Reduction of autophagy and increase in apoptosis correlates with a favorable clinical outcome in patients with rheumatoid arthritis treated with anti-TNF drugs.

BACKGROUND: Autophagy has emerged as a key mechanism in the survival and function of T and B lymphocytes, and its activation was involved in apoptosis resistance in rheumatoid arthritis (RA). To investigate whether the relationship between autophagy and apoptosis may impact the response to the therapy, we analyzed ex vivo spontaneous autophagy and apoptosis in patients with RA subjected to treatment with anti-tumor necrosis factor (TNF) drugs and in vitro the effects of TNFα and anti-TNF drugs on cell fate. METHODS: Peripheral blood mononuclear cells (PBMCs) from 25 RA patients treated with anti-TNF drugs were analyzed for levels of autophagy marker LC3-II by western blot and for the percentage of annexin V-positive apoptotic cells by flow cytometry. The same techniques were used to assess autophagy and apoptosis after in vitro treatment with TNFα and etanercept in both PBMCs and fibroblast-like synoviocytes (FLS) from patients with RA. RESULTS: PBMCs from patients with RA responsive to treatment showed a significant reduction in LC3-II levels, associated with an increased apoptotic activation after 4 months of therapy with anti-TNF drugs. Additionally, the expression of LC3-II correlated with DAS28. TNFα was able to induce autophagy in a dose-dependent manner after 24 h of culture in RA PBMCs and FLS. Moreover, etanercept caused a significant reduction of autophagy and of levels of citrullinated proteins. CONCLUSIONS: Our results show how the crosstalk between autophagy and apoptosis can sustain the survival of immune cells, thus influencing RA progression. This suggests that inhibition of autophagy represents a possible therapeutic target in RA.

Association of fission proteins with mitochondrial raft-like domains.

It was shown that receptor-mediated apoptosis involves a cascade of subcellular events including alterations of mitochondria. Loss of mitochondrial membrane potential that follows death receptor ligation allows the release of apoptogenic factors that result in apoptosis execution. Further important mitochondrial changes have been observed in this regard: mitochondrial remodeling and fission that appear as prerequisites for the occurrence of the cell death program. As it was observed that lipid rafts, glycosphingolipid-enriched structures, can participate in the apoptotic cascade being recruited to the mitochondria under receptor-mediated proapoptotic stimulation, we decided to analyze the possible implication of these microdomains in mitochondrial fission. We found that molecules involved in mitochondrial fission processes are associated with these domains. In particular, although hFis1 was constitutively included in mitochondrial raft-like domains, dynamin-like protein 1 was recruited to these domains on CD95/Fas triggering. Accordingly, the disruption of rafts, for example, by inhibiting ceramide synthase, leads to the impairment of fission molecule recruitment to the mitochondria, reduction of mitochondrial fission and a significant reduction of apoptosis. We hypothesize that under apoptotic stimulation the recruitment of fission-associated molecules to the mitochondrial rafts could have a role in the morphogenetic changes leading to organelle fission.

Lipid microdomains contribute to apoptosis-associated modifications of mitochondria in T cells.

Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltage-dependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-beta-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.

Conjugates of aberrant gangliosides in antiglioma vaccine: toxicological assay.

We studied sterility and toxicity of vaccine LS1 containing aberrant gangliosides isolated from brain bioptates of 48 patients with gliomas of different malignancy and covalently bound to keyhole limpet hemocyanin. The vaccine was safe. This preparation produced no side effects in experimental animals. Our findings substantiated the necessity of father development of this method of vaccination. The vaccine should undergo clinical tests in patients with malignant gliomas.

GD3 glycosphingolipid contributes to Fas-mediated apoptosis via association with ezrin cytoskeletal protein.

Efficiency of Fas-mediated apoptosis of lymphoid cells is regulated, among other means, by a mechanism involving its association with ezrin, a cytoskeletal protein belonging to the 4.1 family of proteins. In the present work, we provide evidence for a further molecule that associates to ezrin in Fas-triggered apoptosis, the disialoganglioside GD3. In fact, as an early event, GD3 redistributed in membrane-associated domains in uropods and co-localized with ezrin. Co-immunoprecipitation analyses confirmed this result, indicating a GD3-ezrin association. Altogether, these results are suggestive for a role of GD3 in Fas/ezrin-mediated apoptosis, supporting the view that uropods contain a multimolecular signaling complex involved in Fas-mediated apoptosis.

Prosaposin treatment induces PC12 entry in the S phase of the cell cycle and prevents apoptosis: activation of ERKs and sphingosine kinase.

We report that prosaposin treatment induced extracellular signal-regulated kinases (ERKs) and sphingosine kinase activity, increased DNA synthesis, and prevented cell apoptosis. Prosaposin treatment induced pheochromocytoma cells (PC12) to enter the S phase of the cell cycle; this effect was inhibited by the MEK inhibitor PD98059, indicating that prosaposin-induced ERK phosphorylation is required for stimulation of DNA synthesis. The prosaposin effect was also inhibited by pertussis toxin, indicating that the prosaposin receptor is a G-protein-coupled receptor. Prosaposin rescued PC12 cells from apoptosis induced by staurosporine or ceramide. Sphingosine kinase activity was increased by prosaposin treatment. We propose that this effect is a mechanism underlying the proliferative and anti-apoptotic functions of prosaposin. Prosaposin appears to be a key regulatory factor in the ceramide-S-1-P rheostat, which regulates cell fate.

Association between GM3 and CD4-Ick complex in human peripheral blood lymphocytes.

The aim of this study was to further elucidate our previous observation on molecular interaction of GM3, CD4 and p56Ick in microdomains of human peripheral blood lymphocytes (PBL). We analyzed GM3 distribution by immunoelectron microscopy and the association between GM3 and CD4-p56Ick complex by scanning confocal microscopy and co-immunoprecipitation experiments. Scanning confocal microscopy analysis showed an uneven signal distribution of GM3 molecules over the surface of human lymphocytes. Nearly complete colocalization areas indicated that CD4 molecules were distributed in GM3-enriched plasma membrane domains. Co-immunoprecipitation experiments revealed that CD4 and p56Ick were immunoprecipitated by IgG anti-GM3, demonstrating that GM3 tightly binds to the CD4-p56Ick complex in human PBL. In order to verify whether GM3 association with CD4 molecules may depend on the presence of p56Ick, we analyzed this association in U937, a CD4 + and p56Ick negative cell line. The immunoprecipitation with anti-GM3 revealed the presence of a 58kDa band immunostained with anti-CD4 Ab, suggesting that the GM3-CD4 interaction does not require its association with p56Ick. These findings support the view that GM3 enriched-domains may represent a functional multimolecular complex involved in signal transduction and cell activation.

Prosaposin-derived peptides enhanced sprouting of sensory neurons in vitro and induced sprouting at motor endplates in vivo.

Prosaposin exhibits neurotrophic factor properties that are localized to a 12-amino acid sequence located in the amino terminal portion of the saposin C domain. Prosaptides are peptides derived from the neurotrophic portion of prosaposin; these have been previously reported to be bioactive in neuroblastoma cell lines in vitro. We report that prosaptides were also bioactive in explants of adult primary sensory neurons by dose-dependently increasing both the number (3- to 4-fold) and elongation of these neurites by 50%. Local injection of prosaptides into the gluteus muscle of adult mice also induced sprouting at the motor endplate. Our results indicate that prosaptides are potent neuritogenic factors for both sensory and motor neurons of adult peripheral nerve.

Glycosphingolipid domains on cell plasma membrane.

In this study we analyzed by immunofluorescence, laser confocal microscopy, immunoelectron microscopy and label fracture technique the ganglioside distribution on the plasma membrane of several different cell types: human peripheral blood lymphocytes (PBL), Molt-4 lymphoid cells, and NIH 3T3 fibroblasts, which mainly express monosialoganglioside GM3, and murine NS20Y neuroblastoma cells, which have been shown to express a high amount of monosialoganglioside GM2. Our observations showed an uneven distribution of both GM3 and GM2 on the plasma membrane of all cells, confirming the existence of ganglioside-enriched microdomains on the cell surface. Interestingly, in lymphoid cells the clustered immunolabeling appeared localized over both the microvillous and the nonvillous portions of the membrane. Similarly, in cells growing in monolayer, the clusters were distributed on both central and peripheral regions of the cell surface. Therefore, glycosphingolipid clusters do not appear confined to specific areas of the plasma membrane, implying general functions of these domains, which, as structural components of a cell membrane multimolecular signaling complex, may be involved in cell activation and adhesion, signal transduction and, when associated to caveolae, in endocytosis of specific molecules.

Colocalization and complex formation between prosaposin and monosialoganglioside GM3 in neural cells.

Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor in vitro as well as in vivo. Its neurotrophic activity has been localized to a linear 12-amino acid sequence located in the NH2-terminal portion of the saposin C domain. In this study, we show the colocalization of prosaposin and ganglioside GM3 on NS20Y cell plasma membrane by scanning confocal microscopy. Also, TLC and western blot analyses showed that GM3 was specifically associated with prosaposin in immunoprecipitates; this binding was Ca2+-independent and not disassociated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The association of prosaposin-GM3 complexes on the cell surface appeared to be functionally important, as determined by differentiation assays. Neurite sprouting, induced by GM3, was inhibited by antibodies raised against a 22-mer peptide, prosaptide 769, containing the neurotrophic sequence of prosaposin. In addition, pertussis toxin inhibited prosaptide-induced neurite outgrowth, as well as prosaptide-enhanced ganglioside concentrations in NS20Y cells, suggesting that prosaposin acted via a G protein-mediated pathway, affecting both ganglioside content and neuronal differentiation. Our findings revealed a direct and tight GM3-prosaposin association on NS20Y plasma membranes. We suggest that ganglioside-protein complexes are structural components of the prosaposin receptor involved in cell differentiation.

A novel mechanism of CD4 down-modulation induced by monosialoganglioside GM3. Involvement of serine phosphorylation and protein kinase c delta translocation.

In this report the molecular mechanism(s) involved in the rapid and selective endocytosis of cell surface glycoprotein CD4 induced by exogenous monosialoganglioside GM3 in human peripheral blood lymphocytes have been investigated. Inhibition of the GM3-induced CD4 down-modulation was observed in the presence of specific protein kinase C (PKC) inhibitors. Scanning confocal microscopy revealed the translocation and clustering on the cell surface of PKC isozymes delta and theta (more evidently than alpha and beta) after GM3 treatment, suggesting the involvement of these isozymes in the ganglioside-induced CD4 down-modulation. Exogenous GM3 induced phosphorylation of CD4 molecule, which then dissociated from p56(lck), as early as after 5 min. Moreover, addition of GM3 resulted in a rapid (1 min) cytosolic phospholipase A2 activation with consequent arachidonic acid release, whereas no phosphatidylinositol-phospholipase C activity was observed. Both PKC translocation and CD4 down-modulation were blocked by the trifluoromethylketone analog of arachidonic acid, a selective inhibitor of cytosolic phospholipase A2 and by mitogen-activated protein kinase inhibitor PD98059. Taken together, these findings strongly suggest that GM3 may trigger a novel mechanism of modulation of the CD4 surface expression through the activation of enzyme(s) involved in the regulation of cellular functions.

Identification of a neurotrophic sequence in erythropoietin.

Erythropoietin (Epo) is a hematopoietic factor that facilitates erythroid progenitor cell proliferation and differentiation. Recently, trophic effects of Epo have been observed in central cholinergic neurons. We have confirmed the neurotrophic factor activity of Epo and moreover, demonstrated sprouting and signaling by Epo in neural cells. Further, we have identified a 17-mer peptide sequence (epopeptide AB) in Epo (AEHCSLNENITVPDTKV) with activity similar to that of the holoprotein. This peptide induces differentiation and prevents cell death in both murine NS20Y and human SK-N-MC neuroblastoma cell lines. However, epopeptide AB does not promote the proliferation of erythropoietic cell lines or mouse primary spleen cells. The biological activities in neural cells were blocked by the addition of an antibody to the extracellular domain of the Epo receptor, indicating that the bioactive effects of epo-peptide AB in neural cells are Epo receptor mediated. Both epopeptide AB and Epo stimulated phosphorylation of ERKs in PC12 cells. When epopeptide AB or Epo was locally injected into mice, the frequency of motor end plate sprouting in adjacent muscles increased in a manner similar to that induced by CNTF. These findings indicate that neural cells and not hematological cells respond to a peptide sequence within erythropoietin and suggests that Epo may have separate domains for neurotrophic and hematotrophic function.

Prosaptide prevents paclitaxel neurotoxicity.

Paclitaxel (Taxol), a chemotherapeutic agent used to treat breast and ovarian tumors, has been reported to induce a predominantly sensory neuropathy. Co-treatment with neurotrophic factors and paclitaxel has been proposed for preventing or reversing paclitaxel-induced peripheral neuropathy. Prosaposin, the precursor of saposins A, B, C and D was recently identified as a neurotrophic factor and was reported to facilitate nerve regeneration in vivo. Peptides (prosaptides) encompassing the neurotrophic sequence located in the saposin C domain, have neurotrophic activity similar to the holoprotein (O'Brien et al. 1995). In the present study, we investigated the effect of a 14-mer prosaptide, TX14(A), or a 22-mer prosaptide, 769P, on paclitaxel-induced neutrotoxicity in vitro and in vivo. Paclitaxel treatment (1 microM) decreased cell viability of both PC12 and Schwann cells. TX14(A) (10 nM) prevented paclitaxel-induced loss of cell viability in PC12 cells but not in Schwann cells. Systemic injections (i.p.) of paclitaxel (1.2 mg/kg/day) given five times per week for three weeks (cumulative dose 18 mg/kg) or given every third day (25, 12.5 and 12.5 mg/kg) for 10 ten days (cumulative dose 50 mg/kg) in adult rats induced thermal hypoalgesia that was not accompanied by morphological changes in the sciatic nerve or changes of nerve conduction velocity. Co-administration of paclitaxel with prosaptides (cumulative dose 3 or 10 mg/kg) prevented paclitaxel-induced thermal hypoalgesia. In the short-term high dose study, paclitaxel treated rats lost 10% of their body weight, had reduced erythrocyte counts, hematocrit and hemoglobin concentrations which were not prevented by treatment with prosaptide. TX14(A) did not diminish paclitaxel cytotoxicity of breast cancer cells in vitro. These findings suggest that prosaptide prevents the neurotoxic effects of paclitaxel while not interfering with its anti-tumor activity.

Gangliosides and autoimmune diabetes.

Gangliosides are sialic acid-containing glycolipids which are formed by a hydrophobic portion, the ceramide, and a hydrophilic part, i.e. the oligosaccharide chain. First described in neural tissue, several studies have shown that gangliosides are almost ubiquitous molecules expressed in all vertebrate tissues. Within cells, gangliosides are usually associated with plasma membranes, where they can act as receptors for a variety of molecules and have been shown to take part in cell-to-cell interaction and in signal transduction. In addition, gangliosides are expressed in cytosol membranes like those of secretory granules of some endocrine cells (adrenal medulla, pancreatic islets). As far as the role of gangliosides in diseases is concerned, there are some cases in which an aberrant ganglioside expression plays a crucial role in the disease pathogenetic process. These diseases include two major forms of ganglioside storage, namely GM2-gangliosidosis (Tay-Sachs and its beta-hexosaminidase deficiency) and GM1-gangliosidosis (beta-galactosidase deficiency), where the most prominent pathological characteristic is the lysosomal ganglioside accumulation in neurons. Other inflammatory or degenerative diseases both within and outside the nervous system have been shown to be associated with an altered pattern of ganglioside expression in the target organ. Since monoclonal antibodies have been discovered and used in immunology, a large variety of ganglioside antigens has been described both as blood group antigens and as tumour-related antigens. Several studies have also indicated that gangliosides can act not only as antigens, but also as autoantigens. As a matter of fact, auto-antibodies to gangliosides, detected by immunostaining methods performed directly on TLC plates or by ELISA, have been described in several autoimmune disorders such as Guillain-Barré syndrome, multiple sclerosis, lupus erythematosus, Hashimoto's thyroiditis and, last but not least, insulin-dependent (type 1) diabetes mellitus. This last disease is caused by the autoimmune destruction of insulin-producing pancreatic islet cells in genetically predisposed individuals. Autoantibodies and T lymphocytes directed towards multiple islet autoantigens have been detected in the circulation, well before the clinical onset of the disease, in a prodromal phase during which pancreatic islet beta-cells are presumably destroyed. Among the target autoantigens, some are of protein nature but others are acidic glycolipids such as sulphatides158 and the gangliosides GT3, GD3 and especially GM2-1. This last component is specifically expressed in pancreatic islets and has been shown to represent a target of IgG autoantibodies highly associated with diabetes development in first-degree relatives of type 1 diabetic individuals. In addition, the GM2-1 ganglioside appears to be one of the antigens recognized by cytoplasmic ICA, a heterogeneous group of antibodies which specifically react with islets on pancreatic frozen sections. In conclusion, studies performed in the last decade have clearly indicated that gangliosides represent a heterogeneous class of molecules that are involved in several cellular processes that are of crucial importance in physiological as well as in pathological conditions. Interestingly, these molecules, despite their small size, have been shown to represent not only important antigens in tumour immunology but are also able to elicit a specific autoimmune response, thus representing important autoantigens in some autoimmune disorders. It is of interest that, in addition to neurological autoimmune disorders where autoimmunity to gangliosides is frequent and usually of considerable magnitude, an autoimmune response to this class of molecules has been observed in autoimmune diabetes. (ABSTRACT TRUNCATED)

Prosaposin and prosaptide, a peptide from prosaposin, induce an increase in ganglioside content on NS20Y neuroblastoma cells.

Prosaposin has been recently identified as a neurotrophic factor eliciting differentiation in neuronal cultured cells (NS20Y). In this paper we investigate whether prosaposin and its active peptide (prosaptide) may modify the ganglioside pattern in neuroblastoma cells. The analysis by high performance thin layer chromatography did not reveal qualitative changes in the ganglioside pattern of NS20Y cells incubated in the presence of prosaposin, compared to control cells, but it did reveal an increase of the content of all three major resorcinol positive bands (GM3, GM2, GD1a). Cytofluorimetric and immunofluorescence microscopic analysis revealed that the increase of the ganglioside content was at the plasma membrane level. These findings suggest that the neurotrophic activity of prosaposin on NS20Y neuroblastoma cells might be mediated in part by the increase of cell surface gangliosides.

[Congenital cervico-vaginal atresia and pelvic endometriosis. Clinical case and review of the literature]. / Angeborene zerviko-vaginale Atresie und Beckenendometriose. Klinischer Fall und Literaturübersicht.

Congenital cervico-vaginal atresia is a rare malformation; the presence of a functioning endometrium combined with this anomaly causes a retrograde menstruation and then a greater likelihood of endometriosis. Up to the present literature relates only thirteen cases in which congenital cervico-vaginal atresia was combined with a functioning endometrium. Primary amenorrhea and cyclic, cramping lower abdominal pain was the common symptoms of all the patients. In this work we report a new case of such pathology in a 23 old woman affected also by harmonic nanism. After the review of the literature the embryologic mechanisms involved in lower Mullerian tract malformations, the surgical treatment and the mechanism of formation of endometriosis in association with an outflow tract obstruction are discussed.