Flt-3 and its ligand are expressed in neural crest-derived tumors and promote survival and proliferation of their cell lines.

Flt-3 ligand (FL) is a cytokine that promotes the survival, proliferation, and differentiation of hematopoietic progenitors in synergy with other growth factors, such as stem cell factor. Previously we have demonstrated that stem cell factor and its receptor c-kit are expressed in neural crest-derived tumor cells and that a c-kit block induces their apoptosis. Here we have evaluated the expression of flt-3 and its ligand in 12 neuroectodermal tumor cell lines from neuroblastoma (NB), neuroepithelioma (NE), Ewing sarcoma (ES), and peripheral neuroectodermal tumor (PNET) and in 38 biopsies: 19 from NB and 19 from ES and PNET. RT-PCR demonstrated the expression of flt-3 and FL in all lines. Coexpression was observed in 42% of NB and in 74% of ES and PNET biopsies. Flow cytometry confirmed the presence of membrane and cytoplasmic flt-3 and membrane FL in all lines, whereas soluble FL protein was not measurable in their supernatants. Microphysiometric demonstration of acidification of the medium provided evidence of the specific response of cell lines to FL stimulation. Specific flt-3 phosphorylation after FL treatment was also demonstrated by Western blotting analysis. In cells growing in RPMI plus 1% fetal calf serum, FL revealed a significant proliferating activity, more evident in NB and NE lines (mean increase of viable cells, 73 +/- 26% after 1 day). Treatment with flt-3 antisense oligonucleotides significantly inhibited cell growth. FL also displayed an antiapoptotic activity: after a 12-hour culture in the presence of 0.1% fetal calf serum, FL caused a 50% reduction of apoptotic cells. These results provide further evidence that neuroectodermal and hematopoietic cells share common regulatory pathways, and could be of interest in the clinical management of neuroectodermal tumors.

Proteasomes are a target of the anti-tumour drug vinblastine.

Proteasomes, the proteolytic machinery of the ubiquitin/ATP-dependent pathway, have a relevant role in many processes crucial for cell physiology and cell cycle progression. Proteasome inhibitors are used to block cell cycle progression and to induce apoptosis in certain cell lines. Here we examine whether proteasomal function is affected by the anti-tumour drug vinblastine, whose cytostatic action relies mainly on the disruption of mitotic spindle dynamics. The effects of vinblastine on the peptidase activities of human 20 S and 26 S proteasomes and on the proteolytic activity of 26 S proteasome were assessed in the presence of specific fluorogenic peptides and (125)I-lysozyme-ubiquitin conjugates respectively. The assays of ubiquitin-protein conjugates and of inhibitory kappa B alpha (I kappa B alpha), which are characteristic intracellular proteasome substrates, by Western blotting on lysates from HL60 cells incubated with or without vinblastine, illustrated the effects of vinblastine on proteasomes in vivo. We also evaluated the effects of vinblastine on the signal-induced degradation of I kappa B alpha. Vinblastine at 3--110 microM reversibly inhibited the chymotrypsin-like activity of the 20 S proteasome and the trypsin-like and peptidyl-glutamyl-peptide hydrolysing activities of both proteasomes, but only at 110 microM vinblastine was the chymotrypsin-like activity of the 26 S proteasome inhibited; furthermore, at 25--200 microM the drug inhibited the degradation of ubiquitinated lysozyme. In HL60 cells exposed for 6 h to 0.5--10 microM vinblastine, the drug-dose-related accumulation of polyubiquitinated proteins, as well as that of a high-molecular-mass form of I kappa B alpha, occurred. Moreover, vinblastine impaired the signal-induced degradation of I kappa B alpha. Cell viability throughout the test was approx. 95%. Proteasomes can be considered to be a new and additional vinblastine target.

Integrin expression and IgA nephropathy: in vitro modulation by IgA with altered glycosylation and macromolecular IgA.

BACKGROUND: Signal transduction by mesangial cell (MC) integrins regulates cell growth and survival, extracellular matrix production, and organization. The aim of the study was to investigate human MC integrin modulation by differently glycosylated IgA and macromolecular IgA, which are thought to play a pathogenetic role in IgA nephropathy (IgAN). METHODS: MCs were incubated with purified human polymeric IgA, heat-aggregated IgA, IgA glycoforms generated by enzymatic hydrolysis of saccharide residues and serum fractions from IgAN patients, and controls isolated by lectin affinity and containing IgA with peculiar glycan patterns. Integrins were quantitated by flow cytometry. RESULTS: Cultured MCs highly expressed alphavbeta3 and some alpha3beta1; alphavbeta3 was up-regulated by matrix components (P < 0.02). In vitro desialylated and degalactosylated polymeric human IgA enhanced alphavbeta3 expression on cultured MCs (P < 0.001). Serum IgA glycoforms isolated from IgAN patients with high exposure of internal sugars, GalNAc, Neu5Ac2,6GalNAc, and Man enhanced alphav expression on cultured MCs more than healthy controls. CONCLUSIONS.: These data support the hypothesis that IgA glycation plays a role in modulating the cell-matrix interaction, and that this mechanism can be operating in IgAN.

Individual and combined effect of granulocyte-macrophage colony-stimulating factor and prolactin on maturation of dendritic cells from blood monocytes under serum-free conditions.

Prolactin (PRL) shares structural and functional features with haemopoietic factors and cytokine peptides. Dendritic cells (DC) are involved in both initiating the primary and boosting the secondary host immune response and can be differentiated in vitro from precursors under the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus other factors. Because PRL has been shown to functionally interact with GM-CSF, we have addressed its role on GM-CSF-driven differentiation of DC. Monocytic DC precursors from peripheral blood mononuclear cells (PBMC) were enriched either by adhesion to a plastic surface or CD14-positive selection and cultured for 7 days in serum-free medium containing GM-CSF, interleukin (IL)-4 and PRL, alone or in combination. Cells with large, veiled cytoplasm, expressing major histocompatibility complex (MHC) class II and the costimulatory molecules CD80, CD86 and CD40 and lacking the monocyte marker CD14, were considered as having the phenotype of cytokine-generated DC. Functional maturation was assessed by proliferation and interferon-gamma (IFN-gamma) release of allogeneic T lymphocytes. Physiological (10-20 ng/ml) concentrations of PRL interacted synergistically with GM-CSF and the effect was similar to that induced by IL-4 on GM-CSF-driven DC maturation. When used alone, the physiological concentrations of PRL were inhibitory, whereas higher concentrations (80 ng/ml) were stimulatory. The synergistic effect of PRL may in part be caused by its ability to counteract the down-modulation of the GM-CSF receptor observed in serum-free conditions. These data provide further evidence of the significance of PRL in the process of T lymphocyte activation.

Nitric oxide mediates cyclosporine-induced apoptosis in cultured renal cells.

BACKGROUND: The clinical use of cyclosporine (CsA) is limited by its nephrotoxicity. Apoptosis, perhaps instigated by increased nitric oxide synthase (NOS) activity, may play a role in such toxicity. METHODS: Human mesangial cells, human tubular cells, human umbilical vein endothelial cells, or murine endothelial cells were cultured with CsA at final concentrations of 0 to 1000 ng/mL for 4 to 24 hours. As inhibitors of apoptosis, 0.01 mol/L L-nitromethylarginine (L-NAME) or 1 microg/mL cycloheximide (CHX) was added, whereas 0.01 mol/L sodium nitroprusside (as a nitric oxide donor) was used as a positive control. Apoptosis was assessed by using TUNEL method and by DNA fragmentation by electrophoresis. In addition, NOS enzymatic activity, Northern blots for inducible NOS (iNOS) mRNA, and immunohistochemically demonstrable iNOS protein were evaluated. RESULTS: Within 12 to 24 hours, CsA significantly increased the fraction (8 to 35%) of apoptotic cells in each cell line, according to the dose. Fragmentation of DNA confirmed apoptosis. L-NAME and CHX inhibited the phenomenon, whereas sodium nitroprusside enhanced it. Each cell line significantly increased NOS activity in response to CsA, an effect blunted by L-NAME and CHX. Neither inhibitor modified the increased iNOS mRNA expression elicited by CsA. Positive staining for both iNOS and p53 proteins was observed in all cell lines incubated with CsA that were inhibited by CHX; L-NAME inhibited only p53 staining. CONCLUSIONS: CsA induces apoptosis in various renal cell lines, and this effect is mediated by the induction of iNOS via p53. These effects may contribute to the acellular fibrosis characteristic of late CsA nephrotoxicity.

Stem cell factor is not essential for cell survival and proliferation of soft tissue sarcoma of neuroectodermal origin.

BACKGROUND AND OBJECTIVE: Stem cell factor (SCF), and its receptor (c-kit) play key roles in the expansion and differentiation of hematopoietic progenitor cells, in melanoblasts and primordial germ cells, making it possible that SCF and c-kit are involved in neoplastic processes deriving from these cells. C-kit has been described to be expressed at different levels in neuroblastoma and in soft tissue sarcoma of neuroectodermal origin, and seems to be required for survival processes. In this study we investigate how c-kit expression is regulated and whether a SCF autocrine loop is essential for survival of sarcoma cell lines. DESIGN AND METHODS: C-kit modulation and internalization was evaluated incubating cells with rhSCF. Cell differentiation and proliferation experiments were performed to test whether c-kit expression is related to cell cycle progression or to differentiation processes. Cell cultures were treated with neutralizing antibody and antisense oligonucleotides in order to assess the possible significance of the SCF autocrine loop. RESULTS: In vitro SCF stimulation induces c-kit down-regulation; this phenomenon could be connected with receptor internalization, and new protein synthesis is necessary for its re-expression. The cell proliferation arrest in G0/G1 does not modify c-kit expression while down-regulation of c-kit was demonstrated after cells had been treated with differentiating agents. SCF neutralization does not influence either the S phase or apoptosis in sarcoma cell lines. INTERPRETATION AND CONCLUSIONS: In sarcoma cell lines, c-kit is regulated by differentiation processes; moreover our results suggest that c-kit activity, but probably not the SCF autocrine loop, is essential for survival of these cell lines.

c-kit is expressed in soft tissue sarcoma of neuroectodermic origin and its ligand prevents apoptosis of neoplastic cells.

During development, mice with mutations of stem cell factor (SCF) or its receptor c-kit exhibit defects in melanogenesis, as well as hematopoiesis and gonadogenesis. Consequently, accumulating evidence suggests that the c-kit/SCF system plays a crucial role in all of these processes and in tumors which derive from them. Especially in neuroblastoma (infant tumors of neuroectoderm crest derivation such as melanocytes) it would appear that an autocrine loop exists between c-kit and SCF, and that the functional block of the c-kit receptors with monoclonal antibodies (MoAbs) results in a significant decrease in cellular proliferation. We studied the expression and role of c-kit and SCF in cell lines of soft tissue sarcoma of neuroectodermic origin, such as Ewing's sarcoma (ES) and peripheral neuro-ectodermal tumors (PNET). Using flow cytometry with MoAb CD117 PE, c-kit expression was highlighted in all six of the cell lines examined. This receptor was specifically and functionally activated by SCF, as shown by the binding experiments and the intracellular phosphotyrosine and immunoprecipitation studies that were performed. Using reverse transcriptase polymerase chain reaction analysis, five of the six cellular lines expressed the mRNA of SCF. In the medium measured by using an enzyme- linked immunosorbent assay, low concentrations of SCF were found: only the TC32 cellular line produced significantly higher levels (32 pg) than control. In serum-free culture the addition of SCF reduced the percentage of apoptotic cells from 25% to 90% in five out of the six cellular lines. This observation was confirmed by (1) the functional block of c-kit with MoAb: after 7 days of culture more than 30% of the cells were apoptotic (range 31.5% to 100%) in five out of six cell lines and there was also a decrease in the percentage of cells in phase S, and (2) c-kit antisense oligonucleotides: in the cellular lines treated with oligonucleotides (in relation to the untreated lines) there was a notable reduction (P < .001) both in the absolute number of cells and the 3H-thymidine uptake. These results indicate that ES and PNET express c-kit and its ligand SCF and that SCF is capable of protecting the tumor cells against apoptosis. Furthermore, the reverse transcriptase-polymerase chain reaction performed on the biopsies revealed the presence of mRNA both of SCF and c-kit in practically all of the samples studied. Our in vitro data lead us to assume that SCF may also inhibit tumor cell apoptosis in vivo.

Stem cell factor suppresses apoptosis in neuroblastoma cell lines.

Stem cell factor (SCF) is a glycoprotein growth factor produced by marrow stromal cells that acts after binding to its specific surface receptor, which is the protein encoded by the protooncogene c-kit. SCF synergizes with specific lineage factors in promoting the proliferation of primitive hematopoietic progenitors, and has been administered to expand the pool of these progenitors in cancer patients treated with high-dose chemotherapy. SCF and its c-kit receptor are expressed by some tumor cells, including myeloid leukemia, breast carcinoma, small cell lung carcinoma, melanoma, gynecological tumors, and testicular germ cell tumors. Previous studies of SCF in neuroblastoma have produced conflicting conclusions. To explore the role of SCF in neuroblastoma, we studied five neuroblastoma lines (IMR-5, SK-N-SH, SK-N-BE, AF8, and SJ-N-KP) and the neuroepithelioma line CHP-100. All lines expressed mRNA for c-kit and c-kit protein at low intensity as measured by flow cytometry, and secreted SCF in medium culture as shown by ELISA. Exogenous SCF did not modify 3H thymidine uptake in the neuroblastoma and neuroepithelioma cell lines. After 6 days' culture in the presence of anti-c-kit, the number of viable neuroblastoma cells was significantly lower than the control, and terminal deoxynucleotidyl transferase assay showed a substantial increase of apoptotic cells: The percentage of positive cells was 1-3% in the control lines, whereas in the presence of anti c-kit it varied from 29% of SK-N-BE to 92% of CHP-100. After 9 days' culture in the presence of anti-c-kit, no viable cells were detectable. These data indicate that SCF is produced by some neuroblastoma cell lines via an autocrine loop to protect them from apoptosis.

The common TEL/AML1 rearrangement does not represent a frequent event in acute lymphoblastic leukaemia occuring in children with Down syndrome.

Individuals with constitutional trisomy 21 (Down syndrome) are at increased risk of developing acute leukaemias, both of myeloid and lymphoid lineage. Although the cause of leukaemia in Down syndrome (DS) remains unknown, potential candidate genes include the ones on chromosome 21, and in particular AML1, the rearrangement of which in the t(8,21) is associated with the French-American-British (FAB) classification M2 subtype of acute myeloid leukaemia (AML) in the general population and has been described in Down patients with AML-M2. Recently, a new rearrangement involving AML1, the t(12;21), producing the TEL/AML1 hybrid transcript, has been described by molecular analysis as the most recurrent genetic lesion in childhood acute lymphoblastic leukemia (ALL). In order to investigate whether the t(12;21) could give a molecular clue as to the precise basis of the etiologic association between DS and acute lymphoblastic leukemia, we tested a series of 11 consecutive cases of ALL in DS children for the presence of the TEL/AML1 transcript, by RT-PCR analysis. We report absence of the TEL/AML1 rearrangement among the 11 cases tested. This data may be suggestive of alternative pathways involved in the pathogenesis of ALL in children with constitutional trisomy 21.

Outcome and lineage involvement in t(12;21) childhood acute lymphoblastic leukaemia.

The t(12;21)(p13;q22) translocation has been described recently as the most recurrent genetic lesion in paediatric acute lymphoblastic leukaemias (ALLs). It has also been associated with B-precursor lineage involvement and good outcome. We tested 51 diagnostic paediatric ALLs and found 11 cases with molecular evidence of the t(12;21). Interestingly, amongst t(12;21) positive patients, we report three cases with hybrid phenotype, and two cases showing an aggressive and fatal disease. Our data show that the t(12;21) does not represent an independent good-risk indicator. Long follow-ups and additional molecular investigations are needed to assess the prognostic and pathogenetic relevance of t(12;21) in childhood ALLs.

Human immunodeficiency virus infection as risk factor for mother-to-child hepatitis C virus transmission; persistence of anti-hepatitis C virus in children is associated with the mother's anti-hepatitis C virus immunoblotting pattern.

To determine the rate of vertical transmission of hepatitis C virus (HCV), we prospectively studied 45 babies born to anti-HCV-positive women with or without concomitant infection with the human immunodeficiency virus (HIV). We performed a second-generation recombinant immunoblotting assay, alanine transaminase (ALT) evaluation, and HCV-RNA testing on sera from 27 infants of HCV+, HIV- mothers and 18 babies of HCV+, HIV+ women, at birth and thereafter. After birth, HCV antibodies progressively disappeared within 12 months in all children but one, whose mother was HCV+, HIV+; this child was the only one who showed detectable levels of HCV-RNA and abnormal ALT values throughout the follow-up (range, 12 to 27 months). Viremia was persistently negative, and ALT levels were continuously normal in the remaining infants, showing that "seronegative" infection with HCV was absent in both groups. Clearance of passively acquired anti-HCV antibodies was found to be slower among babies born to HIV+ mothers (22.3% vs. 3.8% at 12 months, P = .03) and children whose mothers showed three or four anti-HCV reactivities by immunoblotting maintained anti-HCV for longer periods compared with babies born to mothers with one or two anti-HCV reactivities (P = .0001). Seventeen of 27 babies born to HCV+, HIV- mothers were breast-fed, and none of them was infected, confirming the apparent safety for HCV of breast milk. In summary, according to our study, vertical transmission of HCV is an infrequent event, and the presence of HIV in the mother is not an important co-factor for transmission of HCV infection.

[Long lasting conjunctivitis: research of etiological factors]. / Congiuntiviti a lenta risoluzione: ricerca degli agenti etiologici.

Forty children with long-lasting or recurrent conjunctivitis were included in this etiological study. It is well known that purulent conjunctivitis is mainly bacterial, with a major source of infection from Chlamydia; recently, however, a greater percentage of viral forms, with the exception of conjunctivitis without secretion, has been reported. The authors focused their attention on the clinical symptoms and on bacteriological studies of the forms of Chlamydia and Mycoplasma conjunctivitis, highlighting their marked sensitivity to antibiotics and the clinical response and recommending the importance of an etiological study in all cases in which conjunctivitis does not resolve within a short period of time.