Serum Fetuin-A levels in obese children with biopsy proven nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Fetuin-A has been proposed as a marker of liver damage in adults with obesity-related NAFLD. The aim of this study was to test serum fetuin-A concentrations in obese children with NAFLD diagnosed either by ultrasonography or by liver biopsy and to determine its applicability as predictive tool in pediatric NAFLD. METHODS AND RESULTS: Metabolic parameters and fetuin-A levels were investigated in 81 obese children with NAFLD diagnosed by biopsy, 79 obese children with NAFLD defined by liver ultrasonography and 23 lean subjects. Serum fetuin-A correlated significantly with age, waist circumference, systolic blood pressure, fasting insulin and 2-h postload insulin during OGTT, HOMA-IR, ISI, CRP, and apo B levels. Obese children with NAFLD detected by ultrasonography had significantly higher fetuin-A levels compared to those with normal liver. In obese children who underwent liver biopsy, no significant differences were detected in fetuin-A levels between subject with nonalcoholic steatohepatitis and those with simple steatosis. Fetuin-A was not different between obese and lean children. CONCLUSION: Fetuin-A is not related with the degree of liver damage in obese children with NAFLD and its routine measurement as marker of liver disease severity is therefore not recommended.

Mesenchymal cell precursors of peritubular smooth muscle cells of the mouse testis can be identified by the presence of the p75 neurotrophin receptor.

In the mouse embryo, at approximately 11.5 days postcoitum (dpc), cells migrate from the mesonephros into the developing testis to contribute to the somatic population of the interstitial compartment (i.e., peritubular myoid cells, Leydig cells, and endothelial cells). Studies from this laboratory have shown that the interstitial population of mesenchymal cells in fetal and newborn mouse testis express the p75 neurotrophin receptor (p75NTR, formerly known as the low-affinity nerve growth factor receptor); part of the cell population progressively congregates around testis cords, later to be replaced by contractile peritubular myoid cells, which express smooth muscle cell markers. In the present study, we show that the migrating cells and the p75NTR-expressing cells are the same population. We also show that the neurotrophin receptor is a useful endogenous marker to follow cell migration within the urogenital ridge and to identify and isolate mesenchymal precursors of myoid cells. A time-course immunolocalization study of the location of p75NTR-bearing cells within the urogenital ridge of mouse embryos between 10.5 and 12.5 dpc showed that the interstitium of the fetal testis was progressively occupied by p75NTR+ cells. The progressive increase of p75NTR expression within the developing testis was confirmed by immunoblot analysis of proteins isolated from the fetal gonads. Organ cultures of isolated testes or testis-mesonephros grafts confirmed that p75NTR+ cells do not appear in the testis unless a mesonephros is attached to it. Cells bearing the p75NTR receptor, purified from 12.5-dpc male mouse mesonephroi by immunomagnetic sorting, were able to differentiate in vitro into myoid cells. Immunofluorescence analysis of postnatal testis sections confirmed the presence around the tubules of cells coexpressing p75NTR and alpha-smooth muscle actin. The ability to identify and purify precursors of myoid cells may be of considerable help for studying the mechanisms regulating their differentiation.

IGF-I stimulates chemotaxis of human neuroblasts. Involvement of type 1 IGF receptor, IGF binding proteins, phosphatidylinositol-3 kinase pathway and plasmin system.

SH-SY5Y human neuroblastoma cells express IGF receptors, IGFs and IGF binding proteins (IGFBPs), and provide a model for studying the role of the IGF system in human neuronal development. We investigated the effect of IGF-I and des(1-3)IGF-I on the motility of SH-SY5Y cells by a cell migration assay based on the assessment of the number of cells which migrated across 8 microm pore size membranes and around an agarose drop. IGF-I and des(1-3)IGF-I stimulated neuroblast chemotaxis in a dose-dependent manner. Treatment of cells with these agents for 24 h resulted in a significant increase (IGF-I by 70% and des(1-3)IGF-I by 90%; P<0. 0001) in cell motility relative to control conditions. Addition of monoclonal antibody against type 1 IGF receptor (alpha-IR3), significantly (P<0.05) reduced the cell motility induced by IGF-I (by 30%) and des(1-3)IGF-I (by 70%). Wortmannin, a specific inhibitor of phosphatidylinositol (PI)-3 kinase intracellular signalling, also reduced the IGF-stimulated cell migration (by over 40%, P<0.01), indicating a key role of the PI-3 kinase pathway in mediating the IGF effect on neuroblast migration. Finally, cell treatment with plasminogen (PLG) markedly enhanced neuroblast migration (by over 200%, P<0.01), whereas incubation with the PLG inhibitor 4-(2-aminoethyl)-benzenesulphonyl fluoride reduced cell motility (by 80%, P<0.01), thus suggesting an involvement of PLG-dependent IGFBP proteolysis in the regulation of neuroblast motility. In conclusion, IGF-I is a potent stimulator of neuroblast migration through the activation of type 1 IGF receptor and the PI-3 kinase intracellular pathway. IGFBPs and the plasmin system seem to play a role in cell motility, although the nature and the extent of their involvement has yet to be elucidated.

HSP70 production and inhibition of cell proliferation in Molt-4 T-cells after cell-to-cell transmission of HTLV-I: effect of PGA1.

Infection with HTLV-I is associated with leukemic transformation of mature CD4+ T lymphocytes. PGA1, a powerful inhibitor of tumour cell proliferation, can prevent the clonal expansion of HTLV-I-infected cells following acute infection of cord blood-derived mononuclear cells. Since the antiproliferative effect of PGA1 on HTLV-I transformed, chronically infected MT-2 cell line was associated with induction of HSP70, we have investigated the effect of PGA1 on cell cycle progression and HSP70 production in a leukemic T-cell line (Molt-4) shortly after exposure to HTLV-I in a cell-to-cell transmission model. Rate of cell proliferation and HSP70 expression were studied within one duplication cycle of Molt-4 cells after exposure to HTLV-I. Growth of both control and virus-exposed cultures was inhibited by treatment with PGA1 (4 micrograms/ml) and cell cycling was arrested preferentially at the G1/S interphase. Synthesis of HSP70 was induced within 3 h by PGA1 in control and virus-exposed Molt-4 cells and became undetectable from overnight onward, though the protein accumulated in the cells. The arrest of growth was observed from overnight up to 48 h so that treated cells almost missed one cycle. Interestingly, HSP70 transcript and protein persisted at remarkably high levels in Molt-4 cells exposed to HTLV-I in the absence of PGA1, showing that HSP70 expression can be directly activated during primary infection with this human retrovirus. Moreover, in these cocultures, treatment with PGA1 or heat shock was not able to increase further the elevated level of HSP70 found in untreated cocultures, suggesting that during the early period of the virus-transmission phase, HTLV-I could interfere with HSP70 induction by other inducers.

Cell-cycle progression and apoptosis in K562 and Molt-4 cells after cell-to-cell transmission of HTLV-I: modulation by interferons.

Human T-cell leukemia virus type I (HTLV-I) is mainly propagated by cell division and therefore the virus-driven proliferation of infected cells can represent a predisposing condition to final development of adult T-cell leukemia (ATL) in vivo. To correlate virus expression and cell cycle progression of recipient cells after acute infection with HTLV-I, K562 multipotent erytholeukemia and Molt-4 T-lymphoma cells were used as recipient cells in a cell-to-cell virus transmission model. Cell cycle progression was studied by flow cytometry during one duplication cycle of recipient cells and transcription of HTLV-I was evaluated during the same time course. The antiproliferative and antiviral effects of recombinant interferons alpha, beta and gamma were also evaluated on cell cycle progression and HTLV-I expression. Transcription of HTLV-I in immortalised virus-donor MT-2 T-cells was found to be related to cell cycle. After coculturing recipient K562 or Molt-4 cells with lethally irradiated, non-dividing virus-donor MT-2 cells, progression into cell cycle of recipient cells was delayed. A pre-G(1) peak, corresponding to 6-11 % apoptotic cells, was identified in cocultured Molt-4/MT-2 cells and not in Molt-4 controls, and was not affected by treatment with IFNs. Notably, no such peak was identified either in control or in cocultured K562 cells. During this time course, transcription of the viral subgenomic mRNA encoding for the env-pX region was prevalently observed. Treatment with IFNalpha and especially with IFNbeta at the onset of the cultures inhibited the growth of both control and virus-exposed recipient cells. IFNgamma was less effective. A clearcut reduction of the percentage of cells entering the S phase was observed only after treatment with IFNbeta. At the same time, in IFNbeta-treated cocultures a marked inhibition of transcription of viral mRNA was observed, suggesting that, during acute infection, treatment with IFNbeta contributes to reduce the infection of recipient cells by down-regulating both the cellular proliferation rate and virus transcription in infected cells.

Effects of cyclopentenone prostaglandins on myeloid cells during early infection with HTLV-I. I. Cell differentiation determines sensitivity to prostaglandins and virus infection.

Human myeloid cell lines at different stages of differentiation (K562, HL60 and U937) were used to analyze the permissivity of the myelomonocytic lineage to acute infection with human T-cell leukemia virus type-I (HTLV-I) after cell-to-cell transmission and to evaluate the effect of cyclopentenone prostaglandins (PG)A1 and PGJ2 on virus transmission, proliferation of recipient cells and cell-mediated cytotoxicity against virus-donor cells. Exposure to HTLV-I delayed the growth rate of recipient cells, especially in U937 cells. This effect was related to the phase of cell cycle when cells were exposed to HTLV-I. Treatment of control and virus-exposed cells with these PGs, both inducing growth arrest prevalently at the G1/S interphase of the cell cycle, inhibited cell proliferation in a concentration-dependent way. The antiproliferative effect of both PGs increased progressively from pluripotent K562 to promyelocytic HL60 and monoblastoid U937 cells, suggesting that differentiated cells were more susceptible to PG-mediated inhibition of growth than pluripotent cells. PG treatment influenced the permissivity of recipient cells to HTLV-I, with different effects on less differentiated myeloid cells in comparison with more differentiated monoblastoid cells. In fact, the percentage of cells positive for the p19gag protein was increased among PG-treated K562 or HL60 cells, although it was reduced in PG-treated U937 cells. To this respect, PGA1 was more effective on asynchronous and PGJ2 on synchronous U937 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiviral activity of individual versus combined treatments with interferon alpha, beta and gamma on early infection with HTLV-I in vitro.

We have shown previously that infection of mononuclear cells derived from neonatal cord (CBMC) or adult peripheral (PBMC) blood with HTLV-1 can be controlled in vitro by treatment with interferon (IFN) alpha, beta or gamma. The activity of IFNs was mainly related to the induction of an active antiviral competence in host's immune effector cells. The antiviral activity of IFN-boosted CBMC could be ascribed both to a positive regulation of cell-mediated immunity and to inhibition of viral infection. Data described herein provide further information on the mechanisms of the antiviral activity of IFNs and compare the activity of each type of IFN with the association of alpha + beta, alpha + gamma and beta + gamma IFNs, at a concentration of 100 or 1000 IU/ml. When added at the onset of the co-culture of CBMC with lethally irradiated, virus-donor MT-2 cells, IFNs could protect host CBMC by inhibiting HTLV-1 infection in terms of reduced proviral integration and a lower percentage of virus-positive cells, until 4 weeks of culture. Infection of CBMC was inhibited at a comparable extent by either individual or combined IFN treatments. However, a clearcut inhibition of HTLV-I transcription was found only when alpha 100 + beta 1000 IU/ml and especially alpha 1000 + gamma 100 IU/ml combined treatments were tested. When the chronically infected, virus-producing MT-2 cells were treated with IFNs, a remarkable inhibition of HTLV-I transcription was found only after multiple treatments. However, MT-2 cells became resistant to the antiviral activity of IFN gamma, but not to that of IFN alpha or beta. These data provide further information on the control of HTLV-I replication mediated by IFNs at different steps of the viral life cycle, being therefore relevant to the clinical use of combined IFNs in the treatment of acute infection. Moreover, IFNs could be used to prevent the establishment of a persistent infection, which is a prerequisite for developing adult T-cell leukemia (ATL) and/or virus-associated myelopathy.

Comparative anti-viral and anti-proliferative activity of PGA1 and PGJ2 against HTLV-I-infected MT-2 cells.

Prostaglandin (PG) A and J exert anti-viral and anti-proliferative effects in a number of experimental models. In particular, multiple treatments with PGAs prevent in vitro the clonal selection of HTLV-I-infected and potentially transformed cord-blood-derived mononuclear cells. Proliferation of HTLV-I-infected leukemic T cells is refractory in most cases to conventional anti-blastic therapy. We examined whether these cyclopentenone PGs might control cell proliferation and/or alter virus replication also in HTLV-I-transformed cells. We show that PGA1 and PGJ2 can exert powerful control of proliferation of the HTLV-I-immortalized, virus-producing MT-2 cell line, in a concentration-dependent fashion. Cells were preferentially arrested at the G1/S interface by treatment with PGA1 or PGJ2 without any detectable cellular toxicity. The anti-proliferative effect of PG treatment was independent of the growth phase of MT-2 cells, since both asynchronous and synchronous cells were sensitive to treatment. This effect was accompanied by an increase in the synthesis of a 70 kDa heat-shock protein (HSP70). However, synthesis of HSP70 was induced to a much greater extent by PGJ2 than by PGA1 at the same concentration. Neither PGA1 or PGJ2 inhibited the transcription of HTLV-I in MT-2 cells, but treatment with PGJ2, and not with PGA1, moderately inhibited the synthesis of viral proteins, i.e., p40 Tax and p19 core proteins. Moreover, infection of recipient K562 cells was significantly inhibited after pre-treatment of MT-2 cells with PGJ2 14 hr before or co-treatment at the onset of the co-culture with K562 cells. This effect was not obtained when MT-2 cells were repeatedly pre-treated with PGJ2 for 1 week before co-culturing. This suggests that reduced infection could be related to impairment of some step in virus-transmission phase.