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It has been demonstrated that the third component of complement (C3)(1) and its peptides increase normal and tumour cell proliferation. However, the signal cascade responsible for this phenomenon is still unknown. In this study, we elucidate some of the mechanisms involved in the signalling of C3 stimulation of cell proliferation. We have first investigated the in and out traffic of C3 peptides, then we have identified the subcellular localisation of internalised C3 and, finally, we have explored the role of protein phosphorylation in C3 traffic and in the proliferation of the Lewis lung carcinoma (3LL) cells. Our results indicate that traffic of C3 is not dependent on cytoskeletal integrity and requires protein kinase C-dependent phosphorylation. In addition, proliferation of 3LL cells stimulated by C3 depends on both C3 internalisation and protein-kinase C phosphorylation., (Copyright 2004 Wiley-Liss, Inc.)

In this investigation we show that the death-inducing signaling complex (DISC) associates with glycosphingolipid-enriched microdomains (GEM) upon CD95/Fas engagement. We primarily analyzed the ganglioside pattern and composition of GEM after triggering through CD95/Fas and observed that GM3 is the main ganglioside constituent of GEM. Stimulation with anti-CD95/Fas did not cause translocation of gangliosides within or from the GEM fraction. Scanning confocal microscopy showed that triggering through CD95/Fas induced a significant GM3-caspase-8 association, as revealed by nearly complete colocalization areas. Coimmunoprecipitation experiments demonstrated that GM3 and GM1 were immunoprecipitated by anti-caspase-8 only after triggering through CD95/Fas. This association was supported by the recruitment of caspase-8, as well as of CD95/Fas, to GEM upon CD95/Fas engagement, as revealed by the analysis of linear sucrose gradient fractions. It indicates that the DISC associates with GEM; no changes were observed in the distribution of caspase-9. The disruption of GEM by methyl-beta-cyclodextrin prevented DNA fragmentation, as well as CD95/Fas clustering on the cell surface, demonstrating a role for GEM in initiating of Fas signaling. These findings strongly suggest a role for gangliosides as structural components of the membrane multimolecular signaling complex involved in CD95/Fas receptor-mediated apoptotic pathway.

In this report we demonstrated that cellular prion protein is strictly associated with gangliosides in microdomains of neural and lymphocytic cells. We preliminarily investigated the protein distribution on the plasma membrane of human neuroblastoma cells, revealing the presence of large clusters. In order to evaluate its possible role in tyrosine signaling pathway triggered by GEM, we analyzed PrPc presence in microdomains and its association with gangliosides, using cholera toxin as a marker of GEM in neuroblastoma cells and anti-GM3 MoAb for identification of GEM in lymphoblastoid cells. In neuroblastoma cells scanning confocal microscopical analysis revealed a consistent colocalization between PrPc and GM1 despite an uneven distribution of both on the cell surface, indicating the existence of PrPc-enriched microdomains. In lymphoblastoid T cells PrPc molecules were mainly, but not exclusively, colocalized with GM3. In addition, PrPc was present in the Triton-insoluble fractions, corresponding to GEM of cell plasma membrane. Additional evidence for a specific PrPc-GM3 interaction in these cells was derived from the results of TLC analysis, showing that prion protein was associated with GM3 in PrPc immunoprecipitates. The physical association of PrPc with ganglioside GM3 within microdomains of lymphocytic cells strongly suggests a role for PrPc-GM3 complex as a structural component of the multimolecular signaling complex involved in T cell activation and other dynamic lymphocytic plasma membrane functions.

In this study we analyzed the signaling pathway triggered by GM3 in lymphoblastoid T-cells. In these cells, GM3 induced cPLA2 activation, arachidonic acid release, and PKC-delta translocation. In order to clarify the upstream molecular signals triggered by GM3, we analyzed the activation of extracellular signal-regulated kinase (ERK)s, a downstream effector of Ras-regulated cytoplasmic kinase cascade. Our results showed that GM3 treatment led to rapid ERK phosphorylation in lymphoblastoid T-cells, as detected by anti-phospho-p44/42 MAP kinase. Similar findings were found in human peripheral blood lymphocytes. Moreover, we showed that GM3 specifically phosphorylated ERK-2, as revealed by anti-phosphotyrosine reactivity on both cell free lysates and ERKs immunoprecipitates. The role of the CD4 cytoplasmic domain in GM3-triggered signaling pathway was investigated using A2.01/CD4-cyt399 cells, which had been transfected with a mutant form of CD4 lacking the bulk of the cytoplasmic domain. In these cells GM3 induced cPLA2 activation, arachidonic acid release, and PKC-delta translocation, but not CD4 endocytosis, indicating that the CD4 cytoplasmic domain plays a key role in GM3-triggered CD4 endocytosis and the GM3-triggered biochemical pathway is upstream of CD4 phosphorylation. These findings strongly suggest that GM3 triggers a novel signaling pathway involved in the regulation of cellular functions.

Recent evidence demonstrated that T cell activation leads to the redistribution of membrane and intracellular kinase-rich raft microdomains at the site of TCR engagement. In this investigation we demonstrated by high performance thin layer chromatography, gas chromatographic, and mass spectrometric analyses that GM3 is the main ganglioside constituent of these microdomains in human lymphocytes. Then we analyzed GM3 distribution and its interaction with the phosphorylation protein Zap-70. Human T lymphocytes were stimulated with anti-CD3 and anti-CD28. Immunofluorescence microscopy analysis revealed a clustered GM3 distribution over the cell surface and an intracellular localization resembling specific cytoplasmic compartment(s). Scanning confocal microscopy showed that T cell activation induced a significant association between GM3 and Zap-70, as revealed by nearly complete colocalization areas; very few colocalization areas were detected in unstimulated cells. Coimmunoprecipitation experiments revealed that GM3 was immunoprecipitated by anti-Zap-70 only after co-stimulation through CD3 and CD28 as detected by both thin layer chromatography and immunoblotting. Therefore, T cell activation does not promote a redistribution of glycosphingolipid-enriched microdomains but induces Zap-70 translocation in selective membrane domains in which Zap-70 may interact with GM3. These findings suggest that GM3 is a component of a multimolecular signaling complex involved in T cell activation.

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.

This study provides evidence that cardiolipin (CL) molecules are expressed on the surface of apoptotic cells and are recognized by antiphospholipid antibodies, purified from patients with the antiphospholipid antibody syndrome (APS). CL expression on cell surface was demonstrated by high performance thin layer chromatography analysis of phospholipids from plasma membrane purified fractions and by the positive staining with the CL-specific dye nonyl-acridine orange. This finding was complemented with the observation that aCL IgG purified from patients with APS bind to the surface of apoptotic cells. This staining shows a clustered distribution mostly localized on surface blebs. Interestingly, CL exposure on the cell surface preceded the DNA fragmentation, as shown by cytofluorimetric analysis. These findings demonstrate that exposure of CL molecules on the cell plasma membrane is an early event of the apoptotic cellular program that may represent an in vivo trigger for the generation of aCL.

This study was undertaken to analyze the role of disialoganglioside GD3 in HIV infection and disease progression. We report here the results obtained by both ex vivo and in vitro experiments on (1) surface and cytoplasmic expression and distribution of GD3 in HIV-infected cells, (2) the presence of anti-GD3 antibodies in sera of patients with HIV infection in various stages of the disease, and (3) the association of GD3 expression with HIV-related apoptotic events. GD3 expression was determined by high-performance thin-layer chromatography (HPTLC) and lipid-bound sialic acid and by static and flow cytometric analyses in peripheral blood lymphocytes from 22 AIDS patients, 20 anti-HIV Ab(+) asymptomatic subjects, and 25 healthy donors. Results obtained clearly indicated a significantly higher expression of plasma membrane GD3 content in lymphocytes from HIV-infected patients with respect to healthy controls. These HIV-induced perturbations of glycosphingolipid metabolism could be detected in all stages of the disease, including asymptomatic individuals. In addition, a significant percentage of patients showing disease progression displayed in serum samples an increased presence of anti-GD3 antibodies. Interestingly, ex vivo studies of lymphocytes from patients with HIV infection also indicated that GD3 expression is strictly associated with annexin V binding, an early marker of apoptosis. Moreover, cytofluorimetric analysis showed that virtually all anti-p24 Ab-positive cells were also immunolabeled with anti-GD3 antibodies. Accordingly, in vitro studies showed a significant redistribution and increase in GD3 expression in cultured U937 cells chronically infected with HIV-1 with respect to uninfected counterparts. In conclusion, our data clearly indicate that a significant increase in GD3 content in HIV-infected lymphocytes can occur and that this GD3 overexpression is paralleled by the presence of anti-GD3 antibodies in the plasma of patients. This is the first demonstration that disialoganglioside GD3, independent of the therapeutic schedule employed, can be considered as one of the early markers of HIV infection and can contribute to the early events leading to T cell depletion by apoptosis.

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.

C3 molecules from normal murine serum are mainly bound to Lewis lung carcinoma cells (3LL) that do not express CRs, mainly through covalent binding as determined by the appearance of bands stained with anti-C3 and larger than 190 kD in immunoblots of proteins in whole cell extracts. Methylamine-treated, or zymosan-treated normal mouse serum, heat inactivated, or EDTA-treated murine serum resulted in low C3 deposition on 3LL cells, as indicated by fluorescence tests and immunoblotting. Cytofluorimetric studies showed that C3 molecules bound to 3LL cells were internalized in a time- and temperature-dependent process. This was confirmed by electronmicroscopic studies. The conditions allowing C3 fixation to acceptor sites and subsequent internalization increased cell proliferation. This was also true, when serum from mice genetically deficient in C5 was used which stresses the role of C3 in contrast to effects of membrane attack complex formation.

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.

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.

Gangliosides may play functional roles in platelet physiology, therefore this study has been designed to evaluate whether changes in ganglioside composition may occur as a consequence of platelet activation. The results obtained indicate that lactosylceramide and GM3 are the major glycosphingolipids of human platelets. The lipid-bound sialic acid (LBSA) content was 1.27 +/- 0.04 micrograms/mg of protein. Resting platelets did not express GD3; GD3 was synthesized upon platelet activation (24 +/- 8 ng/mg of protein). The stimulation of platelets with adenosine diphosphate showed the appearance of GD3 even in the absence of degranulation. Finally, incorporation of pyrene-labeled GM3 into platelet membranes, followed by stimulation with adenosine diphosphate, resulted in the appearance of a fluorescent band comigrating with GD3. The present studies indicate that sialytransferase activation may occur as an early event following platelet stimulation, leading to GD3 synthesis mainly from the GM3 pool.

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.

This study has been undertaken to assess whether anticardiolipin and anti-beta 2-GPI are two distinct populations of (auto)antibodies, and to clarify whether the beta 2-GPI region critical for phospholipid binding is also crucial for anti-beta 2-GPI reactivity. Fourteen of the 62 anticardiolipin (aCL) ELISA positive sera (22.6%) were positive for anti-beta 2-GPI by immunoblotting, 42 (67.7%) for aCL using TLC immunostaining. IgG fractions from 5 sera gave the same anticardiolipin reactivity detected by TLC immunostaining in the corresponding sera. All anti-beta 2-GPI-positive sera were reactive with the phenylthiocarbamyl derivative of the protein, indicating that binding of phenylisothiocyanate with lysine residues does not modify the molecule antigenicity. In addition, incubation of IgG fractions with the phospholipid binding site did not modify reactivity with beta 2-GPI. These findings demonstrate that: a) "true" antiphospholipid antibodies are detectable in patients' sera; b) aCL and anti-beta 2-GPI have a different immunological profile; c) the beta 2-GPI phospholipid-binding site is not the region recognized by the antibodies.

PGE2 has been shown to be able to interfere with various lymphocyte and macrophage functions, but its effects on macrophage activation are still unclear. In this study, carried out on peritoneal macrophages obtained from healthy, tumour-bearing and Corynebacterium parvum-treated mice, we demonstrated that PGE2 is involved in the down-regulation of macrophage activation, but it cannot exert its inhibiting effect when macrophages are further stimulated with activating cytokines, such as IFN gamma and TNF alpha. Our findings provide new insight into how macrophage tumoricidal activity may be induced and maintained even in presence of significant levels of PGE2.

Gangliosides modulate the expression of CD4 molecules on the cell surface of T lymphocytes. We report here that treatment of human peripheral blood lymphocytes with exogenous monosialoganglioside GM3 induces a rapid down-modulation of the CD4 molecules on the plasma membrane of CD4+ T lymphocytes, as assessed by cytofluorimetric analysis and quantitative immunoelectron microscopy. The CD4 down-modulation was ganglioside-dose dependent and was already evident after 5 min of treatment, reaching the maximum after 20 min. The expression of other surface antigens was not affected by GM3 treatment. The immunoelectron microscopic analysis showed that, following GM3 addition, gold labelled CD4 molecules were rapidly redistributed on the cell surface, clustered and internalized via endocytic pits and vesicles. These results indicate that CD4 down-modulation induced by GM3 occurs through an endocytic mechanism. A persistent low level of CD4 expression on the cell surface up to 24 h after GM3 treatment, compared with a stable expression of either CD4 in untreated cells and CD3 in GM3-treated cells, suggests intracellular degradation of the internalized CD4 molecules.

The possible correlation(s) between platelet proaggregating activity, and sialic acid content and ganglioside expression of six human colorectal tumour cell lines (CBS, GEO, HT-29, WiDr, MIP and DLD-1) was evaluated. The three cell lines (HT-29, WiDr and DLD-1) capable of inducing remarkable in vitro platelet aggregation, had significantly higher amounts of lipid-bound sialic acid than those cell lines characterised by a lower platelet proaggregating activity (GEO, CBS and MIP). High performance thin-layer chromatography demonstrated the presence of one band comigrating with GM3 in all cell lines, while GD1a and GT1b comigrating gangliosides were present only in HT-29, WiDr and DLD-1 cells. Finally, an increased platelet pro-aggregating activity of GEO and CBS cell lines was observed after the incorporation of exogenous gangliosides. The present data support the hypothesis that lipid-bound sialic acid may be involved in platelet-tumour cell interactions.

There is increasing interest in the role of antiphospholipid antibodies in the so-called 'antiphospholipid antibody syndrome' (APS). The two major methods currently employed for detecting the autoantibodies are the solid phase ELISA and the LAI test (inhibition of phospholipid dependent coagulation assay). In our study we have tested the possibility of detecting antiphospholipid antibodies by immunostaining on thin layer chromatography (TLC) plates, since this technique permits the use of pure phospholipid molecules as antigen. Sera were collected from 20 patients with SLE without APS, 20 patients with APS, 20 anti-HIV positive subjects, ten patients with signs of APS but antiphospholipid negative (ELISA), 20 patients with syphilis and 40 matched blood donors. Results showed that only 72.3% of sera containing detectable levels of aCL antibodies in solid phase ELISA were also positive for aCL in TLC immunostaining; these discrepancies may be due to the presence of antibodies reacting with a protein complexed with phospholipid (beta 2-glycoprotein-I) or, alternatively, to the different antigenic presentation of phospholipids on chromatograms compared to the surface of microtitre wells. Furthermore, aCL monoclonal antibody CAL-3, as well as nine sera positive for aCL, also reacted with PS and PE. Previous absorption of these sera with CL micelles completely abolished the reactivity with PS and PE, demonstrating cross-reactivity among these three phospholipids. In conclusion, our findings reveal that TLC immunostaining is more specific, but less sensitive, than ELISA for the detection of antiphospholipid antibodies in human sera.

In this study we analysed the relationship between anti-lymphocytic ganglioside antibodies and anti-lymphocyte antibodies in AIDS patients. Anti-lymphocytic ganglioside antibodies were detected by thin layer chromatography (TLC) immunostaining; three colour flow cytometry was used to analyse circulating antibodies against different lymphocyte subsets. Anti-lymphocytic ganglioside antibodies were detected in 23 out of 49 AIDS patients sera (46.9%). All positive sera reacted selectively with the GM3 comigrating band from AIDS lymphocytes. Twenty two out of the 23 anti-lymphocytic GM3 positive sera also had antibodies against CD4+T cells, versus 17/26 anti-GM3 negative. Furthermore, patients with lymphocytic GM3 antibodies showed a significantly higher antibody reactivity against CD4+ T cells than patients in which these antibodies were not detected. The absorption tests revealed that preincubation of positive sera with GM3 was followed by a decrease in the reaction with target lymphocytes. These findings suggest that anti-GM3 antibodies are a portion, but not the majority, of antibodies reacting with CD4+ T cells.

Raji cells, a CR2-positive Burkitt lymphoma cell line, incubated in normal human serum, activate C3 and fix C3-derived fragments. The presence of these molecules on the cell surface does not affect subsequent Epstein-Barr virus (EBV) binding but it prevents superinfection. On the other hand, EBV superinfection is enhanced if Raji cells were incubated with heat-inactivated serum whose C3 fragments may bind only through receptor-binding sites. These results indicate that the region on cell surface offering the covalent site to C3 fragments would be essential for EBV superinfection. Incubation of Raji cells for 1 min with EBV results in the phosphorylation of CR2 and of a high-molecular-weight protein followed by their dephosphorylation, completed already after 20 min. This finding ascribes to EBV a prompt action through its receptor, different from that of other compounds causing a prolonged CR2 phosphorylation. Our data suggest that at least two binding sites are required for EBV superinfection of Raji cells or that specific patterns of CR2 phosphorylation may modulate Raji superinfection by EBV.

IgG antibodies reacting with the GM3-comigrating band extracted from pooled AIDS lymphocytes were detected in 33.3% of AIDS patients sera, in 8% of asymptomatic anti-HIV-positive subjects, in none of the sera obtained from asymptomatic anti-HIV-negative drug abusers, from patients with acute B and chronic C hepatitis, and from healthy donors. All positive sera reacted selectively with the GM3-comigrating band obtained from AIDS lymphocytes but not with the corresponding band from normal lymphocytes. The lymphocytic ganglioside autoantigen was revealed as GM3. In addition, two main data were shown: (a) AIDS lymphocytes have an increased concentration of GM3 and (b) the ceramide of AIDS lymphocytic GM3 has a different percentual composition of fatty acids in contrast to control cells. It is suggested that these quantitative and qualitative changes might be responsible for the appearance of circulating anti-lymphocytic GM3 antibodies.

Islet cell antibodies (ICA) bind antigens expressed in both human and rat pancreatic islets. Biochemical studies have shown that an ICA-autoantigen has the properties of a monosialo-ganglioside migrating between GM2 and GM1 standards (GM2-1). We therefore aimed to isolate and characterize gangliosides from whole pancreas and isolated islets of bio breeding diabetes-prone (BB-DP), bio breeding diabetes-resistant (BB-DR), and Wistar Furth (WF) rat strains. Gangliosides were characterized by TLC, HPLC, diode array analysis, and ganglioside-specific staining. ICA binding was studied by indirect immunostaining. The GM2-1 fraction was present in BB-DP, BB-DR, and WF rat pancreases (11, 17, and 9.5%, respectively, of total ganglioside content). Substantial differences were found in other fractions: in BB-DP pancreas, in addition to GM2-1, the main fractions were GM3 (49%), GD1a (12%), GT1b (5%), and a ganglioside migrating between GM1 and GD3 standards (23%), while in BB-DR pancreas the above components were 71, 5.5, 2, and 4.5%, respectively; in WF pancreas, the main fractions were GM3, GD3, GD1a, GT1b and a trisialoganglioside (GT*) migrating above the GT1b standard (42.7, 7, 20.2, 13.8, and 6.8, respectively). A different pattern of ganglioside expression was found in isolated islets of BB-DP, BB-DR, and WF rats: the GM2-1 fraction represented, respectively, 29.1, 30.4, and 31.6% of total ganglioside content; GM3 51.1, 66, and 68.4%. A fraction migrating between GM1 and GD3 standards was present only in BB-DP and BB-DR islets (19.8 and 3.6%, respectively). ICA-positive human sera reacted with pancreas of all rat strains studied, with similar end-point titers. In conclusion, (1) the GM2-1 ganglioside, in the same way as a putative target antigen of ICA, is equally expressed in BB-DP, BB-DR, and WF rat pancreata; and (2) the GM1-GD3 is expressed in higher amounts in BB-DP than in BB-DR pancreas and islets and is absent in WF.

Peritoneal macrophages obtained from Lewis Lung carcinoma (3LL) tumor bearing mice release high amounts of soluble factors such as C3,H2O2 and lysosomal enzymes but fail to exert cytotoxic activity on tumor cells. In the present work we show that they acquire this property and become fully activated after in vitro incubation with supernatants derived from cultures of splenocytes from tumor bearing syngeneic mice. The presence of IFN gamma in the above supernatants was detected by immunoblotting analysis and by bioassay. The role played by IFN gamma in macrophage activation was investigated.

Thymocytes (T) from mice bearing the syngeneic tumor 3LL inhibit the immune response to SRBC by syngeneic splenocytes derived from control mice and cultured in Mishell and Dutton's antibody forming systems. T from control mice are devoid of this capacity but acquire it after in vitro incubation with tumor cells. It has been shown that metabolites of arachidonic acid (PGE-2, LTB-4 and LTC-4) synthesized in excess by tumor cells act as mediators for the acquisition of the above capacity by thymic cells in vitro. Surface phenotypical analysis by flow-cytometry demonstrated that in the thymus of tumor bearing mice the proportion of both single positive thymocytes (L3T4+/LY2- and L3T4-/LY2+) and of double negative thymocytes (L3T4-/LY2-) increased with a parallel decrease in the proportion of double positive cells (L3T4+/LY2+). In the mean time the total number of thymocytes markedly decreased. An attempt was done to mimic in vitro what happens in vivo. For this purpose T from control mice were incubated with conditioned culture medium in which 3LL cells had grown or with arachidonic acid metabolites. After one or the other of these treatments T from control mice did not modify their phenotypic antigenic pattern but acquired the capacity to negatively interfere with the in vitro immune response of normal spleen cells to SRBC. Since serum from tumor bearing mice contains large amounts of PGE-2, LTB-4 and LTC-4 we tested its effects on normal syngeneic T. Serum from tumor bearing mice, but not serum from normal mice, induces T from control syngeneic animals to acquire both immunosuppressive capacity and differentiation pattern clusters similar to those that characterize T derived from the thymus of tumor bearing mice. We demonstrated that metabolites of arachidonic acid are active as inducers of immunosuppressive capacity in T. On the other hand, a yet unknown factor present in serum of tumor bearing mice plays a role as inducer of differentiation of these cells.

Platelet hyperactivity, one of the commonest findings associated with migraine, has been related to increased release of biologically active substances such as catecholamines and arachidonic acid metabolites, which seem to play a role in the pathogenesis of migraine. In this study, in vitro platelet aggregation tests were performed on samples from patients with different types of headache. The presence of platelet hyperactivity was clearly demonstrated in 11 patients with classical migraine between attacks, but not in 4 patients between attacks of common migraine. Nevertheless, the presence of a marked platelet hyporesponsivity was found during the attack phase of both classical and common migraine. No difference in platelet aggregability was found between attack and post-attack phases in 5 patients with cluster headache. Blood leukotrienes were analyzed in 8 patients with classical migraine and in the 5 patients with cluster headache. During the attack phase of classical migraine both LTC4 and LTB4 were present in the peripheral blood, while the post-attack phase was characterized by the disappearance of LTC4 and the presence of LTB4 and its transisomer delta 6-trans-LTB4. Blood leukotrienes were constantly absent during both phases of cluster headache. Incubation of normal platelets with LTC4 or delta 6-trans-LTB4 was followed by inhibition of platelet response to epinephrine. delta 6-trans-LTB4, at higher concentrations, induced the opposite effect. A possible role of blood leukotrienes in the changes occurring in platelet aggregability during the different phases of classical migraine, is discussed.

The thermal stability of the enzyme beta-D-galactosidase varies among different organs in normal C57Bl/6 mice, and increases in the same organs in mice with Lewis Lung carcinoma. Thermal stability of this enzyme is also increased by treatment of the mice with cell-free extracts of tumour cells or with inflammatory compounds such as carrageenan or orosomucoid. After desialylation, orosomucoid more effectively increases the heat stability of the enzyme. By contrast talc, which has no galactosyl groups, is without effect on the stability of the enzyme in vivo. Macrophages of tumour bearing mice release into the culture medium a more heat resistant enzyme than macrophages from control mice. In both cases the heat resistance of the secreted enzyme is higher when fetal calf serum is present in the culture medium. Bovine serum does not modify the thermal stability of beta-D-galactosidase in this system. Incubation of lysosomal fractions of various organs with the synthetic beta-D-galactosidase substrate, p-nitrophenyl-galactopyranoside, also strongly increases the heat resistance of the enzyme. The results suggest that one factor influencing the heat resistance of this enzyme may be complex formation between the enzyme and its substrates, an example of substrate protection of the enzyme. This may not be the only factor involved in enzyme stabilization in vivo.

Rat mastocyte plasma membrane possess two major classes of calcium binding sites which have widely different affinity. At pH 7.5 the low affinity sites can bind about 88 nmol of Ca2+ per milligram of membrane protein and are half-saturated at 125 micro M Ca2+, whereas the high-affinity sites bind about 12 nmol of Ca2+ per milligram of membrane protein and are half-saturated at 5.5 micro M Ca2+. Membrane phospholipids and neuraminic acid residues account for approximately 87% of calcium binding.

The role of macrophages in tumor-mediated immunosuppression was examined, using C57B1/6 strain mice bearing four different immunosuppressive transplantable syngeneic tumors (Lewis Lung Carcinoma, B16 Melanoma, and two fibrosarcomas induced by methylcholanthrene in our laboratory). When tested for immunosuppressive activity, in inhibiting the induction of antibody formation by normal spleen cells in response to SRBC in vitro, the splenic and peritoneal macrophages from tumor-bearing mice were all significantly suppressive. The degree of suppression correlated with immunosuppression in tumor-bearing mice challenged in vivo with SRBC. Direct action of tumor cells on normal splenic macrophages in vitro caused them to become suppressive, the extent of suppression dependent on the time of interaction and on the immunosuppressive activity of the tumor cells in vivo. Pretreatment of suppressive splenic macrophages with indomethacin, a potent inhibitor of the synthesis of prostaglandins (PG), reduced significantly their immunosuppressive activity. Also, peritoneal macrophages from tumor-bearing mice produced significantly more PGE in culture than control macrophages. Thus, tumor-activated macrophages, presumably those macrophages that infiltrate the tumor in a host reaction against the tumor, serve to amplify the level of immunosuppression in the host by producing relatively large amounts of PGE that is a key physiological mediator in the activation and function of suppressor T lymphocytes. The stimulation of PGE synthesis in macrophages, as a result of their interaction with syngeneic tumors, is initiated by PGE produced in relatively large amount by the tumor cells.

The different percentages of acid hydrolases released by peritoneal macrophages obtained from C57B1/6 mice injected i.m. with talc, carrageenan or 3LL cells, and also the demonstration that hydrolase secretion from in vitro macrophage monolayers of tumor-bearing mice undergoes a further increase when zymosan is added to the culture, strongly suggest the possibility that more than one membrane surface receptor is involved in the triggering of lysosomal enzyme release. Furthermore, there is evidence from the reported results that, besides immune complexes, neoplastic cells or their metabolic product(s) could act as inducers of the lysosomal enzyme secretion by macrophages.

Recent biochemical studies have shown that the cytoplasmic islet cell-antibody autoantigen has properties of a monosialoganglioside (GM). To characterize islet glycolipids and ascertain whether islets express unique gangliosides, we determined the pattern of ganglioside expression in whole human pancreas and isolated human islets using high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC). The major gangliosides detected in glycolipid extracts of whole human pancreas were GM3, GD3 (disialoganglioside), and in a lesser amount, a GD1a-comigrating ganglioside. In contrast to whole human pancreas, isolated human islets were found to predominantly express GM3, an acidic glycolipid comigrating with GM2, and a ganglioside with mobility between GM2 and GM1 by both HPLC and HPTLC. Quantitation of the major ganglioside UV peaks seen on HPLC gave the following results. In whole pancreas, GM3 represented 66.7% of total gangliosides detected; an asialoglycolipid comigrating with GM2, 2.0%; a ganglioside migrating between GM2 and GM1, 2.6%; GD3, 22.6%; and a GD1a-comigrating ganglioside, 6.1%. In isolated islets, these components were found at the following levels: GM3, 14.9%; GM2-comigrating glycolipid, 74.2%; a ganglioside migrating between GM2 and GM1, 9.8%; GD3, 1.1%; and the GD1a-comigrating ganglioside, not detectable.

We demonstrated that rat peritoneal cells synthesize, and release into the culture medium, substances that elicit a chemotactic responsiveness of rat macrophages. These substances were identified by HPLC analysis as two diastereoisomers of LTB4, precisely delta-6-trans-LTB4 and delta-6-epi-trans-LTB4. Peritoneal cells release, also, other lipoxygenase metabolites such as LTD4 and LTB4, which were shown not to be active in eliciting chemotaxis of rat macrophages, in agreement with data of other authors. Quantitative assays for the measurement of PMNs or macrophage chemotaxis were carried out in Boyden chambers. Release of leukotrienes by rat peritoneal cells into the culture medium was strongly enhanced upon stimulation with calcium ionophore A23187, an agent known to increase cytoplasmic Ca2+ concentration, thus leading to the activation of Ca2+-dependent phospholipase and a consequent increase in the amount of endogenous free arachidonic acid available for biotransformation. The action of several inhibitors of arachidonate metabolism at concentrations more selective for the lipoxygenase than the cycloxygenase pathway, was also studied. alpha-Tocopherol and ASA were shown to be the most selective in inhibiting the synthesis of both the LTB4 diasteroisomers active as enhancers of rat macrophage chemotaxis.

We investigated the effect of Lewis Lung carcinoma cells on the production of C3 by murine macrophages and examined the capacity of secreted C3 to opsonize Lewis Lung carcinoma cells. C3 released in culture from macrophages obtained from tumor-bearing C57Bl/6 mice as well as from normal macrophages exposed to Lewis Lung carcinoma cells in vitro was measured by hemolytic assays and by Western blot. We found that contact with tumor cells in vivo as well as in vitro enhanced the amount of C3 secreted by murine macrophages by a factor of 2-3. The inflammatory agent carrageenan caused only a small increase in the amount of secreted C3. On Western blots of concentrated macrophage supernatants, there was partial cleavage of secreted C3 which was, however, not more pronounced in the case of C3 from tumor-stimulated macrophages than from normal macrophages. Supernatants from normal as well as tumor-stimulated macrophages were capable of opsonizing Lewis Lung carcinoma cells as shown by their capacity to bind human erythrocyte in an immune adherence reaction. Pretreatment of the tumor cells with a protease inhibitor, PMSF, inhibited the capacity of the tumor cells to bind C3, suggesting that a tumor cell-associated protease might be involved in the binding of C3 to the tumor cell surface.

The immunosuppressive activity of tumor cells was studied in vivo and in vitro using C57BL/6 mice and Lewis lung carcinoma (3LL) cells. The SRBC immunization of tumor-bearing mice in vivo gave a lower number of PFC than the control mice. In vitro, employing the Mishell and Dutton technique, the primary immune response of splenocytes from tumor-bearing mice was significantly reduced. The in vitro primary immune response of normal splenocytes was also reduced when the tumor cells or supernatants of tumor cell cultures were present during SRBC immunization. 3LL cells synthesize a large quantity of PGE2 which was also demonstrated in the supernatants of 3LL cell cultures. Nevertheless, as the addition of indomethacin, a potent inhibitor of the prostaglandin synthesis, only partially reduces the tumor cell immunosuppressive action, prostaglandins are conceivably only one of the factors responsible for the immunodepression exerted by the tumor cells.