Agonistic anti-human Fas monoclonal antibody induces fibroblast-like synoviocyte apoptosis in haemophilic arthropathy: potential therapeutic implications.

Haemophilic arthropathy (HA) is characterized by chronic proliferative synovitis leading to cartilage destruction and shares some pathological features with rheumatoid arthritis (RA). Apoptosis has been implicated in RA pathogenesis, and an agonistic anti-Fas monoclonal antibody (mAb) was found to induce RA fibroblast-like synoviocyte (FLS) apoptosis and suppress synovial hyperplasia in animal models of RA. The aim of this study was to evaluate the effect of anti-Fas mAb on HA-FLS. FLS were isolated from knee synovial biopsies from six HA patients, six RA patients and six healthy subjects. The expression of Fas in synovial biopsies was investigated by immunohistochemistry. FLS were stimulated with anti-Fas mAb at different concentrations, alone or in combination with tumour necrosis factor-α (TNF-α) and basic fibroblast growth factor (bFGF). Fas expression in FLS was assessed by Western blot. Cell viability was studied with the WST-1 assay. Active caspase-3 levels were measured using ELISA and Western blot. A strong Fas-immunoreactivity was observed in different cells of HA synovium, including FLS, inflammatory cells and endothelial cells. Fas antigen was constitutively overexpressed in cultured HA-FLS. Anti-Fas mAb had a significant cytotoxicity on HA-FLS in a dose-dependent manner, either alone or in combination with TNF-α and bFGF. These cytotoxic effects were due to the ability of anti-Fas to induce HA-FLS apoptosis, as shown by the increased active caspase-3 levels. Anti-Fas mAb exhibited a more pronounced pro-apoptotic effect on HA-FLS than RA-FLS. Fas antigen is highly expressed on HA-FLS and its stimulation by anti-Fas mAb may be an effective strategy to induce HA-FLS apoptosis.

Chemically Modified Variants of Fenofibrate with Antiglioblastoma Potential.

Anticancer effects of a common lipid-lowering drug, fenofibrate, have been described in the literature for a quite some time; however, fenofibrate has not been used as a direct anticancer therapy. We have previously reported that fenofibrate in its unprocessed form (ester) accumulates in the mitochondria, inhibits mitochondrial respiration, and triggers a severe energy deficit and extensive glioblastoma cell death. However, fenofibrate does not cross the blood brain barrier and is quickly processed by blood and tissue esterases to form the PPARα agonist fenofibric acid, which is practically ineffective effective in triggering cancer cell death. To address these issues, we have made several chemical modifications in fenofibrate structure to increase its stability, water solubility, tissue penetration, and ultimately anticancer potential. Our data show that, in comparison to fenofibrate, four new compounds designated here as PP1, PP2, PP3, and PP4 have improved anticancer activity in vitro. Like fenofibrate, the compounds block mitochondrial respiration and trigger massive glioblastoma cell death in vitro. In addition, one of the lead compounds, PP1, has improved water solubility and is significantly more stable when exposed to human blood in comparison to fenofibrate. Importantly, mice bearing large intracranial glioblastoma tumors demonstrated extensive areas of tumor cell death within the tumor mass following oral administration of PP1, and the treated mice did not show any major signs of distress, and accumulated PP1 at therapeutically relevant concentrations in several tissues, including brain and intracranial tumors.

Regulation of Id gene expression by type I insulin-like growth factor: roles of Stat3 and the tyrosine 950 residue of the receptor.

Id proteins are known to play important roles in the proliferation and differentiation of many cell types. The type 1 insulin-like growth factor receptor (IGF-IR), activated by its ligand, induces the differentiation of 32D IGF-IR cells, a murine hematopoietic cell line, expressing a human IGF-IR. Expression in 32D IGF-IR cells of a dominant negative mutant of Stat3 (DNStat3) inhibits IGF-I-mediated differentiation. DNStat3 causes a dramatic increase in Id2 gene expression. This increase, however, is IGF-I dependent and is abrogated by a mutation at tyrosine 950 of the IGF-IR. These results indicate that in 32D cells, the IGF-IR regulates the expression of the Id2 gene and that this regulation is modulated by both positive and negative signals. Our results also suggest that in this model, Id2 proteins influence the differentiation program of cells but are not sufficient for the full stimulation of their proliferation program.

Multiple signaling pathways of the insulin-like growth factor 1 receptor in protection from apoptosis.

The type 1 insulin-like growth factor receptor (IGF-1R), activated by its ligands, protects several cell types from a variety of apoptotic injuries. The main signaling pathway for IGF-1R-mediated protection from apoptosis has been previously elucidated and rests on the activation of phosphatidylinositol 3-kinase, Akt/protein kinase B, and the phosphorylation and inactivation of BAD, a member of the Bcl-2 family of proteins. In 32D cells (a murine hemopoietic cell line devoid of insulin receptor substrate 1 [IRS-1]), the IGF-1R activates alternative pathways for protection from apoptosis induced by withdrawal of interleukin-3. One of these pathways leads to the activation of mitogen-activated protein kinase, while a third pathway results in the mitochondrial translocation of Raf and depends on the integrity of a group of serines in the C terminus of the receptor that are known to interact with 14.3.3 proteins. All three pathways, however, result in BAD phosphorylation. The presence of multiple antiapoptotic pathways may explain the remarkable efficacy of the IGF-1R in protecting cells from apoptosis.

Anti-apoptotic signaling of the IGF-I receptor in fibroblasts following loss of matrix adhesion.

The type 1 insulin-like growth factor receptor (IGF-IR) is known to protect cells from a variety of apoptotic injuries. In several instances, the anti-apoptotic effect of the wild type IGF-IR is more evident under conditions of anchorage-independence than in cells in monolayer cultures. We have investigated IGF-IR signaling in cells in anoikis, a form of apoptosis that occurs when cells are denied attachment to the extra-cellular matrix. IGF-I protects mouse embryo fibroblasts (MEF) from anoikis caused by withdrawal of growth factors. Survival is dependent on the concentration of IGF-I and a sufficient number of functional IGF-I receptors. In this model, IGF-I protection correlates best with ras activation and cell-to-cell aggregation, while PI3-kinase, Akt and MAP kinases seem to play a lesser, alternative role.

Mechanisms of regulation of cell adhesion and motility by insulin receptor substrate-1 in prostate cancer cells.

LNCaP cells are human prostatic cancer cells that have a frame-shift mutation of the tumor suppressor gene PTEN and do not express the insulin receptor substrate-1 (IRS-1), a major substrate of the type 1 insulin-like growth factor receptor (IGF-IR). Ectopic expression of IRS-1 in LNCaP cells increases cell adhesion and decreases cell motility by an IGF-I-independent mechanism. We show now that these effects of IRS-1 are accompanied by serine phosphorylation of IRS-1 and are inhibited by inhibitors of phosphatidylinositol 3-kinase (PI3K). We have confirmed the requirement for PI3K activity and serine phosphorylation by the use of IRS-1 mutants, expressed in LNCaP cells. Serine phosphorylation inhibits IGF-I-induced tyrosyl phosphorylation of IRS-1, which is restored by the expression of wild-type PTEN or by inhibition of PI3K activity. Finally, IRS-1 in LNCaP cells co-immunoprecipitates with integrin alpha 5 beta 1, and the association is again IGF-I-independent. We conclude that in LNCaP cells, IRS-1 is serine phosphorylated by PI3K, generating effects that are different, and even opposite, from those generated by IGF-I.

Activation of the IGF-IR system contributes to malignant growth of human and mouse medulloblastomas.

Insulin-like growth factor I receptor (IGF-IR) has been implicated in the normal and malignant growth of many cell types including cells from the central nervous system. In the cerebellar cortex IGF-IR mRNA is found in granular cells and IGF-I stimulation is mitogenic and protects cells from low-potassium-induced apoptosis. Since primitive neuroectodermal tumors/medulloblastomas (PNETs/medulloblastomas) are suspected to originate from the external cerebellar granular layer, it is reasonable to postulate that IGF-IR and/or its signaling molecules may contribute to the transformation of these poorly differentiated cells. To study activation of the IGF-IR system in medulloblastomas, we have utilized an antibody (anti-pY1316) that specifically recognizes the phosphorylated (active) form of the IGF-IR. Medulloblastoma biopsy specimens were positive when examined immunohistochemically with anti-Y1316 antibody. Further analysis of the IGF-IR system was performed in three human (Daoy, TE-671, D283 Med) and four mouse (BsB8, BsB13, Bs-1b, Bs-1c) medulloblastoma cell lines. All the murine cell lines examined express IGF-IR and PI3-kinase at relatively normal levels, and grossly overexpress IRS-1, when compared with normal mouse cerebellum. Within 15 min following IGF-I stimulation both mouse and human cell lines phosphorylate the beta subunit of the IGF-IR, IRS-1, Akt, and MAP kinases. They respond with cell proliferation when stimulated solely with IGF-I and are strongly inhibited when challenged with a dominant negative mutant of the IGF-IR (486/STOP), or with antisense oligonucleotides against the IGF-IR mRNA.

Intracellular association of a mutant insulin-like growth factor receptor with endogenous receptors.

The type 1 insulin-like growth factor receptor (IGF-IR) is emerging as a powerful survival factor against a variety of apoptotic agents in many cell types. A mutant IGF-IR designated 486/STOP is known to induce apoptosis and inhibit the growth of human tumor cells in mice. We have investigated the mechanism of action of 486/STOP. To study it, we have developed a new retroviral vector in which we have combined a self-inactivating 5'-long terminal repeat with an inducible heat-shock promoter (heat shock protein 70) from Drosophila. Using this technique, we find that the polypeptide encoded by 486/STOP is partially retained within the cell and partially secreted. However, the secreted polypeptide is subsequently taken up by the cells. In both cases, a specific intracellular interaction of 486/STOP with the endogenous IGF-IRs can be demonstrated by coimmunoprecipitation.

Domains of the insulin-like growth factor I receptor required for the activation of extracellular signal-regulated kinases.

The type 1 insulin-like growth factor receptor (IGF-IR) activates the extracellular signal-regulated kinases (ERK1 and -2). The two major substrates of the IGF-IR, insulin receptor substrate-1 (IRS-1) and the Shc proteins, are known to contribute to this activation. We investigated the domains of the IGF-IR required for the activation of the ERK proteins. To facilitate this study, we used a cell line (32D cells) that lacks IRS-1. In the absence of IRS-1, ERK activation is inhibited if the IGF-IR is mutated at two domains: tyrosine Y950 and a serine quartet at 1280-1283. Expression of IRS-1 in 32D cells expressing the double mutant IGF-IR restores ERK activation. The importance of the C-terminus of the IGF-IR in ERK activation (in the absence of IRS-1) is confirmed by the failure of the insulin receptor to give a sustained activation of ERK. In this model system, there is a good, but not exact, correlation between ERK activation and cell survival after withdrawal of growth factors.

Antigenicity and immunogenicity of the V3 domain of HIV type 1 glycoprotein 120 expressed on the surface of Streptococcus gordonii.

Five different V3 domains of HIV-1 gp120 were expressed on the surface of the gram-positive bacterium Streptococcus gordonii, a model live vector for vaccine delivery. Sera of HIV-1-infected individuals and human monoclonal antibodies specifically recognized the gp120 sequences on the bacterial surface. Recombinant V3 from the reference HIV-1 strain MN was also shown to retain a conformation that allowed reaction with a conformation-specific monoclonal antibody. A V3-specific serum antibody response was detected in mice immunized both by subcutaneous injection and by vaginal colonization. V3-specific IgG2a antibodies, suggestive of a Th1 response, were found in the sera of mice colonized by recombinant bacteria.

Polyomaviruses and cancer--interplay between viral proteins and signal transduction pathways.

Polyomaviruses are highly suspected to be involved in the development of cancer. A strong correlation has been established between the activity of an early viral genome and the development of a transformed phenotype. Polyomavirus transforming antigens (T-antigens) are the major suspects in the process of deregulating cellular equilibrium. Multiple interactions between T-antigens and cellular regulatory proteins have been detected at different regulatory levels including signal transduction, gene expression, cell cycle progression, and possible DNA repair. In this context, we are reviewing the most recent experimental evidence which, in combination with more than thirty years of studies of polyomaviruses, could help us understand whether and how viral infection contributes to the development of malignant transformation.

Gliomas and seizures.

Glial neoplasms account for nearly 50% of all adult primary brain tumors. They originate from glial cells in the brain and/or spinal cord and include low-grade diffuse astrocytomas, anaplastic-astrocytomas, and glioblastomas. Of all brain tumors, glioblastoma multiforme (GBM) is the most aggressive and is characterized by rapid glial cell growth, resistance to radio- and chemo- therapies, and relentless infiltration and spreading throughout the central nervous system (CNS). In glioblastomas, primary tumor growth and CNS invasion are associated with the activation of complex structural molecular and metabolic changes within the tumor tissue, which profoundly affect the surrounding neuronal networks and may in part explain induction of epilepsy. In fact, epileptic seizures are very common among patients with glial tumors, reaching nearly 50% in glioblastoma patients and almost 90% in low-grade astrocytomas. The overall hypothesis presented here discusses the possibility that the aberrant tumor cell metabolism may act directly on neuronal network, and this leads to seizure susceptibility. Further invasion and growth of the malignant glial cells exacerbate this initial pathologic state which promotes recurrent seizures (epileptogenesis).

IGF-IR-dependent expression of Survivin is required for T-antigen-mediated protection from apoptosis and proliferation of neural progenitors.

The insulin-like growth factor-1 receptor (IGF-IR) and the human polyomavirus JCV protein, T-antigen cooperate in the transformation of neuronal precursors in the cerebellum, which may be a contributing factor in the development of brain tumors. Because it is not clear why T-antigen requires IGF-IR for transformation, we investigated this process in neural progenitors from IGF-IR knockout embryos (ko-IGF-IR) and from their wild-type nontransgenic littermates (wt-IGF-IR). In contrast to wt-IGF-IR, the brain and dorsal root ganglia of ko-IGF-IR embryos showed low levels of the antiapoptotic protein Survivin, accompanied by elevated numbers of apoptotic neurons and an earlier differentiation phenotype. In wt-IGF-IR neural progenitors in vitro, induction of T-antigen expression tripled the expression of Survivin and accelerated cell proliferation. In ko-IGF-IR progenitors induction of T-antigen failed to increase Survivin, resulting in massive apoptosis. Importantly, ectopic expression of Survivin protected ko-IGF-IR progenitor cells from apoptosis and siRNA inhibition of Survivin activated apoptosis in wt-IGF-IR progenitors expressing T-antigen. Our results indicate that reactivation of the antiapoptotic Survivin may be a critical step in JCV T-antigen-induced transformation, which in neural progenitors requires IGF-IR.

p73 modulates HIV-1 Tat transcriptional and apoptotic activities in human astrocytes.

HIV-1 Tat is a potent transcriptional activator of the viral promoter with the ability to modulate a number of cellular regulatory circuits including apoptosis. Tat exerts its effects through interaction with viral as well as cellular proteins. Here, we studied the influence of p73, a protein that is implicated in apoptosis and cell cycle control, on Tat apoptotic function in the central nervous system. We recently demonstrated the ability of Tat to associate with p73, and that this association modulates Tat transcriptional activity (Amini et al., Mol Cell Biol 2005; 18: 8126-8138). We demonstrated that p73 interferes with Tat-mediated apoptosis by preventing the up-regulation of Bax and down-regulation of Bcl-2 proteins in astrocytes. Thus, the interplay between Tat and p73 may affect Tat contribution to apoptotic events in the brain, limiting its involvement in the neuropathology often observed in the brains of HIV-1 patients.

[Fragmentation of pulmonary emboli (author's transl)]. / Frammentazione degli emboli polmonari.

Preliminary results of attempts of pulmonary artery thrombi fragmentation after venous embolism are presented. Fragmentation is directly obtained by angiographic catheters (F 9 or 10). By this method we attempted to remove the block of some pulmonary branches, in order to obtain a perfusion of them and to improve fibrinolysis. This method is useful in patients who are not candidated for surgical embolectomy.

[Achilles tendon ossification and achillodynia]. / Ossificazione del tendine di Achille e achillodinia.

Achilles tendon ossification is a rare and asymptomatic condition. The possible sequelae of tendinitis, bursitis and fracture are some of the causes of achillodynia. Achillodynia is often unrelieved by conservative management and excision of the ossification may be necessary. Moreover, ossification were found in 3 of 41 patients with spontaneous rupture of the Achilles tendon. Predisposing degenerative changes of the tendon were found in all patients. Thus there is no evidence that ossification by itself may increase the risk of tendon rupture.

[Changed diastolic function at rest in patients with perfusion defects in exertion myocardial scintigraphy with TI-201]. / Alterata funzione diastolica a riposo in pazienti con difetti di perfusione alla scintigrafia miocardica da sforzo con TI-201.

Myocardial perfusion scintigraphy with TI-201 after exercise allows distinction between areas of scar (irreversible defect) and areas of ischemia (reversible defects). Accordingly 4 major groups of patients can be identified: with normal perfusion pattern (Group I); with reversible ischemia (Group II); with scar of previous myocardial infarction (Group III); with both evidence of scar and ischemia (Group IV). Sixty-nine patients (59 m; 10 f; mean age 55.7 +/- 9 years) with suspected or demonstrated ischemic heart disease underwent stress TI-201 myocardial scintigraphy and on the basis of the scintigraphic results were assigned as follows: 11 to group 1, 14 to group II, 31 to group III and 13 to group IV. In order to investigate the behaviour of ventricular diastolic function in these different subsets, all the patients underwent subsequently a radionuclide angiography at rest (both first pass and equilibrium gated blood pool studies), which allowed the assessment of left ventricular ejection fraction (EF), peak filling rate (PFR)--as expression of diastolic function--and regional wall motion pattern. The values of EF and PFR were significantly reduced (p less than 0.05) in the patients with defects of perfusion (Groups II, III and IV) in comparison to the patients with normal perfusion (Group I); abnormal wall motion was found in 0 (I), 8 (II), 22 (III) and 7 (IV) patients. The diastolic function was more frequently altered (PFR less than 2.5 EDV/sec) than the systolic function (EF less than 50%) or regional wall motion, mainly in patients with reversible scintigraphic defects (prevalence of alterations in the groups II and IV: PFR: 78%, EF: 22%, abnormal wall motion: 56%).(ABSTRACT TRUNCATED AT 250 WORDS)

[Treatment of hot thyroid nodule with percutaneous ethanol injection: indications, complications, and prognostic factors]. / La cura del nodulo caldo della tiroide con alcolizzazione percutanea: indicazioni, complicanze e fattori prognostici.

Percutaneous ethanol injection (PEI) has recently been proposed as an alternative therapy for toxic thyroid adenomas, instead of conventional treatments (pharmacological, surgical and radiometabolic therapies). The aim of this study was to investigate efficacy, complications and prognostic factors of PEI treatment in a group of 74 patients, 14 men and 60 women, treated from May, 1991, to December, 1994. Twenty-seven patients had nontoxic (pre-toxic) nodules (normal T3 and T4 and undetectable TSH serum levels) and 47 toxic nodules (high serum levels of thyroid hormones). A mean of 1.6 ml ethanol/cc of nodule volume was injected in 3-14 sessions (mean = 6). Ten subjects were treated twice, and 2 patients three times. Results were defined as: 1) complete cure: normalization of T3, T4 and TSH levels and appearance of extranodular thyroid tissue at scintigraphy; 2) partial cure: reduction in thyroid hormones within the normal range but still undetectable TSH levels and still suppressed extranodular thyroid tissue at scintigraphy; 3) failure. Complete cure was obtained in 96% of nontoxic (pretoxic) nodules and 65% of toxic ones. Moreover, partial cure was seen in 27.5% more toxic nodules and failure in 7.5%. The most significant complications were a case of transient dysphonia and two cases of common jugular vein thrombosis, both resolved spontaneously. The most important prognostic factor was the degree of hyperthyroidism (as FT4 and T3 serum levels), while nodule volume was rather useless to predict the final result. In conclusion, PEI can be an alternative, effective and low-cost treatment for autonomous thyroid nodules, without any severe complication and well tolerated by the patients.

Insulin and IGF-I receptors signaling in protection from apoptosis.

R-cells are mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor I receptor (IGF-IR) genes. Because R-cells do not express the IGF-IR, they are ideal for studying the biological effects of the insulin receptor (IR), independently from any contribution by the IGF-IR. By stably transfecting R-cells with constructs expressing the IR, we show here the IR can protect cells from apoptosis induced by anoikis or by okadaic acid. The IR, however, is not as efficient as the IGF-IR in protecting mouse embryo fibroblasts from apoptosis, even when IRS-1, one of its major substrates, is over-expressed. In addition, the protection by the IGF-IR is resistant to inhibitors of phosphatidylinositol 3-kinase (PI 3-ki), while the anti-apoptotic effect of the IR is sensitive. These experiments suggest that the IGF-IR uses an alternative anti-apoptotic pathway, not shared with the IR, which is PI3-ki-independent.