Long-term effects of somatostatin analogues in rat GH-secreting pituitary tumor cell lines.

PURPOSE: First-generation somatostatin analogs, octreotide (OCT) and lanreotide, are the cornerstone for the medical treatment of growth hormone (GH)-secreting pituitary tumors. A new multireceptor analog, such as pasireotide (PAS), showed better activity than OCT in long-term treatment of patients with acromegaly, but modulation of intracellular key processes is still unclear in vitro. In this study, we evaluated the antitumor activity of OCT and PAS in two GH-secreting pituitary tumor cell lines, GH3 and GH4C1, after a long-term incubation. METHODS: The effects of PAS and OCT on the cell viability, cell cycle, apoptosis, GH secretion, and tumor-induced angiogenesis have been evaluated through a colorimetric method (MTS Assay), DNA flow cytometry with propidium iodide, and Annexin V-FITC/propidium iodide staining, ELISA assay and zebrafish platform, respectively. RESULTS: PAS showed a more potent antitumor activity compared to OCT in GH3 cell line exerted through inhibition of cell viability, perturbation of cell cycle progression, and induction of apoptosis after 6 days of incubation. A concomitant decrease in GH secretion has been observed after 2 days of incubation only with PAS. No effect on tumor-induced angiogenesis has been reported after treatment with OCT or PAS in zebrafish/tumor xenograft model. CONCLUSION: Long-term incubation with PAS showed a more potent antitumor activity than that reported after OCT in GH3 cells, mainly modulated by a cell cycle perturbation and a relevant induction in apoptosis.

Antitumor activity of interferon-ß1a in hormone refractory prostate cancer with neuroendocrine differentiation.

PURPOSE: Type I interferons (IFN-α and IFN-ß) are a class of cytokines that exert several biological activities, such as modulation of cell proliferation and differentiation and of the immune system. Although these cytokines interact with a common receptor complex, IFN-ß showed a more potent antitumor activity than IFN-α in several tumor models. New recombinant human IFN-ß products, such as IFN-ß1a and IFN-ß1b, have been produced in order to improve the stability and bioavailability of natural IFN-ß. In this report, we analyzed the effects of recombinant IFN-ß1a on the cell proliferation of two human androgen-resistant prostate cancer cell lines with neuroendocrine differentiation (DU-145, PC-3) and related mechanisms of action. METHODS: The effects of IFN-ß1a on the cell growth proliferation, cell cycle, and apoptosis have been evaluated in DU-145 and PC-3 cells through MTT assay, DNA flow cytometry with propidium iodide, and Annexin V-FITC/propidium iodide staining, respectively. Moreover, the expression of neuron-specific enolase (NSE), cleaved caspase-3, caspase-8, and PARP was evaluated through Western blotting. RESULTS: IFN-ß1a showed a significant anti-proliferative activity in both androgen-resistant cell lines. This effect was related to cell cycle perturbation and induction in apoptosis, as shown by flow cytometric analysis, the activation of caspase-3 and caspase-8 and PARP cleavage during incubation with IFN-ß1a. Moreover, this cytokine reduced the expression of NSE in both cell lines. CONCLUSIONS: Recombinant IFN-ß1a (Rebif) showed a potent in vitro anti-proliferative activity in androgen-resistant prostate cancer cells, and it could represent a promising tool for the treatment of this tumor.

Everolimus is an active agent in medullary thyroid cancer: a clinical and in vitro study.

Everolimus, an mTOR inhibitor, which has been demonstrated to induce anti-tumour effects in different types of neuroendocrine tumours, has never been evaluated in patients with medullary thyroid cancer (MTC). The aim of this study was to evaluate the in vitro and in vivo effects of everolimus in combination with octreotide in MTC. Two patients with progressive metastatic MTC and high calcitonin levels were treated with everolimus 5-10 mg/day. Both patients were under treatment with octreotide LAR at the study entry. An in vitro study was also performed to assess everolimus effects on MTC cell lines (TT and MZ-CRC-1 cells). A tumour response was observed in both patients. Serum calcitonin decreased by 86% in patient 1 and by 42% in patient 2. In TT and MZ-CRC-1 cells, everolimus induced a significant dose-dependent inhibition in cell proliferation. This effect seems to be related to a cell cycle arrest in G(0) /G(1) phase in both cell lines and to the induction of cellular senescence in TT cells. Everolimus in combination with octreotide may be active as anti-tumour therapy in patients with progressive metastatic MTC, suggesting to further evaluate this agent in MTC patients in a large prospective study.

Pro-inflammatory genotype as a risk factor for aPL-associated thrombosis: Report of a family with multiple anti-phospholipid positive members.

OBJECTIVE: Inflammation might represent a second hit for anti-phospholipid antibody (aPL)-mediated thrombosis. Inflammatory responses have been linked to gene polymorphisms of several cytokines and Toll Like Receptors (TLRs). We examined IL1 beta, TNFalpha, TGFbeta, IL6, IFN gamma, IL10, tlr4 gene polymorphisms in a family with several members positive for IgG anti-beta2 glycoprotein I (beta 2GPI) antibodies but with recurrent thrombosis in one member only. METHODS: Lupus anticoagulant, anti-cardiolipin, anti-beta 2GPI IgG/IgM antibodies, IL1beta, TNFalpha, TGF beta1, IL6, IL10, IFN gamma, tlr4 gene polymorphisms (by allele-specific polymerase chain reaction) in addition to standard thrombophilic risk factors and cytokine serum levels (IL-1 beta, TNFalpha, IL-10) were evaluated. RESULTS: Recurrent thrombotic events was reported only in the proband, but not in three healthy siblings persistently positive for IgG anti-beta2GPI antibodies, respectively. The wild type tlr4 gene and cytokine polymorphisms associated with a high pro-inflammatory response (IL-1 beta promoter-511C/T; TNFalpha G/A; TGFbeta+10T/C, +25C/G; IL-6 -174C/G) were found only in the proband. Serum cytokine levels were normal. CONCLUSION: This case report confirms that protective tlr4 gene polymorphisms are more frequent in asymptomatic aPL carriers. In line with the role of inflammatory mediators as second hits for aPL-associated thrombosis, the polymorphisms of cytokines linked to higher inflammatory response were found in the proband only.

Human monoclonal anti-endothelial cell IgG-derived from a systemic lupus erythematosus patient binds and activates human endothelium in vitro.

Our objectives were to obtain monoclonal anti-endothelial cell antibodies (AECA) from systemic lupus erythematosus (SLE) patients, to characterize their antigen specificity, and their capability to induce a pro-inflammatory and pro-adhesive endothelial phenotype, and to investigate the mechanism of endothelial cell (EC) activation in vitro. Monoclonal IgG AECA were generated by hybridoma formation with human SLE B cells. Antigen specificity was characterized by immunoblotting with enriched cell membrane fractions, by cytofluorimetry and by cell solid-phase ELISA. Endothelial activation was evaluated by measuring increases in U937 cell adhesiveness, adhesion molecule (E-selectin and ICAM-1) expression and IL-6 production. In addition, mechanisms of endothelial activation were investigated by assessment of NF-kappaB by measuring the loss of its inhibitor I-kappaB. mAb E-3 bound live EC and recognized a 42 kDa EC membrane protein, it enhanced U937 adhesiveness, E-selectin and ICAM-1 expression and IL-6 production, and caused the loss of I-kappaB. We conclude this is the first in vitro demonstration that a human monoclonal AECA from a SLE patient reacts with a constitutive endothelial membrane antigen and induces a pro-inflammatory endothelial phenotype through NF-kappaB activation.

Interaction between chronically HIV-infected promonocytic cells and human umbilical vein endothelial cells: role of proinflammatory cytokines and chemokines in viral expression modulation.

HIV type 1 expression was significantly up-regulated in chronically infected promonocytic cell line (U1) co-cultured with human umbilical vein endothelial cells (HUVEC). Virus replication, evaluated as supernatant p24 release, was higher when U1 were co-cultured with IL-1beta-activated HUVEC than with unstimulated HUVEC. When non-adherent U1 were removed from co-cultures, the remaining U1 cells adherent to the endothelial monolayer still showed enhanced HIV replication in comparison with an equal number of U1 cultured alone. While addition of adhesion molecule blocking antibodies (anti-intercellular adhesion molecule-1 (ICAM-1), -vascular cell adhesion molecule-1 (VCAM-1), -CD18 and -very late antigen-4 (VLA-4)) strongly inhibited adherence of U1 cells to endothelial monolayers, such treatment resulted in only a partial reduction in p24 release. Furthermore, HIV replication in U1 cells was enhanced on culture in HUVEC-conditioned media. Such data suggest that soluble mediators secreted by endothelial monolayers may modulate HIV-1 expression. Indeed, addition of cytokine and chemokine antagonists to both U1/HUVEC co-cultures and to U1 cultured in HUVEC-conditioned media clearly down-regulated p24 release. Anti-IL-6, anti-tumour necrosis factor-alpha (TNF-alpha) and, particularly, anti-MCP-1 MoAbs reduced p24 release, while anti-IL-8 polyclonal antiserum and IL-1 receptor antagonist (IL-1Ra) had no significant effect. Thus, the interaction between HUVEC and infected monocytic cells up-regulates HIV-1 replication predominantly through production of endothelium-derived soluble factors including MCP-1, TNF-alpha and IL-6. This phenomenon may influence the passage of HIV-1 from latency to productive replication and enhance virus spreading during physiological and/or pathological contact of monocytes with endothelium.

Anti-endothelial cell IgG fractions from systemic lupus erythematosus patients bind to human endothelial cells and induce a pro-adhesive and a pro-inflammatory phenotype in vitro.

Affinity purified immunoglobulin G (IgG) fractions from systemic lupus erythematosus (SLE) patients positive for anti-endothelial cell antibodies (AECA) bind human umbilical vein endothelial cell (HUVEC) monolayers. In vitro incubation of serial protein concentrations of SLE AECA IgG induces a dose-dependent endothelial activation: i) increase of functional adhesion of the monocytic cell line U937; ii) upregulation of E-Selectin, ICAM-1, VCAM-1 expression evaluated by a cell solid-phase enzyme linked immunoassay; and iii) increased secretion of interleukin (IL)-6 in the culture supernatants. Control experiments carried out with HUVEC monolayers incubated with IgG fractions from normal healthy controls or from AECA negative SLE sera do not affect at all endothelial adhesion molecule expression or pro-inflammatory cytokine secretion. The AECA IgG effects are not related to both anti-phospholipid or anti-DNA activities. Taken together the findings suggest that these autoantibodies might be important in recruiting and in activating mononuclear leukocytes responsible for vessel wall infiltration and raise the possibility that AECA might display a pathogenic role in SLE vessel damage.

In vitro type-1 and type-2 cytokine production in systemic lupus erythematosus: lack of relationship with clinical disease activity.

OBJECTIVE: To investigate the relationship between disease activity and in vitro cytokine, soluble(s)CD23 and polyclonal and anti-DNA antibody production by PBMC from patients with active systemic lupus erythematosus (SLE). METHODS: Cytokines, sCD23 and immunoglobulins were estimated by ELISA in unstimulated and polyclonal mitogen-stimulated culture supernatants. RESULTS: PHA-induced IL-2 and IFN-gamma production were decreased, whereas spontaneous and PHA-induced IL-6 and IL-10 production were increased in cultures of SLE lymphocytes. Conversely, spontaneous and PHA-stimulated IL-4 and sCD23 production was comparable between patients and controls. Finally, we found an increase in in vitro spontaneous polyclonal and anti-DNA IgG secretion. CONCLUSIONS: We demonstrated an expanded type-2 cytokine profile with no correlation with parameters of disease activity.

In vitro production of type 1 and type 2 cytokines by peripheral blood mononuclear cells from high-risk HIV-negative intravenous drug users.

OBJECTIVE: To study type 1 and type 2 cytokine patterns in HIV-negative high-risk intravenous drug users (IVDU). DESIGN: We investigated interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-6 and IL-10 production by phytohaemagglutinin (PHA)-stimulated and unstimulated peripheral blood mononuclear cell (PBMC) cultures from HIV-negative high-risk IVDU, HIV-negative controls and HIV-positive subjects. METHODS: Cytokine production was measured in supernatants using enzyme-linked immunosorbent assay (ELISA) in 10 HIV-negative high-risk IVDU, 25 HIV-negative controls, and 12 HIV-positive IVDU. We also determined spontaneous in vitro immunoglobulin (Ig) G and IgM production. RESULTS: HIV-negative high-risk IVDU showed increased IFN-gamma and decreased IL-4, IL-10 and IL-2, although the latter was not significant compared with HIV-negative controls. Further, HIV-negative high-risk IVDU had reduced IgG production and impaired IgM-IgG switch. CONCLUSIONS: The reduced IL-2 and IL-4 production suggest an impaired CD4+ T-cell function in HIV-negative high-risk IVDU. The increased IFN-gamma production along with the decreased type 2 cytokine profile is consistent with the hypothesis that protective immunity against HIV may reside in type 1 responses and cell-mediated immunity.

Protection from experimental autoimmune thyroiditis in CBA mice with the novel immunosuppressant deoxyspergualin.

The effects of the novel immunosuppressant Deoxyspergualin (DSP) on the development of experimental autoimmune thyroiditis (EAT) in CBA mice were studied. For EAT induction, the mice were immunized with 100 micrograms of porcine thyroglobulin (p Tg) emulsified in CFA on day 0 and in IFA, for boosting, on day 14. Twenty-eight days after primary immunization, histological and serological signs of EAT occurred in control mice treated with PBS which showed marked lymphoid infiltration of the thyroid glands along with increased serum titres of anti-pTg antibodies. Development of both these EAT features was significantly suppressed when the mice were treated with 2.5 mg/kg body weight DSP, given daily, five times a week, from day -2 to day +28 after immunization. The effect appeared to be dose-dependent and DSP was ineffective when given under the same experimental conditions at the dose of 0.5 mg/kg body weight. No DSP-toxic effects could be observed during the experiment. These results provide further evidence for the powerful immunosuppressive properties of DSP and suggest that this drug may be used in the treatment of autoimmune thyroid diseases and other T-cell mediated autoimmune disorders in humans.

Enrichment of IgG anti-DNA-producing lymphoblastoid cell lines by antigen-coated immunomagnetic beads.

In this paper we describe the establishment of four anti-DNA-producing lymphoblastoid cell lines (LCL) by Epstein-Barr virus (EBV) infection of peripheral blood B-cells from a patient with systemic lupus erythematosus. The LCL showed a heterogeneous cell composition: the frequencies of cells in active proliferation, cells secreting IgG or IgM, and cells effectively producing IgG or IgM anti-DNA were estimated by limiting dilution analysis. The cells producing anti-DNA antibodies were a small fraction of the whole cell population constituting the LCL. In order to enrich the fraction of cells producing the desired antibody we performed a positive selection by DNA-coated immunomagnetic beads. Results show that in two out of three IgG anti-DNA-secreting LCL the frequency of DNA-producing cells increased after incubation with DNA-coated beads. At variance, IgM anti-DNA-secreting cells were completely lost after immunomagnetic separation. This approach could represent a further tool to obtain better fusion partners to construct stable hybrids secreting human monoclonal antibodies. The advantages of the presented technique would be: (a) removing of the fraction of low-affinity IgM-producing cells, which is often prevalent in EBV-induced LCL; and (b) the enrichment of IgG anti-DNA producing cells, which better represent the antibodies involved in the pathogenesis of the disease.

In vivo treatment with a monoclonal antibody to interferon-gamma neither affects the survival nor the incidence of lupus-nephritis in the MRL/lpr-lpr mouse.

The effects of the in vitro treatment with a mAb (DB-1) that neutralizes mouse IFN-gamma on the development of the SLE-like syndrome in MRL/lpr-lpr (MRL-lpr) mice were studied. The results show that the i.p. administration of 2.6 mg/week of DB-1 from the 12th to the 20th week of age neither affected the survival nor the incidence and severity of lupus nephritis in MRL-lpr mice. This study argues against the pathogenic relevance of IFN-gamma in this experimental model of human SLE.

Antibodies to endothelial cells in primary vasculitides mediate in vitro endothelial cytotoxicity in the presence of normal peripheral blood mononuclear cells.

Twenty-eight out of 62 patients with Wegener's granulomatosis and micropolyarteritis display circulating antiendothelial cell antibodies (AECA) detectable by a cell surface radioimmunoassay. These antibodies do not induce an in vitro endothelial damage either alone or in the presence of fresh complement; however, 50% of IgG-AECA positive sera can be cytotoxic in the presence of human normal peripheral blood mononuclear cells (PBM) at high effector/target ratios. The specificity of the PBM-mediated cytotoxicity is supported by the absence of the phenomenon in AECA negative sera, by the disappearance of the lytic effect after absorption of AECA, and by the finding that cellular-mediated cytotoxicity can be reproduced by purified IgG-AECA positive fractions. On the contrary, polymorphonuclear leukocytes or adherent mononuclear cells are not involved in such a cytotoxic activity. AECA seem to be directed against determinants consitutively expressed on the endothelial surface since the activation of endothelial cells by interleukin-1 beta or interferon-gamma affects neither the antibody binding nor their ability to mediate 51Cr release in the presence of PBM. These findings favor the hypothesis for a possible direct pathogenetic role of circulating AECA in the in vivo vascular damage.

Combination of mutant EL-4 thymoma cells and EBV-infection in B lymphocyte activation a) EBV-infection of EL-4-activated lymphocytes. b) Use of EL-4 cells as feeders for lymphoblastoid cell lines.

The combination of mutant EL-4 thymoma cells and Epstein-Barr virus (EBV)-infection in the activation of human B lymphocytes was studied with two approaches: a) limiting numbers of B cells were first activated by EL-4 contact in order to expand the B cell population and then infected with EBV. Results show that EBV could induce further Ig synthesis, although was unable to determine proliferation or to generate immortalized lines from EL-4 activated cells. b) EL-4 cells were compared to conventional PBM as feeders for cultures of established lymphoblastoid cell lines (LCL) at low cell densities. EL-4 feeder activity was strictly dependent on the presence of phorbol-myristate-acetate (PMA) and supernatant from stimulated T-lymphocytes (T-SN). EL-4 feeders induced earlier proliferation peak and greater Ig synthesis by LCL. The latter effect could be also mediated by the addition of PMA and T-SN, even if a cooperating cell-to-cell signal by PMA and T-SN-sensitized-EL-4 cells could not be excluded. Altogether results indicate that EL-4 cells do not represent a clear advantage over classic PBM as feeders for cultures of established LCL at low cell densities.

Anti-endothelial cell antibodies in patients with Wegener's granulomatosis and micropolyarteritis.

Anti-endothelial cell antibodies (AECA) have been detected by cell surface radioimmunoassay in nine out of 15 patients with micropolyarteritis (MPA) and in two out of five patients with Wegener's granulomatosis. AECA mostly belonged to the IgG isotype and were present in the active phase of the diseases. These antibodies were not detectable in 10 sera from patients with essential mixed cryoglobulinaemia, suggesting that they were not a mere epiphenomenon consequent to the inflammatory vascular injury. The binding activity was not related to ABH antigens or to HLA class I antigens displayed by resting human endothelial cells in culture and was not influenced by removing immune complexes. Absorption of the anti-neutrophil cytoplasmic antibodies (ANCA), present in MPA and Wegener's granulomatosis sera, did not affect the endothelial binding. AECA-positive sera did not display lytic activity against endothelial cells, neither alone nor after addition of fresh complement or normal human peripheral blood mononuclear cells. Although AECA are not cytolytic for endothelial cell monolayers in vitro, the reactivity against intact endothelial cells suggests their possible involvement in in vivo pathological processes affecting vascular structures in small vessel primary vasculitides.

Interleukin 2 enhancement of anti-T3-induced lymphocyte activation: standardization and use of this model in immunocompromized elderly subjects.

Anti-T3-induced T cell activation was standardized in human peripheral blood lymphocytes with respect to dose response, time kinetics and effect of exogenous IL-2 on blastogenesis and on IL-2 receptor expression, identified by anti-Tac monoclonal antibody. IL-2 was able to significantly increase 3H-thymidine uptake and Tac antigen expression in unstimulated and anti-T3 activated lymphocytes. This enhancement was more evident in cultures stimulated with non-mitogenic concentrations of anti-T3, supporting the hypothesis that these concentrations sensitize T cells to the Tac-inducing effects of IL-2. This model of lymphocyte activation was then applied to selected immunocompromized elderly subjects. The aged subjects showed depressed blastogenetic responses and Tac antigen expression, which were not corrected by prolonging the time of cultures. Aged lymphocytes showed a good response to costimulation with anti-T3 and IL-2, suggesting that at least for the anti-T3 activation pathway they are able to respond to IL-2 signals.