Excimer laser technology in percutaneous coronary interventions: Cardiovascular laser society's position paper.

Excimer Laser Coronary Atherectomy (ELCA) is a well-established therapy that emerged for the treatment of peripheral vascular atherosclerosis in the late 1980s, at a time when catheters and materials were rudimentary and associated with the most serious complications. Refinements in catheter technology and the introduction of improved laser techniques have led to their effective use for the treatment of a wide spectrum of complex coronary lesions, such as thrombotic lesions, severe calcific lesions, non-crossable or non-expandable lesions, chronic occlusions, and stent under-expansion. The gradual introduction of high-energy strategies combined with the contrast infusion technique has enabled us to treat an increasing number of complex cases with a low rate of periprocedural complications. Currently, the use of the ELCA has also been demonstrated to be effective in acute coronary syndrome (ACS), especially in the context of large thrombotic lesions.

Knowledge and attitudes on influenza vaccination among Italian physicians specialized in respiratory infections: an Italian Respiratory Society (SIP/IRS) web-based survey.

INTRODUCTION: Influenza epidemics are one of the main causes of morbidity and mortality worldwide. Influenza vaccination is considered the most important public health intervention to prevent seasonal influenza infection. European health authority policies focus on patient protection by vaccinating both these subjects and their care-givers, including health-care workers (HCWs). The aim of this survey is to investigate knowledge about influenza vaccination and intention to get vaccinated among Italian HCWs who take care patients with respiratory disease. METHODS: An anonymous web-based survey was addressed to members of the Italian Respiratory Society (IRS). RESULTS: Among the 1,776 IRS members who have been invited to the survey, 144 (8.1%) completed the survey (97 men; median age 59 years; 85.4% Respiratory Disease). The vast majority recommended vaccination to all their patients (81%). More than two thirds of respondents considered influenza vaccination safe for immunocompromised patients. More than 50% of respondents underwent seasonal influenza vaccination in 2015 and 68% declared the intention to undergo vaccination in 2016 epidemic season. Reasons for having vaccination mainly referred to 'protect oneself from influenza' (63%), 'protect patients' (31%) or household members' (6%). The main reasons for vaccination refusal were 'lack of time' (45%), 'concerns about side effects' (22%), 'do not get influenza easily and/or not afraid of influenza infection' (22%) and 'disagreement with indication of vaccination for HCWs' (9%). CONCLUSIONS: The promotion of better knowledge and attitude towards influenza vaccination among Italian specialists remains an unmet goal and should be addressed by appropriate multifaceted interventions.

Ex vivo host response to gastrointestinal cancer cells presented by autologous dendritic cells.

BACKGROUND: Dendritic cells (DCs) capture apoptotic tumors and cross-present their antigens in the MHC class I and class II pathways for recognition by CD4+ and CD8+ T lymphocytes. Here we have tested the ability of fresh surgically resected colon and gastric cancer tumors to specifically activate host T lymphocytes when presented by autologous DCs. METHODS: DCs derived from adherent blood mononuclear cells of five patients, after a 7-day culture with GM-CSF and IL-4, were exposed to apoptotic autologous tumor (AAT) or apoptotic autologous peritumor normal (AAN) cells and cultured 24 h with monocyte-conditioned medium to achieve full DC maturation. Tumor-specific response was evaluated as single-cell cytokine release in an enzyme-linked immunospot (ELISPOT) and as cytotoxicity in a cold target inhibition (51)Cr-release assay. RESULTS: AAT-DCs induced specific IFN-gamma by T lymphocytes of two patients (rectal and gastric cancer), whereas in another two patients (rectal and gastric cancer) this response was depressed with a similar tumor-specific pattern and in one patient (rectal cancer) there was no response. Activation of IFN-gamma release was accompanied by tumor cytotoxicity and both responses were enhanced by IL-12, indicating the functional integrity of patients' lymphocytes. CONCLUSION: These data show that T-cell memory against rectal/gastric carcinoma antigens can be triggered by tumor-loaded autologous DCs. However, escape mechanisms may exist among tumors of the same histological origin that can inhibit this host response. A DC-based antitumor immunological monitoring assay with autologous tumor biopsies may allow patients to be screened to determine those who are suitable candidates for immune-based immunotherapy.

Individual and combined effect of granulocyte-macrophage colony-stimulating factor and prolactin on maturation of dendritic cells from blood monocytes under serum-free conditions.

Prolactin (PRL) shares structural and functional features with haemopoietic factors and cytokine peptides. Dendritic cells (DC) are involved in both initiating the primary and boosting the secondary host immune response and can be differentiated in vitro from precursors under the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus other factors. Because PRL has been shown to functionally interact with GM-CSF, we have addressed its role on GM-CSF-driven differentiation of DC. Monocytic DC precursors from peripheral blood mononuclear cells (PBMC) were enriched either by adhesion to a plastic surface or CD14-positive selection and cultured for 7 days in serum-free medium containing GM-CSF, interleukin (IL)-4 and PRL, alone or in combination. Cells with large, veiled cytoplasm, expressing major histocompatibility complex (MHC) class II and the costimulatory molecules CD80, CD86 and CD40 and lacking the monocyte marker CD14, were considered as having the phenotype of cytokine-generated DC. Functional maturation was assessed by proliferation and interferon-gamma (IFN-gamma) release of allogeneic T lymphocytes. Physiological (10-20 ng/ml) concentrations of PRL interacted synergistically with GM-CSF and the effect was similar to that induced by IL-4 on GM-CSF-driven DC maturation. When used alone, the physiological concentrations of PRL were inhibitory, whereas higher concentrations (80 ng/ml) were stimulatory. The synergistic effect of PRL may in part be caused by its ability to counteract the down-modulation of the GM-CSF receptor observed in serum-free conditions. These data provide further evidence of the significance of PRL in the process of T lymphocyte activation.

Up-modulation of interferon-gamma mediates the enhancement of spontanous cytotoxicity in prolactin-activated natural killer cells.

Prolactin (PRL) has been shown to participate in lymphocyte activation. In particular, the constitutive natural killer (NK) and the lymphokine-activated killer (LAK) cytotoxicity of CD56+ CD16+ cells is increased by its physiological to supraphysiological concentrations. As PRL has been shown to up-regulate the production of interferon-gamma (IFN-gamma) by peripheral blood mononuclear cells, we studied its effect on IFN-gamma production by NK cells as a possible mechanism of autocrine activation of cytotoxicity. Released and intracellular IFN-gamma, as well as IFN-gamma mRNA expression, were increased by pituitary and recombinant human PRL, which stimulated optimal NK and LAK cytotoxicity. Treatment with blocking anti-IFN-gamma monoclonal antibody (mAb) selectively affected PRL-increased killing of K562 targets, demonstrating that PRL-mediated enhancement of spontaneous cytotoxicity depends, at least in part, on up-regulation of IFN-gamma.

[Tumor samples as a tool for large-scale monitoring of antigen-specific lymphocytes in gastric and colorectal neoplasms]. / L'utilizzo di campioni di tessuto tumorale come mezzo per il monitoraggio su larga scala dei linfociti antigene specifici nelle neoplasie gastriche e colorettali.

The characterization of tumor-associated antigens has enabled to direct the host immune response towards the autologous tumor through appropriate loading and presentation of the antigen. In vivo conditions that generate large numbers of tumor antigens would be an important step in vaccine strategies. In this study we have therefore tested the ability of freshly isolated gastric and colorectal cancer cells to induce a specific anti-tumor response in autologous T lymphocytes. Because dendritic cells (DC) are critically involved in both initiating and boosting host immune responses, they have been used to present apoptotic bodies generated by irradiated tumor cells. Results show that these native antigens stimulate T cytotoxic response against tumor, but not peritumor normal tissues. Induction of IFN-gamma secreting cell activity, which is a standard readout in current cancer vaccine protocols, was also demonstrated by Elispot single-cells assay. These data show the antigenicity of gastric and colorectal tumor cells and open new perspectives in immunotherapy.

Prolactin is an autocrine growth factor for the Jurkat human T-leukemic cell line.

Despite convincing evidence of cooperation between IL-2 and endogenous prolactin (PRL) during T cell activation, the individual role of PRL as a T-cell lineage cytokine remains to be defined. We have examined the production and function of PRL on the Jurkat human T-leukemic cell line, which does not constitutively produce IL-2. The majority of Jurkat cells expressed PRL receptor (R) under standard culture conditions, whereas appearance of the alpha chain of the IL-2-R required PHA-PMA stimulation, as did IL-2 synthesis. Western blotting revealed a predominant band at 23.5 kDa and a weaker band at 25.5 kDa in both Jurkat cell lysates and human (h) pituitary PRL. Metabolic labeling of the cell lysates with 35S-methionine and immunoprecipitation with an antiserum against hPRL showed that both forms of PRL are actively synthesized by the Jurkat cell line. PRL released in the medium was biologically active in the rat Nb2 lymphoma mitogenic assay. Depletion of medium PRL with two polyclonal anti-hPRL antisera inhibited the growth of Jurkat cells in a dose-dependent manner, as evaluated by cell number and 3H-TdR uptake. Purified pituitary or recombinant hPRL at a wide range of concentrations had no significant effect on their growth, but reversed the blocking activity of the anti-hPRL antibody. Recombinant IL-2 had no effect on the antibody-induced growth inhibition. Taken as a whole, these results demonstrate that PRL can act as an autocrine T cell growth factor independently of IL-2 and are the first evidence of its involvement in human leukemic growth and possibly in leukemic transformation.

[Indications for percutaneous atrioseptostomy. Comparison of echocardiographic and fluoroscopic monitoring]. / Indicazioni alla atriosettostomia percutanea. Confronto fra monitorizzazione ecocardiografica e fluoroscopica.

Balloon atrial septostomy (BAS) is of established value in the management of several congenital heart diseases in the neonatal period. This procedure, which leads to creation of a tear in the membrane of the Foramen Ovalis using a balloon catheter, may be undertaken under fluoroscopic monitoring in catheter laboratory as well as under echographic control, even bedside. In this study we present our experience and discuss the indications to these two techniques. 91 neonates underwent to BAS; in 14 of them this was carried out under two-dimensional echocardiographic control in the intensive care unit. In all the patients BAS had good result, with clinical improvement in the majority of cases (97%) and a low rate (6%) of minor complications (such as transient supraventricular arrhythmias), without differences between fluoroscopic and ultrasound monitoring. The higher rate of mortality in the fluoroscopic monitoring group (2/91 = 2.2%) was thought to depend on an extremely critical presentation of the neonates. The echographic monitoring does not seem to offer any real advantage from the technical point of view, but the necessity of a prompt treatment of very ill patients emphasizes the advantages of a quickly and easily feasible procedure. Thus, we recommend the use two-dimensional echocardiographic imaging only in the very ill neonates in whom the septostomy can be more safely performed in intensive care unit bedside.

Arabidopsis thaliana class IV chitinase is early induced during the interaction with Xanthomonas campestris.

Endochitinases are widely distributed among higher plants, including a number of important crop species. They are generally considered to be involved in plant defence against potential pathogens. We have cloned a class IV chitinase gene (AtchitIV) from Arabidopsis thaliana. Southern blot analysis allowed the detection of two cross-hybridising genes in the A. thaliana genome. AtchitIV transcripts are detected in seedpods, but not in roots, inflorescence stems, leaves and flowers of healthy plants. The transcripts accumulated very rapidly in leaves after inoculation with Xanthomonas campestris. Maximum mRNA accumulation was reached one hour after infection and decreased to very low levels 72 hours after induction. This result suggests an involvement of AtchitIV in the initial events of the hypersensitive reaction. Nevertheless, A. thaliana plants transformed with the gus gene under the control of a class IV chitinase bean promoter, showed GUS activity in seed embryos. These data, together with the constitutive expression of the endogenous gene in the seedpods, points to additional physiological roles for this protein.

Role of prolactin in the in vitro development of interleukin-2-driven anti-tumoural lymphokine-activated killer cells.

Exogenous prolactin (PRL) has been shown to synergize with low-dose interleukin-2 (IL-2) and induce the proliferation and lymphokine-activated killer (LAK) maturation of natural killer (NK) cells. PRL itself can also generate LAK activity. Here we show that its local production occurs during, and is necessary for, LAK development. IL-2-stimulated peripheral blood mononuclear cells (PBMC) and purified NK cells were exposed to anti-human (h)PRL antiserum, and residual LAK activity was measured on day 7 against the promyelocytic leukaemia cell line HL-60. Inhibition of LAK activity was much more evident in PBMC compared with NK cell cultures (47% decrease. P - 0.013 and 18.5% decrease. P = 0.048, respectively). Up-modulation of a 32S-methionine-labelled 27,000 MW protein was detected in the lysates and supernatants of IL-2-stimulated PBMC immunoprecipitated with an anti-PRL antiserum. By contrast, the cytoplasmic PRL immunoreactivity observed in freshly isolated NK cells and in IL-2-stimulated, but not unstimulated, NK cell cultures was not associated with PRL gene activation, and can thus be referred to internalized PRL. Preferential re-uptake of externally derived PRL by IL-2-stimulated NK cells was also indicated by up-modulation of the PRL receptor. These data, as a whole, indicate that the PRL promotion of LAK differentiation is mainly mediated by paracrine secretion, with a minor contribution from internalized PRL.

AIDS-related Burkitt's-type lymphomas are a target for lymphokine-activated killers induced by interleukin-2 and prolactin.

The development of non-Hodgkin's lymphomas (NHL) is one of the major complications of AIDS. Although several biologic aspects of AIDS-related NHL have been clarified, their sensitivity to immune system cytotoxic effectors has not been tested. In this study, we have investigated the susceptibility of one major AIDS-related NHL type, Burkitt's-type lymphoma (BL), to the cytotoxic activity of lymphokine-activated killers (LAK) and prolactin-activated killers (PAK), which were generated from peripheral blood mononuclear cells upon stimulation with interleukin-2 (in the case of LAK cells) and prolactin (in the case of PAK cells). The sensitivity of AIDS-related BL to in vitro raised cytotoxic effectors was compared with that of BL variants of the general population, including sporadic BL and endemic BL. The data show that AIDS-related BL is susceptible to cytolysis by LAK cells, whereas both LAK and PAK cells can efficiently kill endemic BL. In contrast, sporadic BL showed resistance to all cytotoxic effectors tested. Intriguingly, in the case of AIDS-related and endemic BL suboptimal doses of interleukin-2 in combination with prolactin displayed a cytotoxic effect similar to that of LAK cells, suggesting a synergistic activity of the two agents. Overall, these data corroborate the notion that the distinct BL variants differ in their biologic features despite their morphologic and genetic similarity.

Prolactin increases the susceptibility of primary leukemia cells to NK and LAK effectors.

Our previous studies have shown that prolactin (PRL), a pituitary and lymphocyte hormone and a ligand of the cytokine/hemopoietin receptors (R) superfamily, acts synergistically with interleukin (IL)-2 on the development of lymphokine activated killer (LAK) cells and enhances the effects of GM-CSF and IL-3 on myeloid progenitors' proliferation and differentiation. More recently, we have demonstrated that GM-CSF and IL-3 increase the sensitivity of acute myeloid leukemic (AML) cells to LAK activity. Together, these findings have prompted us to study the role of PRL on the target arm of the LAK response. We show here that CD33+ blasts from AML patients express membrane PRL-R and that the PRL/PRL-R interaction is followed by increased susceptibility to natural killer (NK) (p < 0.02) and LAK (p < 0.001) cells. As predicted from the dimerization model of PRL-R and in agreement with previous reports, the response of AML blasts to PRL was bell-shaped with a trend peak at 25 ng/ml. Although enhanced lysis occurred at the target recognition level, it was not accompanied by changes in the MHC class I, cellular adhesion molecules, or myeloid differentiation antigens. Cell cycle recruitment and lysis increased concurrently in three cases studied, suggesting a modulatory action of PRL on the expression of putative cycle-related NK/LAK-target structures. Together, these data strengthen the role of PRL in the LAK response.

Independent and synergistic effect of interleukin-2 and prolactin on development of T- and NK-derived LAK effectors.

We have studied the effect of recombinant (r)-Prl on the in vitro-induced MHC-unrestricted cytotoxicity of NK and T cells. A 4-day treatment with r-Prl in serum-free medium enhanced the cytotoxicity of NK cells to the NK-susceptible cell lines K562 and U937, but did not induce de novo NK cytotoxicity in T lymphocytes. By contrast, development of cytotoxicity against the LAK-susceptible cell lines HL60, Jurkat, Daudi and Supt-1 occurred in both NK and T cells. The effect of r-Prl on NK cells was bi-phasic with peaks at 25 ng/ml (1.2 nM), the upper physiological level, and 200 ng/ml (9.6 nM). By contrast, LAK activation of T cells only occurred at the highest r-Prl concentration. In addition to its intrinsic stimulatory activity, r-Prl was also capable of modulating in a dose-dependent manner distinct stages of the IL2-driven LAK/T differentiation pathway. Physiological concentrations of r-Prl interacted with low doses r-IL2 to significantly enhance generation of NK- and T-LAK activities. By contrast, pathological concentrations had opposite effects on generation of optimal LAK response, depending on the kind of LAK progenitor. The T-derived LAK activity was reversibly inhibited at the effector level, while the mature NK-LAK cells were stimulated. These data confirm our previous findings of a co-operative effect of Prl and IL2 on NK cell proliferation and reinforce the view that the signals conveyed by the two factors may be functionally related.

Synthesis of a prolactin-like peptide by natural killer cells: positive regulation by CD16 and exogenous prolactin.

We have previously shown that Prolactin (PRL) activates the native and the in vitro acquired cytotoxicity and the DNA synthetic activity of Natural Killer (NK) cells. Here we show that the supernatant and the cell lysate of NK cells express a 35S-labelled 50 kDa peptide specifically immunostained by two different PRL-antisera. The supernatant of NK cells was biologically active in a Nb2 assay and the activity could be adsorbed by an anti-PRL antiserum. The production of the PRL-like peptide only occurred when NK cells were isolated through binding to immobilized immunocomplexes, the biological ligand for CD16, and was positively modulated by exogenous PRL. These results indicate that PRL, produced by NK cells following stimulation, may act in an autocrine fashion to maintain and/or activate the NK cell function.