The histone deacetylase inhibitor givinostat (ITF2357) exhibits potent anti-tumor activity against CRLF2-rearranged BCP-ALL.

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

An immediate transcriptional signature associated with response to the histone deacetylase inhibitor Givinostat in T acute lymphoblastic leukemia xenografts.

Despite some success with certain hematological malignancies and in contrast with the strong pro-apoptotic effects measured in vitro, the overall response rate of acute lymphoblastic leukemia (ALL) to histone deacetylase inhibitors (HDACis) is low. With the aim to improve the understanding of how HDACis work in vivo, we investigated the therapeutic efficacy of the clinically approved HDACi Givinostat in a collection of nine pediatric human T-ALL engrafted systemically in NOD/SCID mice. We observed highly heterogeneous antileukemia responses to Givinostat, associated with reduction of the percentage of infiltrating blasts in target organs, induction of apoptosis and differentiation. These effects were not associated with the T-ALL cytogenetic subgroup. Transcriptome analysis disclosed an immediate transcriptional signature enriched in genes involved in cell-cycle regulation and DNA repair, which was validated by quantitative RT-PCR and was associated with in vivo response to this HDACi. Increased phospho-H2AX levels, a marker of DNA damage, were measured in T-ALL cells from Givinostat responders. These results indicate that the induction of the DNA damage response could be an early biomarker of the therapeutic effects of Givinostat in T-ALL models. This information should be considered in the design of future clinical trials with HDACis in acute leukemia.

[Ultrasonic videolaparoscopic cholecystectomy: evolution of a revolutionary technique]. / Colecistectomia videolaparoscopica a ultrasuoni: evoluzione di una metodica rivoluzionaria.

AIM: The present retrospective study, which lasted about six months from the beginning of March to the end of August 2008, involved 60 patients suffering from symptomatic calculosis of the gall bladder. METHODS: The patients were operated on with laparoscopy: 30 with traditional instruments, 30 using ultrasound multifunctional scissors. RESULTS: The numerous advantages for the patient and surgeon are immediately evident; in addition, from the economic viewpoint the procedure is advantageous compared to the traditional method because single-use material is employed exclusively. We found less tissue trauma and a lower incidence of short-term complications, such as reoperation for faulty closure of the cystic duct and the cystic artery. It was never necessary to use permanent haemostatic clips. The use of a single instrument for gripping, sectioning and closing haematic and biliary vessels permitted faster, safer and more accurate surgery in the absence of any production of smoke. CONCLUSIONS: In lithiasic pathology of the gall bladder, videolaparoscopy for cholecystectomy is presently considered the operation of first choice. The technique enables the surgeon to respect to the utmost the patient's physical and mental integrity. As the third millennium dawns, technological innovation is able to bring a significant improvement to this procedure. The ultrasound dissector Ultracision is symbolic of development and constant progress.

Selective anti-leukaemic activity of low-dose histone deacetylase inhibitor ITF2357 on AML1/ETO-positive cells.

We analysed the in vitro effects of a new hydroxamate derivative, ITF2357, on AML cells. ITF2357 potently induced histone acetylation. ITF2357 0.1 microM blocked proliferation and induced apoptosis in AML1/ETO-positive Kasumi-1 cells, while AML1/ETO-negative HL60, THP1 and NB4 cell lines were sensitive only to 1 microM ITF2357. Apoptosis was induced by 0.1 microM ITF2357 in AML1/ETO-positive primary blasts and U937-A/E cells induced to express AML1/ETO, but not in U937-A/E cells non-expressing AML1/ETO. In Kasumi-1 cells 0.1 microM ITF2357 induced AML1/ETO degradation through a caspase-dependent mechanism. ITF2357 0.1 microM also determined DNMT1 efflux from, and p300 influx to, the nucleus. Moreover, 0.1 microM ITF2357 determined local H4 acetylation and release of DNMT1, HDAC1 and AML1/ETO, paralleled by recruitment of p300 to the IL-3 gene promoter. ITF2357 treatment, however, did not induce re-expression of IL-3 gene. Accordingly, the methylation level of IL-3 promoter, as well as of several other genes, was unmodified. In conclusion, ITF2357 emerged as an anti-leukaemic agent very potent on AML cells, and on AML1/ETO-positive cells in particular. More relevantly, clearly emerged from our results that ITF2357 could be an ideal agent to treat AML subtypes presenting AML1/ETO fusion protein which determine HDAC involvement in leukaemogenesis.

The histone deacetylase inhibitor ITF2357 has anti-leukemic activity in vitro and in vivo and inhibits IL-6 and VEGF production by stromal cells.

We have investigated the activity of ITF2357, a novel hydroxamate histone deacetylase inhibitor, on multiple myeloma (MM) and acute myelogenous leukemia (AML) cells in vitro and in vivo. ITF2357 induced apoptosis in 8/9 MM and 6/7 AML cell lines, as well as 4/4 MM and 18/20 AML freshly isolated cases, with a mean IC(50) of 0.2 microM. ITF2357 activated the intrinsic apoptotic pathway, upregulated p21 and downmodulated Bcl-2 and Mcl-1. The drug induced hyperacetylation of histone H3, H4 and tubulin. When studied in more physiological conditions, ITF2357 was still strongly cytotoxic for the interleukin-6 (IL-6)-dependent MM cell line CMA-03, or for AML samples maximally stimulated by co-culture on mesenchymal stromal cells (MSCs), but not for the MSCs themselves. Interestingly, ITF2357 inhibited the production of IL-6, vascular endothelial growth factor (VEGF) and interferon-gamma by MSCs by 80-95%. Finally, the drug significantly prolonged survival of severe combined immunodeficient mice inoculated with the AML-PS in vivo passaged cell line already at the 10 mg/kg oral dose. These data demonstrate that ITF2357 has potent anti-neoplastic activity in vitro and in vivo through direct induction of leukemic cell apoptosis. Furthermore, the drug inhibits production of growth and angiogenic factors by bone marrow stromal cells, in particular IL-6 and VEGF.

Insoluble and soluble immune complexes activate neutrophils by distinct activation mechanisms: changes in functional responses induced by priming with cytokines.

BACKGROUND: Rheumatoid synovial fluid contains both soluble and insoluble immune complexes that can activate infiltrating immune cells such as neutrophils. OBJECTIVES: To determine if these different complexes activate neutrophils through similar or different receptor signalling pathways. In particular, to determine the circumstances which result in the secretion of tissue damaging reactive oxygen metabolites and granule enzymes. METHODS: Blood neutrophils were incubated with synthetic soluble and insoluble immune complexes and the ability to generate reactive oxidants tested by luminescence or spectrophotometric assays that distinguished between intracellular and extracellular production. Degranulation of myeloperoxidase and lactoferrin was determined by western blotting. The roles of FcgammaRII (CD32) and FcgammaRIIIb (CD16) were determined by incubation with Fab/F(ab')(2) fragments before activation. The effect of cytokine priming was determined by incubation with GM-CSF. RESULTS: Insoluble immune complexes activated unprimed neutrophils, but most of the oxidants produced were intracellular. This activation required FcgammaRIIIb, but not FcgammaRII function. Soluble complexes failed to activate unprimed neutrophils but generated a rapid and extensive secretion of reactive oxygen metabolites when the cells were primed with granulocyte-macrophage colony stimulating factor (GM-CSF). This activity required both FcgammaRII and FcgammaRIIIb function. Insoluble immune complexes activated the release of granule enzymes from primed or unprimed neutrophils, but the kinetics of release did not parallel those of secretion of reactive oxygen metabolites. Only primed neutrophils released enzymes in response to soluble complexes. CONCLUSIONS: Soluble and insoluble immune complexes activate neutrophils by separate receptor signalling pathways. Profound changes in neutrophil responsiveness to these complexes occur after cytokine priming.

Involvement of iron (ferric) reduction in the iron absorption mechanism of a trivalent iron-protein complex (iron protein succinylate).

Iron protein succinylate is a non-toxic therapeutic iron compound. We set out to characterise the structure of this compound and investigate the importance of digestion and intestinal reduction in determining absorption of the compound. The structure of the compound was investigated by variable temperature Mössbauer spectroscopy, molecular size determinations and kinetics of iron release by chelators. Intestinal uptake was determined with radioactive compound force fed to mice. Reduction of the compound was determined by in vitro incubation with intestinal fragments. The compound was found to contain only ferric iron, present as small particles including sizes below 10 nm. The iron was released rapidly to chelators. Digestion with trypsin reduced the molecular size of the compound. Intestinal absorption of the compound was inhibited by a ferrous chelator (ferrozine), indicating that reduction to ferrous iron may be important for absorption. The native compound was a poor substrate for duodenal reduction activity, but digestion with pepsin, followed by pancreatin, released soluble iron complexes with an increased reduction rate. We conclude that iron protein succinylate is absorbed by a mechanism involving digestion to release soluble, available ferric species which may be reduced at the mucosal surface to provide ferrous iron for membrane transport into enterocytes.

Reconstruction of intrathoracic oesophagus using tubular lateral thigh free flap.

The gastric pull-up or pharyngogastroplasty is the most widely used technique in reconstructing the digestive tract in cases of distal oesophageal tumours. This operation consists of drawing the stomach or part of it up through the chest and mediastinic region to the neck where a mucosal anastomosis with the residual pharyngeal tract is made. The most feared complication is proximal necrosis of the gastric stump with salivary fistulae usually followed by a mediastinitis. In the presence of such a complication the surgeon must tackle the challenge of reconstructing the missing part of the intrathoracic digestive tract. We describe the case of a patient in whom the missing intrathoracic oesophagus, following complete necrosis of a previously performed pharyngogastroplasty, was reconstructed using a revascularized lateral thigh free flap.

Plasma levels of interleukin 2, 6, 10 and phenotypic characterization of circulating T lymphocytes in ischemic heart disease.

The purpose of this study was to assess lymphocyte receptors expression in patients with ischemic heart diseases, as well as to measure the plasma levels of interleukin (IL) 2, 6 and 10. T Lymphocytes are found in large numbers in human atherosclerotic plaques, indicating that immune and inflammatory mechanisms are important factors in the pathogenesis of atherosclerosis. Recent data have also implicated T lymphocytes in the pathogenetic mechanism of unstable angina and ischemic heart disease. Three groups of patients were studied: 42 with an acute ischemic syndrome (AIS), 36 with stable angina (SA) and 39 healthy controls. To characterize lymphocyte phenotype, flow cytometry was performed in whole-blood samples. IL-2, IL-6 and IL-10 were measured using the ELISA method. Double fluorescence evaluation showed an increase in CD8+/CD11b+ cells (cytotoxic T lymphocytes) and in CD11b+/CD16+CD56+ cells (NK lymphocytes) in the AIS group and in SA group as compared to the control group (P < 0.05 and P < 0.001, respectively). IL-2 was increased in the AIS and SA groups compared to the control group (AIS 4.5 +/- 0.5 pg/ml; SA 6.3 +/- 0.6 pg/ml; controls 2.4 +/- 0.8 pg/ml, P < 0.05), whereas IL-6 was higher in the AIS group than in the other two groups (AIS 10.8 +/- 1.8 pg/ml; SA 1.8 +/- 0.8 pg/ml; controls 1.2 +/- 0.6 pg/ml, P < 0.0001). These data show that patients with ischemic heart disease have an increase in circulating cytotoxic T lymphocytes and in IL-2 plasma levels, irrespective of their clinical presentation, compared to normal control subjects, whereas IL-6 is elevated only in patients with AIS.

Neutrophil infiltration into human gliomas.

Human gliomas were analysed for the infiltration of neutrophils using immunohistochemistry by staining sections for CD15-positive and myeloperoxidase-positive cells. Over 70% of all glioma samples analysed (n = 105) had significant neutrophil infiltration, but there was a marked and significant correlation between tumour grade and the extent of the neutrophil infiltration. In the low grade tumours only 40-50% had significant infiltration, while in glioblastoma multiforme over 85% of the samples analysed had significant infiltration. Numbers of neutrophils infiltrating glioblastoma multiforme tumours were also greater than in the other tumour groups. Circulating white blood cell counts were elevated above the normal range in all glioma patients, but this elevation was entirely due to increased numbers of circulating neutrophils. Again, the highest numbers of circulating neutrophils were seen in the glioblastoma multiforme patients. These experiments indicate that glioma-derived factors may directly or indirectly affect the number of circulating neutrophils and influence their infiltration into the tumours.

In vitro effects of GM-CSF on mature peripheral blood neutrophils.

GM-CSF can play a crucial role in regulating the neutrophil-mediated inflammatory response. This growth factor is a proliferative stimulus for bone marrow neutrophil stem cell precursors and has at least 3 important roles in regulating neutrophil-mediated immunity: a) a direct effect on the proliferation and development of neutrophil progenitors; b) synergistic activity with other haemopoietic growth factors; c) stimulation of the functional activity of mature neutrophils. The production of GM-CSF may be triggered directly by exogenous factors such as antigens and endotoxins, or indirectly through the release of cytokines by a variety of cells including lymphocytes, activated macrophages and endothelial cells exposed to products of mononuclear phagocytes. Such production of GM-CSF may serve to quickly release mature neutrophils from the bone marrow in response to infections. Moreover, enhancement of the function of mature neutrophils may also augment their ability to migrate to infective sites and then phagocytose and kill pathogens. Increased expression of CD11b/CD18 may play a fundamental part in this mechanism because this receptor is essential for the adhesion of neutrophils to the endothelium. Both phagocytosis and oxidative burst activity increase as a result of the action of GM-CSF and the increased expression of complement- and Fc-receptors can augment opsono-phagocytosis. A further level of neutrophil up-regulation occurs by increasing the functional life span of neutrophils by GM-CSF. Thus, by delaying neutrophil apoptosis, GM-CSF greatly extends the time over which neutrophils may function at inflammatory sites. GM-CSF can thus exert a variety of important regulatory controls of neutrophil function during bacterial infections. Both the number and the functional status of neutrophils is highly regulated by GM-CSF. It is also possible that GM-CSF produced within localised sites of acute inflammation or infection may attract, trap and then activate neutrophils within this site.

[Retroperitoneal tumors. Observations on 8 cases]. / I tumori retroperitoneali. Osservazioni su 8 casi.

The incidence of retroperitoneal tumours is extremely low (0.01-0.2% of all neoplasias). Their onset is atypical and a palpable mass is often clinically evident, together with abdominal pain and fever. A major impetus has been given to diagnosis by instrumental techniques, and in particular CT. Surgical treatment is required for these tumours. The prognosis does not improve following radio and chemotherapy. The authors describe 8 cases referred to their attention between 1980 and 1995.

[beta-Interferon therapy of chronic hepatitis HCV+, 1b genotype]. / La terapia con beta-interferone dell'epatite cronica HCV+, genotipo 1b.

Various papers reported that chronic viral hepatitis is the principal cause of chronic liver disease, as cirrhosis and hepatocarcinoma. Interferon is the only agent known to have a beneficial effect in chronic hepatitis. The response rate has been less than 10 percent in patients with genotype 1b, but in patients with genotype 2 or 3 it has been greater than 40 percent. Aim of our investigation was to study 10 patients suffering from chronic viral hepatitis HCV related, genotype 1b, non responder to interferon-alpha therapy. In these patients we administered beta-interferon at the dose of 6 million units, 3 times a week, for 3 months. A significant reduction of aminotransferase level was reported after 3 months of the start of the therapy. An higher level of beta-interferon plasma rate was found in 3 non responder patients. The interaction of beta-interferon with the immune system was demonstrated with an increase of CD8+ lymphocytes that correlated with decrease of HCVRNA. The treatment with beta-interferon have a beneficial effect in patients with chronic hepatitis HCV related, genotype 1b, no responder to interferon-alpha therapy.

Catheter seeding of hepatocellular carcinoma following placement of a total implantable access port system.

A 61-year-old cirrhotic patient underwent hepatic resection for hepatocellular carcinoma and placement of a total implantable access port system in the hepatic artery for chemotherapy infusion. A year later, he developed a parietal metastasis at the port site as a consequence of tumor seeding along the arterial catheter. The metastasis was excised but the patient died because of disseminated disease two years after the first operation. Tumor seeding along the catheter should be included in the group of potential complications after placement of total implantable access port systems for intrahepatic chemotherapy The possible causes of this rare but life-threatening complication are discussed.

Expression of adhesion molecules and functional stimulation in human neutrophils: modulation by GM-CSF and role of the Bcr gene.

Although devoid of proliferative capacity, polymorphonuclear neutrophils (PMN) express receptors for haemopoietic growth factors and need growth factors for survival and functional stimulation. This study showed that in vitro treatment of human PMN with GM-CSF for up to 48 h increases cell surface expression of the beta 2-integrin molecules CD11b/CD18 and CD11c/CD18 and of the receptor for the chemotactic peptide fMLP. Such modifications are usually expression of PMN activation. PMN treated with GM-CSF also displayed increased phagocytosis of latex particles and enhanced oxidative burst and superoxide anion release. Since integrins mediate PMN adhesion to endothelium, homotypic adhesion, chemotaxis/phagocytosis and the triggering of respiratory burst, our results suggested that functional stimulation of PMN persisted following prolonged exposure of PMN to growth factors and that it was not a temporary phenomenon which lasted only for the first 12-24 h of treatment. We also used oligonucleotides antisense to the Bcr gene mRNA to inhibit expression of the gene and evaluate its function in PMN, following the recent observation that PMN from Bcr-null mutant mice produced increased amounts of reactive oxygen metabolites upon activation. The antisense oligonucleotides had no effect on the parameters investigated. This may indicate that increased production of O2 by neutrophils in which the Bcr gene is not expressed requires either that gene expression is absent in the earlier stages of myeloid differentiation/maturation, so that when inhibition occurs in the terminally differentiated neutrophils their functional status is no longer influenced, or that the residual low-level expression of the gene which may be present in the antisense-treated cells is sufficient to provide a normal response to stimulation.

Mycobacterium tuberculosis chaperonin 10 and N-truncated fragments. Their synthesis and purification by the isoelectric focusing technique carried out in solution.

The Mycobacterium tuberculosis chaperonin 10 protein and fragments corresponding to sequences 59-99, 51-99 and 26-99 were synthesised by the solid-phase methodology using a double coupling protocol and without the aid of capping agents. After the final acid cleavage using the low TFMSA-high HF protocol the polypeptides were purified by either the ion exchange chromatography/RP-HPLC combination or the isoelectric separation carried out in solution and followed by semi-preparative RP-HPLC. Comparison of the results obtained through the two approaches indicated that in general the isoelectricfocusing/HPLC combination was superior both in terms of recovery of final material and its purity. The advantages found were as follows: (i) Unlike ion exchange chromatography, no tailoring of the separation conditions is required, (ii) Several consecutive focusings can be carried out in progressively narrower pH gradients. This increases the separation resolution without the need of changing other separation parameters, (iii) Very little manipulation is needed, and each focusing requires 3-5h. (iv) Full compatibility with non-ionic denaturants such as 8 M urea. This increases solubility so that using the ROTOFOR instrument described here 50-100 mg crude polypeptide can be processed daily. Thus the isoelectric focusing technique carried out in solution is a valid and inexpensive alternative to ion exchange chromatography.

Intrahepatic fluorodeoxyuridine to treat unresectable hepatocellular carcinoma patients.

Of 16 HCC patients who received intrahepatic FdUrd at a dose of 0.3 mg/kg/day for 14 consecutive days by continuous infusion, every 28 days, no one achieved a complete response, while 3 patients achieved partial response (18.75%), lasting 6, 9 and 18 months, respectively. Seven patients (43.75%) exhibited stable disease and their mean time to progression was 4.28 months; in the extant 6 patients (37.5%) the disease progressed. Major biliary toxicity was observed. On the basis of these data, intra-arterial chemotherapy should not yet be considered the standard treatment of unresectable HCCs.

Evidence for GroES acting as a transcriptional regulator.

Cochaperonins (cpn10) assist chaperonins (cpn60) in promoting folding and assembly of other proteins. Upon expression of Mycobacterium tuberculosis cpn10 in Escherichia coli we have purified a polypeptide which, through amino acid sequencing, was identified as the endogenous E. coli 10K-S protein. Subsequent studies showed that its expression was specifically upregulated upon cloning of different members of the cpn10 family, including GroES, the E. coli cpn10. Pulse-chase experiments demonstrated that 10K-S is but one of several proteins whose expression is modulated upon cloning of cpn10. Up-regulation of 10K-S was also observed after exposure of normal cells, but not of groES- mutants, to elevated temperatures (42 degrees C). This allowed us to define 10K-S as a heat-shock protein (hsp) whose expression is dependent on the production of another hsp, GroES. Northern blot experiments showed that enhanced expression of 10K-S was consequent to increased accumulation of transcripts and that groES- mutants were devoid even of baseline levels of transcripts both at 37 degrees C and after temperature upshift. These results show that GroES, in addition to its established role in assisting protein folding may act as a transcriptional regulator and is likely to play an important role in modulating gene expression particularly in those conditions, like the stress response, in which its production is greatly enhanced.