PO-15 - Antiangiogenic small molecule ligands of FGF2 derived from the endogenous inhibitor thrombospondin-1.

INTRODUCTION: Platelet thrombospondin-1 (TSP-1) is a major endogenous regulator of growth factor activity in physiological and pathological processes, including tumor onset, progression and angiogenesis. We previously demonstrated that TSP-1 binds to FGF-2, sequestering the growth factor and inhibiting its angiogenic activity. We also identified a non-peptidic antiangiogenic compound (SM27) that retains the structural and functional properties of the FGF2-binding sequence of TSP-1. AIM: To identify new small molecule inhibitors of FGF2 that recapitulate the structure and functional properties of the FGF-2-binding site of TSP-1, by investigating the chemical space around SM27. MATERIALS AND METHODS: A similarity-based screening of small molecule libraries has been used to identify candidates, followed by docking calculations, and evaluation of the activity of the resulting compounds in biochemical and biophysical assays, to assess interaction with FGF2, and in experimental models of angiogenesis, to assess biological activity. RESULTS: The used integrated approach allowed selecting 7 bi-naphthalenic compounds that bound FGF2 inhibiting FGF2 binding to both heparan sulfate proteoglycans and FGFR1. The compounds inhibited FGF2-induced endothelial cell proliferation, vessel sprouting from aortic rings and angiogenesis in the chorioallantoic membrane assay, with improved potency over SM27. CONCLUSIONS: We have identified new compounds that are valuable as FGF inhibitors for potential therapeutic purposes. Moreover, these compounds are useful chemical tools to identify the minimal stereochemical requirements for FGF2 binding and activity to improve the design of new agents for antineoplastic therapy. ACKNOWLEDGEMENT: Supported by AIRC (Associazione Italiana per la Ricerca sul Cancro).

T-level downstaging and complete pathologic response after preoperative long-term radiochemotherapy for locally advanced rectal cancer.

Advantages of neoadjuvant chemoradiotherapy for locally advanced carcinoma of the middle and the lower third of the rectum are downstaging and downsizing of the tumor. Results of pathologic results are affected by post-treatment tissue changes and may influence the choice of surgical procedure. Forty-three consecutive patients (27 male, 16 female; mean age 64 years) were operated after receiving a long-term chemoradiotherapy during a period of 16 months. The data of initial staging procedure (high resolution magnetic resonance imaging) and results of pathological examination of the surgical specimens were analyzed. Regression of tumor was assessed by the absence of vital tumor cells and the post-treatment fibrotic tissue alterations. Regression of tumor size was seen in 42/43 patients leading to an improved T-stage in 27 patients. R0-resection was possible in all cases, although there was a perirectal tumor infiltration to less than 2 mm to circumference of the surgical specimen in 2 cases and unexpected small liver metastasis in 5 cases. Complete remission rate was 23.3% (10 cases). Detecting small amounts of vital tumor cells in altered tissue after chemoradiotherapy is a major problem of pathological examination procedure and should be taken into consideration by the surgeons. The choice of operation (resection vs. abdominoperineal extirpation vs. local excision) should be committed to the initial imaging procedure and not to any restaging procedure after neoadjuvant chemoradiotherapy.

[Intraoperative neuro-monitoring of the recurrent laryngeal nerve. Experience of 1000 consecutive patients]. / Neuromonitoring intraoperatorio del nervo laringeo ricorrente. Un'esperienza di 1000 pazienti consecutivi.

Intraoperative neuro-monitoring was introduced in thyroid surgery several years ago resulting in a facilitated identification of the recurrent laryngeal nerve and less recurrent laryngeal nerve injuries. Between 1999 and 2005 data of all patients undergoing thyroid resection were recorded and analyzed yearly. The intraoperative identification of recurrent laryngeal nerve succeeded in 99.2% (1768 nerves at risk). The percentage of complete resecting surgical procedures raised from 17% to 84%. Minimal vocal cord dysfunction, associated with hematoma and edema in most cases, was diagnosed laryngoscopically in 2.9%. The permanent palsy rate of 0.8% in the first year decreased down to 0.32%. Routinely introduction of intraoperative neuro-monitoring in thyroid surgery is associated with a demonstrable decreased palsy rate. Anyway, the rate of minimal vocal cord movement disorders and transient recurrent laryngeal nerve palsies is not changed.

An unusual case of synovial cyst of the hip joint presenting as femoral vein compression and severe lower limb edema.

INTRODUCTION: Synovial cyst of the hip joint causing the compression of the femoral vein is a rare occurrence. We carefully reviewed the international literature collecting 26 additional cases. REPORT: A case of a patient affected with synovial cyst of the hip joint causing the compression of the femoral vein and severe lower limb edema is presented. DISCUSSION: The treatment of choice of synovial cyst compressing the femoral vein is surgical removal.

Laparoscopic ultrasound-guided resection of gastric submucosal tumors.

BACKGROUND: The laparoscopic resection of gastric stromal tumors is being performed with increased frequency. Wedge resection of anterior wall lesions is generally performed. The treatment of posterior wall lesions is still controversial. METHODS: We report three cases of gastric submucosal tumors treated by a laparoscopic wedge resection of the stomach. All lesions were localized anterior gastric wall by intraoperative ultrasound on the. In the first patient the resection was performed with an endoscopic stapler; in the other patients, ultrasonic coagulation in association with an intracorporeal suture has been used. RESULTS: All patients were successfully treated laparoscopically; there were no conversions to open surgery. In all cases the operative course was uneventful. The postoperative hospital stay ranged from 6 to 8 days. CONCLUSIONS: The results suggest that laparoscopic surgery is an adequate strategy for gastric submucosal neoplasms including gastrointestinal stromal tumors (GIST). Intraoperative ultrasound is very useful in the selection of the technical approach with or without the endoscopic stapler.

Heparin derivatives as angiogenesis inhibitors.

Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumor neovascularization and in angioproliferative diseases. Tumors cannot growth as a mass above few mm(3) unless a new blood supply is induced. It derives that the control of the neovascularization process may affect tumor growth and may represent a novel approach to tumor therapy. Angiogenesis is controlled by a balance between proangiogenic and antiangiogenic factors. The angiogenic switch represents the net result of the activity of angiogenic stimulators and inhibitors, suggesting that counteracting even a single major angiogenic factor could shift the balance towards inhibition. Heparan sulfate proteoglycans are involved in the modulation of the neovascularization that takes place in different physiological and pathological conditions. This modulation occurs through the interaction with angiogenic growth factors or with negative regulators of angiogenesis. Thus, the study of the biochemical bases of this interaction may help to design glycosaminoglycan analogs endowed with angiostatic properties. The purpose of this review is to provide an overview of the structure/function of heparan sulfate proteoglycans in endothelial cells and to summarize the angiostatic properties of synthetic heparin-like compounds, chemically modified heparins, and biotechnological heparins.

Radiofrequency of hepatocellular carcinoma in patients with liver cirrhosis: a critical appraisal of the laparoscopic approach.

BACKGROUND: The optimal treatment for hepatocellular carcinoma (HCC) is surgical resection. However, only a small percentage of patients are operative candidates. Percutaneous radiofrequency interstitial thermal ablation proved to be effective in the treatment of unresectable HCC. Recent advances in laparoscopic ultrasound have improved the accuracy in detecting small intrahepatic HCC nodules missed by preoperative imaging techniques. Our objective was to evaluate a novel operative combination of laparoscopic ultrasound with laparoscopic radiofrequency (LRF) in the treatment of HCC not amenable to liver resection. The aim of our review was to evaluate the advantages and limits of the laparoscopic approach according the criteria of the evidence-based medicine. CONCLUSIONS: LRF of HCC proved to be a safe and effective technique at least in the short and mid-term. This technique may be indicated in selected cases when the percutaneous approach to the lesion is very difficult or contraindicated.

Regulation of mouse p45 NF-E2 transcription by an erythroid-specific GATA-dependent intronic alternative promoter.

The erythroid-enriched transcription factor NF-E2 is composed of two subunits, p45 and p18, the former of which is mainly expressed in the hematopoietic system. We have isolated and characterized the mouse p45 NF-E2 gene; we show here that, similar to the human gene, the mouse gene has two alternative promoters, which are differentially active during development and in different hematopoietic cells. Transcripts from the distal promoter are present in both erythroid and myeloid cells; however, transcripts from an alternative proximal 1b promoter, lying in the first intron, are abundant in erythroid cells, but barely detectable in myeloid cells. During development, both transcripts are detectable in yolk sac, fetal liver, and bone marrow. Transfection experiments show that proximal promoter 1b has a strong activity in erythroid cells, which is completely dependent on the integrity of a palindromic GATA-1 binding site. In contrast, the distal promoter 1a is not active in this assay. When the promoter 1b is placed 3' to the promoter 1a and reporter gene, in an arrangement that resembles the natural one, it acts as an enhancer to stimulate the activity of the upstream promoter la.

Expression in hematopoietic cells of GATA-1 transcripts from the alternative "testis" promoter during development and cell differentiation.

GATA-1 is a transcription factor expressed both in the hematopoietic system and in the Sertoli cells of the testis, and is essential for correct erythropoiesis. Hematopoietic and Sertoli cells transcribe GATA-1 from two different promoters: the proximal (erythroid) is active in hematopoietic cells; the distal (testis) is active in Sertoli cells. We investigated by RT-PCR the possibility that GATA-1 might be transcribed from the testis promoter also in hematopoietic cells. Testis promoter-derived transcripts are present at low levels in vivo at all stages of hematopoietic development. Purified multipotent progenitors, fractionated into populations expressing low or high levels of GATA-1, do not contain any "testis" transcripts. However, when grown in vitro, they rapidly express GATA-1 from the testis promoter in the presence of Erythropoietin (Epo) but not in that of other growth factors. This result reflects an Epo-dependent differentiation event, rather than a direct effect of Epo. Indeed, immortalized progenitor cell lines which respond to both Epo and SCF, continue to express testis-derived transcripts when switched from Epo to SCF.

Erythroid-specific activation of the distal (testis) promoter of GATA1 during differentiation of purified normal murine hematopoietic stem cells.

To understand the molecular mechanisms of erythroid differentiation, we analyzed by semiquantitative RT-PCR the expression of the transcription factor GATA1, the erythropoietin receptor (EpoR), and erythroid (beta-globin) differentiation markers in purified hematopoietic stem cells (HSCs) after in-vitro-induced differentiation. Whether GATA1 transcription was from the proximal (with respect to the AUG, also known as erythroid) or the distal (also known as testis) promoter was analyzed as well. Low-density marrow cells which bind to wheat germ agglutinin, but not to the antibody 15.1.1, and which either do or do not retain the dye rhodamine-123 (Rho-bright and Rho-dull, respectively), were purified. Rho-dull, but not Rho-bright cells permanently reconstitute lymphomyelopoiesis in W/Wv and severe-combined-immunodeficiency mice and, therefore, contain HSCs. Both Rho-dull and Rho-bright cells give rise to progenitor and differentiated cells (peak values at days 15 and 5, respectively) in liquid culture. Multilineage, erythroid-restricted or myeloid-restricted differentiation is observed when the cultures are stimulated with stem cell factor (SCF) + interleukin (IL)-3, SCF + IL-3 + Epo, or SCF + IL-3 + granulocyte-colony-stimulating factor, respectively. Rho-dull cells have barely detectable reconstitution potential at day 5 of culture. None of the genes examined were expressed in purified Rho-bright or Rho-dull cells. The only exception was GATA1 which was expressed at maximal levels in Rho-bright cells at the onset of culture. Rho-dull cells did not express GATA1 before day 3 of culture (maximal expression at days 10-15). Activation of GATA1 and EpoR was observed in all growth of mRNA for the two genes expressed by the cells. In contrast, beta-globin mRNA was detected only in the presence of Epo. The transcription of GATA1 was exclusively from the proximal promoter in the absence of Epo but both proximal and distal transcripts were observed in its presence. Maximum transcription from the distal promoter (approximately equal to 0.2% of total GATA1 mRNA) coincided with maximal globin mRNA levels (day 5 or day 15 for Rho-bright and Rho-dull cells, respectively). These results indicate that GATA1 is activated at the transition point between HSCs and pluripotent progenitor cells and erythroid-specific GATA1 regulation involves activation of the distal GATA1 promoter.

Immortalization of multipotent growth-factor dependent hemopoietic progenitors from mice transgenic for GATA-1 driven SV40 tsA58 gene.

The transcription factor GATA-1 is required for the normal development of erythroid cells. GATA-1 is also expressed in other hemopoietic cells, suggesting that it might be initially activated in a multipotent progenitor. To immortalize GATA-1-expressing progenitors, we generated mice transgenic for a thermosensitive SV40 T gene, driven by the GATA-1 promoter-enhancer. Immortalized marrow cells grow in culture at 32 degrees C but not at 38 degrees C, and are dependent on erythropoietin (Epo) or interleukin 3 (IL-3). Epo dependent cells express hemoglobin, high levels of GATA-1, GATA-2 and NF-E2 p45 mRNAs, and are positive for stem cell antigen 2 (Sca-2) and the early myeloid marker ER-MP12. IL-3 dependent cells can be derived from Epo dependent lines, and are hemoglobin-, Sca-2- and ER-MP12-negative, have low GATA-1 and NF-E2 p45 mRNA levels, and express myeloid markers Mac-1, F4/80 and Gr-1. Brief treatment of Epo dependent cells with myeloid growth factors (plus Epo) leads to the induction of Mac-1, F4/80 and Gr-1, concomitant with the disappearance from most cells of Sca-2, ER-MP12 and GATA-1 driven T antigen nuclear expression. Thus, the immortalized Epo dependent cells have the property of a progenitor capable of differentiation towards either the erythroid or myeloid lineages. These cells initiate transcription of a proportion of GATA-1 RNA molecules at an upstream promoter, previously known to be expressed only in testis cells.

An erythroid specific enhancer upstream to the gene encoding the cell-type specific transcription factor GATA-1.

The transcription factor GATA-1 is expressed in a subset of hemopoietic cells, where it mediates the cell-type specific expression of several genes. We have cloned the mouse and human GATA-1 genes. A region upstream to the first exon, and highly conserved between mouse and man, acts as an erythroid specific enhancer in transient assays, if linked to the GATA-1 or to the SV40 promoter. The activity of the enhancer is almost completely dependent on the integrity of a dimeric GATA-1 binding site.

[Type-III acrocephalosyndactylia (Saethre-Chotzen syndrome). Description of 2 cases]. / Acrocefalosindattilia tipo III (S. Saethre-Chotzen). Descrizione di due casi.

The acrocephalosyndactylia III is a syndrome, which follows an autosomal dominant mode of inheritance, characterized by premature fusion of the cranial sutures in association with mild cutaneous syndactyly. The authors describe two cases recently come to their observation and point out the usefulness of the imaging diagnostics (CT, MRI) in finding anomalies specific of this affection.

[Non-invasive cardiologic study on a soccer team]. / Studio cardiologico non invasivo in una squadra di calcio.

Fifteen professional football players have been examined with basal electrocardiography, M-mode echocardiography, bicycle ergometry, Holter dynamic electrocardiography; in six of them also, the electrocardiogram has been recorded by during a match. The aim of the research was to evaluate, by non-invasive tests, the cardiovascular status of an athletic team, with particular consideration to rhythm disturbances. The methods employed have given valuable informations. The echocardiographic investigation has frequently shown left ventricular dilation and/or hypertrophy. Dynamic electrocardiography has revealed in many subjects rhythm and conduction disturbances. No athlete has displayed severe rhythm disturbances during the bicycle ergometer test, except one, who had paroxysmal ventricular tachycardia, of complex etiopathogenetic and prognostic significance. Telemetric recording has been very useful to follow the heart action during a match, but its use was often hampered by technical difficulties.

[Pseudo-Conn's syndrome after habitual ingestion of liquorice. Report on various clinical cases]. / Pseudo-sindrome di Conn da ingestione abituale di liquirizia. Rapporto su alcuni casi clinici.

Four female subjects were admitted to our Department for a form of severe systo-diastolic hypertension, recalcitrant to previous anti-hypertensive treatment, accompanied by marked hypokalemia. Patients had a common history of the continuous use, for pleasure or medicinal purposes, of liquorice-based preparations. In all cases, suspension led to normalisation of kalemia in a period varying from six to fifteen days, while arterial pressure values and all other essential parameters examined (plasma reninic activity, aldosteronuria, etc.) recovered their balance more slowly.