Liposomal amphotericin B in the treatment of visceral leishmaniasis in immunocompromised patients.

Leishmaniasis is a zoonosis caused by a protozoan of the Leishmania genus. First-line treatment for all forms is currently represented by the use of antimony derivatives, although toxic effects and the number of resistant strains in both immunocompromised and immunocompetent patients is increasing. Liposomal amphotericin B (L-AMB) is less toxic, more effective, and better tolerated, especially in human immunodeficiency virus-negative immunocompromised patients. We present 2 cases of transplanted patients affected by visceral leishmaniasis treated successfully with L-AMB.

CD44v6 as innovative sarcoma target for CAR-redirected CIK cells.

Purpose of our study was to explore a new immunotherapy for high grade soft tissue sarcomas (STS) based on cytokine-induced killer cells (CIK) redirected with a chimeric antigen receptor (CAR) against the tumor-promoting antigen CD44v6. We aimed at generating bipotential killers, combining the CAR specificity with the intrinsic tumor-killing ability of CIK cells (CAR+.CIK). We set a patient-derived experimental platform. CAR+.CIK were generated by transduction of CIK precursors with a lentiviral vector encoding for anti-CD44v6-CAR. CAR+.CIK were characterized and assessed in vitro against multiple histotypes of patient-derived STS. The anti-sarcoma activity of CAR+.CIK was confirmed in a STS xenograft model. CD44v6 was expressed by 40% (11/27) of patient-derived STS. CAR+.CIK were efficiently expanded from patients (n = 12) and killed multiple histotypes of STS (including autologous targets, n = 4). The killing activity was significantly higher compared with unmodified CIK, especially at low effector/target (E/T) ratios: 98% vs 82% (E/T = 10:1) and 68% vs 26% (1:4), (p<0.0001). Specificity of tumor killing was confirmed by blocking with anti-CD44v6 antibody. CAR+.CIK produced higher amounts of IL6 and IFN-γ compared to control CIK. CAR+.CIK were highly active in mice bearing subcutaneous STS xenografts, with significant delay of tumor growth (p<0.0001) without toxicities. We report first evidence of CAR+.CIK's activity against high grade STS and propose CD44v6 as an innovative target in this setting. CIK are a valuable platform for the translation of CAR-based strategies to challenging field of solid tumors. Our findings support the exploration of CAR+.CIK in clinical trials against high grade STS.

The tyrosine kinase encoded by the MET proto-oncogene is activated by autophosphorylation.

Protein tyrosine kinases are crucially involved in the control of cell proliferation. Therefore, the regulation of their activity in both normal and neoplastic cells has been under intense scrutiny. The product of the MET oncogene is a transmembrane receptorlike tyrosine kinase with a unique disulfide-linked heterodimeric structure. Here we show that the tyrosine kinase activity of the MET-encoded protein is powerfully activated by tyrosine autophosphorylation. The enhancement of activity was quantitated with a phosphorylation assay of exogenous substrates. It involved an increase in the Vmax of the enzyme-catalyzed phosphotransfer reaction. No change was observed in the Km (substrate). A causal relationship between tyrosine autophosphorylation and activation of the kinase activity was proved by (i) the kinetic agreement between autophosphorylation and kinase activation, (ii) the overlapping dose-response relationship for ATP, (iii) the specificity for ATP of the activation process, (iv) the phosphorylation of tyrosine residues only, in the Met protein, in the activation step, (v) the linear dependence of the activation from the input of enzyme assayed, and (vi) the reversal of the active state by phosphatase treatment. Autophosphorylation occurred predominantly on a single tryptic peptide, most likely via an intermolecular reaction. The structural features responsible for this positive modulation of kinase activity were all contained in the 45-kDa intracellular moiety of the Met protein.

Loss of the exon encoding the juxtamembrane domain is essential for the oncogenic activation of TPR-MET.

TPR-MET, a transforming counterpart of the c-MET proto-oncogene detected in experimental and human cancer, results from fusion of the MET kinase domain with a dimerization motif encoded by TPR. In this rearrangement the exons encoding the Met extracellular, transmembrane and juxtamembrane domains are lost. The juxtamembrane domain has been suggested to be a regulatory region endowed with negative feedback control. To understand whether its absence is critical for the generation of the Tpr-Met transforming potential, we produced a chimeric molecule (Tpr-juxtaMet) with a conserved juxtamembrane domain. The presence of the domain (aa 962-1009) strongly inhibited Tpr-Met dependent cell transformation. Cell proliferation, anchorage-independent growth, motility and invasion were also impaired. The enzymatic behavior of Tpr-Met and Tpr-juxtaMet was the same, while Tpr-juxtaMet ability to associate cytoplasmic signal transducers and to elicit downstream signaling was severely impaired. These data indicate that the presence of the juxtamembrane domain counterbalances the Tpr-Met transforming potential and therefore the loss of the exon encoding the juxtamembrane domain is crucial in the generation of the active TPR-MET oncogene.

Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-MET.

The human proto-oncogene c-MET encodes a heterodimeric 190 kDa transmembrane protein (p190c-met) with structural features of a tyrosine kinase receptor. The ligand for this putative receptor has not yet been identified. By Northern blot hybridization we found that, among a restricted number of human tissues, c-MET is highly expressed in the liver. This prompted us to test the hypothesis of a functional interaction between the c-MET receptor and Hepatocyte Growth Factor (HGF), a heparin-binding polypeptide consisting of heavy and light chains of 65 and 35 kDa. Nanomolar concentrations of highly purified HGF added to GTL-16 cells, which overexpress the c-MET receptor, enhanced the phosphorylation on tyrosine of the p190c-met kinase. Addition of other known growth factors or serum was ineffective. The kinase activity of the c-MET receptor was also stimulated by HGF in an in vitro assay, after detergent solubilization and partial purification of p190c-met. Moreover, elution of immunoprecipitates obtained with anti-MET antibodies from GTL-16 cell lysates yielded an HGF-responsive kinase activity. These results suggest that HGF, or a growth factor structurally related to HGF, is a candidate ligand for the receptor encoded by c-MET.

Targeting the oncogenic Met receptor by antibodies and gene therapy.

The receptor for hepatocyte growth factor (HGF), a tyrosine kinase encoded by the Met oncogene, has a crucial role in cancer growth, invasion and metastasis. It is a validated therapeutic target for 'personalized' treatment of a number of malignancies. Therapeutic tools prompting selective, robust and highly effective Met inhibition potentially represent a major step in the battle against cancer. Antibodies targeting either Met or its ligand HGF, although challenging, demonstrate to be endowed with promising features. Here we briefly review and discuss the state of the art in the field.

Preparation and in vitro characterization of chitosan nanobubbles as theranostic agents.

Theranostic delivery systems are nanostructures that combine the modality of therapy and diagnostic imaging. Polymeric micro- and nanobubbles, spherical vesicles containing a gas core, have been proposed as new theranostic carriers for MRI-guided therapy. In this study, chitosan nanobubbles were purposely tuned for the co-delivery of prednisolone phosphate and a Gd(III) complex, as therapeutic and MRI diagnostic agent, respectively. Perfluoropentane was used for filling up the internal core of the formulation. These theranostic nanobubbles showed diameters of about 500nm and a positive surface charge that allows the interaction with the negatively charged Gd-DOTP complex. Pluronic F68 was added to the nanobubble aqueous suspension as stabilizer agent. The encapsulation efficiency was good for both the active compounds, and a prolonged drug release profile was observed in vitro. The effect of ultrasound stimulation on prednisolone phosphate release was evaluated at 37°C. A marked increase on drug release kinetics with no burst effect was obtained after the exposure of the system to ultrasound. Furthermore, the relaxivity of the MRI probe changed upon incorporation in the nanobubble shell, thereby offering interesting opportunity in dual MRI-US experiments. The ultrasound characterization showed a good in vitro echogenicity of the theranostic nanobubbles. In summary, chitosan drug-loaded nanobubbles with Gd(III) complex bound to their shell might be considered a new platform for imaging and drug delivery with the potential of improving anti-cancer treatments.

MACOP-B regimen followed by involved-field radiation therapy in early-stage aggressive non-Hodgkin's lymphoma patients: 14-year update results.

A single-center, retrospective study was conducted to evaluate therapeutic results of the MACOP-B third-generation chemotherapy regimen followed by involved-field radiation therapy in a stage I-II aggressive non-Hodgkin's lymphoma (NHL) patients. From 1986 to 1995, 118 consecutive patients with the diagnosis of aggressive NHL, stage I-IE or II-IIE, with or without bulky disease were treated with MACOP-B regimen followed, when appropriate, by 30-36 Gy involved-field radiation therapy. The complete response (CR) rate was 95% after the combined modality treatment (97% for stage I-IE and 93% for stage II-IIE). Patients with bulky disease had a CR rate of 92%. Treatment was well tolerated and no deaths occurred from acute toxicity. After a median follow-up of 68 months, 24 (21%) patients relapsed. The 14-year projected relapse-free and overall survival rates were 78% and d 69%, respectively. MACOP-B regimen with/without involved-field radiation therapy provides a safe and effective combined modality treatment for early-stage aggressive NHL, with the possibility to definitively cure two thirds of the patients.

Anaplastic thyroid carcinoma in a 49 year-old woman with a long-standing goiter. A case report.

A 49 year-old woman with anaplastic thyroid carcinoma came, for the first time, to our clinic with an enlarged supraclavicular lymph node. Ten years earlier she received a diagnosis of benign nodular goiter and has been followed up with ultrasonography, which never demonstrated any enlargement of her thyroid nodule and with fine needle aspiration biopsies (FNAB), that always proved normal. An ultrasonographic control, performed 10 months before diagnosis, showed the onset of a shell calcification all around the thyroid nodule that forbade the performance of FNAB. At initial examination in our endocrinology centre she was found to have an enlarged thyroid nodule (changing from 3.5 cm to 4.5 cm) and a supraclavicular lymph node 3 cm wide. The patient was therefore sent to the surgeon who performed a total thyroidectomy and a lymph node exenteration. The histological examination reported a thyroid anaplastic carcinoma, composed of osteoclast-like cells and large calcifications, which showed signs of local invasion and vessel infiltration; the supraclavicular lymph node proved to be a large metastasis of anaplastic thyroid cancer. Total body CT scan, bone scintigraphy and brain CT scan showed, respectively, lung, bone and brain metastasis. Postoperative chemotherapy was rapidly interrupted for acute toxicity. The patient died for lung, bone and brain metastasis, 2 months after initial examination.

Ferric chondroitin 6-sulfate (Condrofer): a new potent antianaemic agent with a favourable pharmacokinetic profile.

One and 3 mg/kg iron as Condrofer**, a new soluble formulation of this metal, and 1 mg/kg iron as Proteoferrina*** or ferritin were given orally for 4 weeks to male rats in which severe experimental anaemia had previously been induced (by iron-deficient diet and repeated bleedings). Haematological (erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, reticulocytes and leukocytes) and blood chemistry (sodium, potassium, iron and total protein) parameters were checked weekly and at the end of the drug administration period. Clinical and behavioral signs, body weight, food intake and necroscopic observations were also recorded. Condrofer time- and dose-dependently improved the general blood picture, the clinical data and the autoptic findings to the point of making these animals significantly approach control rats, save for one parameter, sideremia, which after 4 weeks of treatment remained lower than normal. The most plausible explanation would seem that the severe anaemia interfered both with the physiological iron storage and with the iron-dependent mitochondrial enzymatic systems. Iron (1 mg/kg) daily as Proteoferrina or ferritin was significantly less effective than when this metal was administered as Condrofer, since all the haematological parameters and the clinical, behavioral signs and necroscopic observations were less favourable. The more complete reversal of anaemia in the rats that received Condrofer is, most probably, due to the higher bioavailability of iron administered under this formulation, as demonstrated by iron kinetics after equidoses of iron as Condrofer and Proteoferrina.

[Perioperative teicoplanin prophylaxis in patients undergoing breast reconstruction with the abdominal wall. A case-control study]. / Profilassi perioperatoria con teicoplanina in pazienti sottoposte a ricostruzione mammaria con retto addominale. Studio caso-controllo.

The authors report the results of a randomized clinical trial of antibiotic prophylaxis of postoperative infection following breast reconstruction by transposition of rectus abdominis myocutaneous flap (TRAMF). The aim was to evaluate the efficacy and tolerability of a short-term parenteral prophylaxis with Teicoplanin and the end-point of the study was the evaluation of wound contamination assessed by means of microbiologic culture of drainage fluid. From October 1990 to March 1992 38 patients were recruited: 20 patients in the antibiotic prophylaxis arm and 18 patients in the control group. Analysis of drainage fluids showed a higher contamination rate (15/18 = 83%) in the control group as compared to the prophylaxis arm (2/20 = 10%) (p < 0.0001). Moreover, 11 patients in the control arm suffered from fever > 37.5 degrees C for at least 3 days as compared to 1 patient in the antibiotic prophylaxis group; the postoperative stay was 13.3 +/- 4.3 and 9.0 +/- 1.6 in the control and antibiotic arm respectively. No antibiotic related side effects were evidenced through the study. These results seem to confirm the value of parenteral short-term antibiotic prophylaxis of postoperative infection in such kind of "clean" operative procedure.

[A clinico-microbiological assessment of patients who have undergone breast reconstruction with a myocutaneous flap of the rectus abdominis]. / Valutazione clinico-microbiologica delle pazienti sottoposte a ricostruzione mammaria con lembo-miocutaneo di retto addominale.

In plastic surgery a minor complication such as wound infection can cause a lot of damage, because it can put back flap revascularization or it can stop wound healing properly. So the final result is not as good as it should be. In head-neck and in breast reconstruction with prostheses there are set-treatments of antibiotic prophylaxis; while in breast reconstruction with myocutaneous flaps it is very indecisive where antibiotics are concerned. This fact has encouraged us to evaluate bacteria causing infective complications in patients who have had breast reconstruction with TRAMF. In this operation there are 4 conditions which could give rise to microbiological contamination: 1) duration of operation (about 4 hours); 2) wide and prolonged tissue exposure; 3) areas of ischemic tissue or liponecrosis; 4) drainage. In this study we have shown the percentage of bacterial infections and the type of bacteria responsible for these complications in a group of patients who had already undergone breast reconstruction with TRAMF.

Rituximab for the treatment of patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that originates from antigen-experienced B lymphocytes that do not die and hence accumulate due to external survival signals or undergo apoptosis and are replenished by proliferating precursors. These neoplastic lymphocytes exhibit a characteristic immunophenotype of CD5(+)/CD19(+)/CD20(+)/HLA-DR+/CD23(+)/sIgdim. Thus, the CD20 antigen has been an appealing target for therapy. The introduction of the monoclonal antibody rituximab (anti-CD20) enabled an outstanding advance in CLL treatment. The introduction of this monoclonal antibody into chemotherapy regimens has dramatically improved complete response rates and progression-free survival in patients with both untreated and relapsed CLL. Although only preliminary data from phase III confirmatory trials have been reported, the FCR regimen, which combines fludarabine and cyclophosphamide with rituximab, is currently the most effective treatment regimen for CLL patients, and has also been demonstrated to significantly improve overall survival. The success of rituximab and the identification of other CLL lymphocyte surface antigens have spurred the development of a multitude of monoclonal antibodies targeting distinct proteins and epitopes in an attempt to target CLL cells more effectively.

Insights into defibrotide: an updated review.

Defibrotide is a polydisperse oligonucleotide with antiatherosclerotic, anti-inflammatory, anti-ischaemic, pro-fibrinolytic and antithrombotic actions without significant systemic anticoagulant effects. It has been used in the treatment of various cardiovascular disorders, and especially in endothelial complications of allogeneic stem-cell transplantation. We reviewed the published work for the mechanism of action and clinical use of defibrotide to consolidate data and to describe new applications of this drug. We reviewed the most relevant papers on defibrotide published from November 1982 to January 2008. (selected through PubMed), and used recent meeting abstracts as sources for this review. Reports have suggested that defibrotide has clinical efficacy for treatment and prophylaxis of hepatic sinusoidal obstruction syndrome occurring after stem-cell transplantation. Animal models have clearly shown the potential antineoplastic effect of this drug. Further clinical investigations are needed to clarify this new application.

Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy.

Lentiviral vectors are tools for gene transfer derived from lentiviruses. From their first application to now they have been strongly developed in design, in biosafety and in their ability of transgene expression into target cells. Primate and non-primate derived lentiviral vectors are now available and with both types of systems a lot of studies tuned to improve their performances in a large number of tissues are ongoing. Here we review the state of the art of lentiviral vector systems discussing their potential for gene therapy.

Identification of the major autophosphorylation site of the Met/hepatocyte growth factor receptor tyrosine kinase.

The MET proto-oncogene encodes a transmembrane tyrosine kinase receptor for HGF (p190MET). In this work, p190MET was immunoprecipitated, allowed to phosphorylate in the presence of [gamma-32P]ATP, and digested with trypsin. A major phosphopeptide was purified by reverse phase chromatography. The phosphorylated tyrosine was identified as residue 1235 (Tyr1235) by Edman covalent radiosequencing. A synthetic peptide derived from the corresponding MET sequence was phosphorylated by p190MET in an in vitro assay and coeluted in reverse phase chromatography. Tyr1235 lies within the tyrosine kinase domain of p190MET, within a canonical tyrosine autophosphorylation site that shares homology with the corresponding region of the insulin, CSF-1 and platelet-derived growth factor receptors, and of p60src and p130gag-fps. The p190MET kinase is constitutively phosphorylated on tryosine in a gastric carcinoma cell line (GTL16), due to the amplification and overexpression of the MET gene. Metabolic labeling of GTL-16 cells with [32P]orthophosphate followed by immunoprecipitation and tryptic phosphopeptide mapping of p190MET showed that Tyr1235 is a major site of tyrosine phosphorylation in vivo as well. Since phosphorylation activates p190MET kinase, we propose a regulatory role for Tyr1235.

Biological activation of pro-HGF (hepatocyte growth factor) by urokinase is controlled by a stoichiometric reaction.

Hepatocyte growth factor (HGF) is a paracrine inducer of morphogenesis and invasive growth in epithelial and endothelial cells. HGF is secreted by mesenchymal cells as an inactive precursor (pro-HGF). The crucial step for HGF activation is the extracellular hydrolysis of the Arg494-Val495 bond, which converts pro-HGF into alpha beta-HGF, the high-affinity ligand for the Met receptor. We previously reported that the urokinase-type plasminogen activator (uPA) activates pro-HGF in vitro. We now show that this is a stoichiometric reaction, and provide evidence for its occurrence in tissue culture. Activation involves the formation of a stable complex between pro-HGF and uPA. This complex was isolated from the in vitro reaction of pure uPA with recombinant pro-HGF, as well as from the membrane of target cells, after sequential addition of uPA and pro-HGF. On the cell membrane, the uPA-HGF complex was bound to the Met receptor. Monocytic cell lines, and primary monocytes after adhesion, activated efficiently pro-HGF both on their surface and in the culture medium. This activation was inhibited by anti-catalytic anti-uPA antibodies, and occurred by a stoichiometric reaction. The stoichiometry of the activation reaction suggests that the biological effects of HGF can be titrated in vivo by the level of uPA activity. Adequate amounts of uPA can be locally provided by the macrophages, which would condition the tissue microenvironment by rendering HGF bioavailable to its target cells.