN-gamma release was detected in 7 of 11 cases when postvaccination lymphocytes were stimulated by the untransduced allomelanoma cells. However, induction of a specific recognition of autologous melanoma cells by PBLs was obtained after vaccination in only one of six cases studied. This response involved the melanoma peptide Melan-A/MART-1(27-35) that was recognized in an HLA-A2-restricted fashion. These results indicate that vaccination with allogeneic melanoma cells releasing IL-4 locally can expand a T cell response against antigen(s) of autologous, untransduced tumor, although in a minority of patients.

Neuropsychological and neurophysiological assessment of the central effects of interleukin-2 administration.

Neuropsychiatric disturbances may occur following interleukin-2 (IL2) administration. We studied the effects of IL2 infusion on cerebral functions in 7 patients with neuropsychological tests and event-related evoked potentials (P300). We observed a failure in the cognitive performances, an increase in latency, and a decrease in amplitude of P300. These effects followed IL2 administration and were reversible.