Obesity and kidney disease: Beyond the hyperfiltration.

In industrialized countries, overweight and obesity account for approximately 13.8% and 24.9% of the kidney disease observed in men and women, respectively. Moreover, obesity-associated glomerulopathy is now considered as "an emerging epidemic." Kidney function can be negatively impacted by obesity through several mechanisms, either direct or indirect. While it is well established that obesity represents the leading risk factor for type 2 diabetes and hypertension, awareness that obesity is associated with direct kidney damage independently of hypertension and diabetes is still not widespread. In this paper we will discuss the emerging role of adipose tissue, particularly in the visceral depot, in obesity-induced chronic kidney damage.

Resistance to the atypical retinoid ST1926 in SK-N-AS cells selected the subline rAS-ST with enhanced sensitivity to ATRA mediated by not conventional mechanisms: DNA damage, G2 accumulation and late telomerase inhibition.

BACKGROUND AND PURPOSE: 13-cis-Retinoic acid represents a well-established clinical strategy for the management of minimal residual disease of high risk neuroblastoma (NB) patients. However, the clinical efficacy on the overall survival of these patients remains limited, addressing the issue of better understanding the molecular mechanisms and intracellular pathways mediating Retinoic Acid (RA) clinical effects. EXPERIMENTAL APPROACH: This work investigates the mechanism underlying the sensitivity/resistance to RA in NB by taking advantage of the paired SK-N-AS/rAS-ST cells showing different responsivity to ATRA. The subline rAS-ST was selected by inducing resistance to the novel retinoid ST1926 in the NB SK-N-AS cell line. KEY RESULTS: Resistance to ST1926 was neither dependent on cellular uptake nor on multi-drug resistance phenotype. Rather, both delayed/lower DNA damage and apoptosis appeared involved in reduced sensitivity of rAS-ST cells to ST1926. This subline showed enhanced responsivity to ATRA compared to the wt counterpart, that was associated with enhanced RARα/ß expression, DNA damage, G2 accumulation, PI3K/AKT pathway inhibition, cellular differentiation and delayed telomerase inhibition, without involvement of either p27/p53 or caspase-mediated apoptosis. CONCLUSIONS AND IMPLICATIONS: The present data add important information to the understanding of RA sensitivity in NB, providing further insights towards a more efficacious clinical use of this drug.

Regulatory T cells in the immunodiagnosis and outcome of kidney allograft rejection.

Acute rejection (AR) is responsible for up to 12% of graft loss with the highest risk generally occurring during the first six months after transplantation. AR may be broadly classified into humoral as well as cellular rejection. Cellular rejection develops when donor alloantigens, presented by antigen-presenting cells (APCs) through class I or class II HLA molecules, activate the immune response against the allograft, resulting in activation of naive T cells that differentiate into subsets including cytotoxic CD8(+) and helper CD4(+) T cells type 1 (TH1) and TH2 cells or into cytoprotective immunoregulatory T cells (Tregs). The immune reaction directed against a renal allograft has been suggested to be characterized by two major components: a destructive one, mediated by CD4(+) helper and CD8(+) cytotoxic T cells, and a protective response, mediated by Tregs. The balance between these two opposite immune responses can significantly affect the graft survival. Many studies have been performed in order to define the role of Tregs either in the immunodiagnosis of transplant rejection or as predictor of the clinical outcome. However, information available from the literature shows a contradictory picture that deserves further investigation.

Triazene compounds in the treatment of acute myeloid leukemia: a short review and a case report.

Acute myeloid leukemia (AML) is a highly lethal disease, especially in old patients. Chemoresistance and the absence of host immune responses against autochthonous malignancy play a major role in the poor prognosis of AML. The triazene compounds Dacarbazine and Temozolomide are monofunctional alkylators that donate methyl groups to many sites in DNA, including the O(6)-position of guanine producing O(6)-methylguanine (O(6)-MeG). If not repaired, O(6)-MeG frequently mispairs with thymine during DNA duplication. O(6)-MeG:T mismatches can be recognized by the mismatch repair (MMR) system which activates a cascade of molecular events leading to cell cycle arrest and cell death. If MMR is defective, cells continue to divide and GC → AT transition mutations occur. In preclinical models, such mutations can lead to the appearance of abnormal proteins containing non-self peptides ("chemical xenogenization" CX) that can be recognized by host cell-mediated immunity. Repair of O(6)-MeG is achieved by the DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT), which removes the methyl adduct in an autoinactivating stoichiometric reaction. High MGMT levels attenuate the pharmacodynamic effects of triazenes. In the last few years, triazenes, alone or with MGMT inhibitors, have been tested in AML. In view of their potential activity as CX inducers, triazenes could offer the additional advantage of host anti-leukemia immune responses. The present paper describes several studies of leukemia treatment with triazenes and a case of acute refractory leukemia with massive skin infiltration by malignant cells. Treatment with Temozolomide and Lomeguatrib, a potent MGMT inhibitor, produced a huge, although transient, blastolysis and complete disappearance of all skin lesions.

Chronic kidney disease, obesity, and hypertension: the role of leptin and adiponectin.

Chronic kidney disease is a major public health problem and characterized by a progressive loss in renal function over a period of months or years as defined by structural or functional abnormalities of the kidney. Several elements contribute to determine a progression of the kidney injury, inducing a worsening of renal damage and accelerating the decline of renal function: obesity and hypertension are two known factors of kidney progression. Remarkable improvements have been recently achieved in the study of the endocrine features of the adipose tissue and have been able to produce hormone-like peptides named adipokines or adipocytokines. Among these adipocytokines, which represent a link between obesity, hypertension, and chronic nephropathy, leptins and adiponectin appear to play an important role. Leptin not only is a prohypertension element (renal progression factor) through the activation sympathetic nervous, but also is able to induce prosclerotic effects directly on the kidney. In contrast, a decline of adiponectin levels has been shown to be related to a picture of hypertension: an endothelial dysfunction has been described as the main pathogenic mechanism responsible for this phenomenon.

Interfacing Sca-1(pos) mesenchymal stem cells with biocompatible scaffolds with different chemical composition and geometry.

An immortalized murine mesenchymal stem cell line (mTERT-MSC) enriched for Lin(neg)/Sca-1(pos) fraction has been obtained through the transfection of MSC with murine TERT and single-cell isolation. Such cell line maintained the typical MSC self-renewal capacity and continuously expressed MSC phenotype. Moreover, mTERT-MSC retained the functional features of freshly isolated MSC in culture without evidence of senescence or spontaneous differentiation events. Thus, mTERT-MSC have been cultured onto PLA films, 30 and 100 microm PLA microbeads, and onto unpressed and pressed HYAFF-11 scaffolds. While the cells adhered preserving their morphology on PLA films, clusters of mTERT-MSC were detected on PLA beads and unpressed fibrous scaffolds. Finally, mTERT-MSC were not able to colonize the inner layers of pressed HYAFF-11. Nevertheless, such cell line displayed the ability to preserve Sca-1 expression and to retain multilineage potential when appropriately stimulated on all the scaffolds tested.

Effect of Saquinavir on proliferation and telomerase activity of human peripheral blood mononuclear cells.

The present study describes the effect of Saquinavir on proliferation, interferon-gamma production and telomerase activity of non-stimulated, or activated non-adherent mononuclear cells (NAMNC), obtained from peripheral blood of healthy donors. Fresh NAMNC, non-stimulated or activated in vitro with PHA or with a mixture of monoclonal antibodies against CD3 and against CD28 membrane antigens (in order to obtain prevalent T cell responses), were exposed to Saquinavir before or at the time of mitogenic stimulation. Control and treated cells were tested for DNA synthesis (3H-thymidine incorporation), interferon-gamma production and telomerase activity (TRAP assay). The results indicate that Saquinavir is able to increase proliferation and interferon-gamma release in PHA-stimulated NAMNC, and telomerase activity either in non-stimulated and in PHA or antibody-activated cells. These results suggest that the activity against HIV infection afforded by Saquinavir, could be corroborated by its effects on the host. These include its adjuvant activity on mitogen-induced responses of lymphocytes, and its possible antagonistic effects against lymphoid cell senescence, through telomerase activation.

Bacillus Calmette-Guerin down-regulates CD1b induction by granulocyte-macrophage colony stimulating factor in human peripheral blood monocytes.

Non-peptide antigens (e.g. glycolipids of microbial origin) presented by monocyte-associated CD1 molecules to T cells appear to play an important role in host immunity against tuberculosis and other pathogenic bacteria. Since vaccination with Bacillus Calmette-Guerin (BCG) has limited efficacy, the influence of viable BCG organisms on the induction of CD1b antigen by granulocyte macrophage-colony stimulating factor (GM-CSF) has been tested in adherent mononuclear cells obtained from peripheral blood of healthy donors. The results indicate that the vaccine reduces substantially CD1b induction by GM-CSF. On the other hand, BCG was found to promote a slight increase in the expression of this molecule on target cells not exposed to GM-CSF. Attempts to reverse the antagonistic effects of BCG on GM-CSF with high concentrations of GM-CSF, alone, or associated with IL-4, were unsuccessful. Moreover, mycobacteria suppression by 10 microg/ml of rifampin, did not affect BCG influence on CD1b induction. The present results suggest that mycobacterium-induced impairment of the CD1 system could play a role in the unsatisfactory results obtained with BCG vaccination.

Autocrine nerve growth factor is essential for cell survival and viral maturation in HHV-8-infected primary effusion lymphoma cells.

High levels of nerve growth factor (NGF) are found in sera from individuals infected with human herpesvirus 8 (HHV-8). BC-1 and BCBL-1 cells are primary effusion lymphoma-derived B-cell lines; BC-1 cells are infected by HHV-8 and the Epstein-Barr virus (EBV), and BCBL-1 cells are infected only by HHV-8. Both cells express NGF receptors and produce NGF, whereas RAMOS cells (a B-cell line that is negative for HHV-8 and EBV) express NGF receptors but do not produce detectable NGF. Neutralization of endogenous NGF results in cell growth inhibition and apoptosis in BCBL-1 cells and, to a minor extent, in BC-1 cells. When the HHV-8 lytic cycle is induced in BCBL-1 cells by tetradecanoyl phorbol acetate (TPA), an initial reduction of endogenous NGF production is observed, and many cells undergo apoptosis. However, at 48 hours, TPA-treated cells produce significantly more NGF than untreated controls, and a subsequent recovery of cell viability is observed. Consistent with this finding, the addition of exogenous NGF or anti-NGF antibodies to TPA-treated cells reduces or increases, respectively, the rate of apoptosis in response to TPA. Finally, electron microscopy of TPA-treated BCBL-1 cells shows that the addition of exogenous NGF increases the number of cells producing and releasing complete virions as compared with the controls (25% versus 5%). On the contrary, NGF neutralization leads to the production of defective viral progeny in about 2% of cells. These data indicate that NGF is essential for both cell survival and virus maturation in HHV-8-infected cell lines. (Blood. 2000;95:2905-2912)

Bacterial lipopolysaccharide increases prostaglandin production by rat astrocytes via inducible cyclo-oxygenase: evidence for the involvement of nuclear factor kappaB.

This study was set to investigate the mechanisms through which bacterial lipopolysaccharide (LPS) stimulates prostaglandin (PG) production in rat astrocytes. Primary cultures of rat hypothalamic astrocytes were established. Cells were treated with LPS alone or LPS plus antagonists of various pathways, and the subsequent changes in cyclo-oxygenase (COX) activity were monitored by measuring a COX end product, PGE2, released into the incubation medium. It was found that (i) LPS produced a concentration-dependent increase in PGE2 release from astrocytes. The potency of LPS was significantly increased by the addition of serum into the incubation medium; (ii) after 24 h of incubation, inducible COX (COX-2) accounts for most of the LPS-stimulated PG production, as the latter was markedly reduced by dexamethasone and the specific COX-2 inhibitor NS 398; and (iii) nuclear factor kappaB appears to play a role in the activation of COX-2 induced by LPS, since certain inhibitors of this transcription factor were able to antagonize, at least in part, the effects of LPS on PGE2 release.

Detection of high nerve growth factor serum levels in AIDS-related and -unrelated Kaposi's sarcoma patients.

OBJECTIVE: Nerve growth factor (NGF) is an autocrine survival factor for memory B lymphocytes. As functional B-cell deregulation is present during HIV infection, NGF serum levels were measured in HIV-infected patients and compared with the patients' clinical features. DESIGN: Sera from 48 consecutive HIV-infected patients and matched healthy controls were retrospectively and blindly analysed. Sera from seven patients with classical Kaposi's sarcoma (KS) were subsequently included in the study. The effects of NGF on spindle-shaped cells from KS lesions (KS cells) were also investigated. METHODS: NGF titration was performed by enzyme-linked immunosorbent assay (ELISA) and human herpesvirus 8 (HHV-8) antibody testing by immunofluorescent assay (IFA). NGF receptors were assessed by Western blot analysis. Cell growth assays were performed by cell counting. RESULTS: Very high median NGF serum levels were detected in all seven patients with AIDS-related KS (2500 pg/ml) compared with HIV-infected patients without KS (40 pg/ml), as well as in all seven classical KS patients (550 pg/ml) compared with healthy controls (20 pg/ml). In HIV-infected patients, NGF serum levels were significantly related to KS (P=0.0038) by stepwise multiple regression analysis, and HHV-8 seropositivity was significantly associated with KS (P=0.045) and to NGF levels (P=0.001) by logistic regression analysis. KS cells did not produce NGF but expressed both NGF receptors and presented increased proliferation rate after exogenous NGF addition. CONCLUSIONS: These results strongly suggest that increased NGF levels, possibly related to HHV-8 infection, may be involved in KS progression.

Effect of prostaglandin A1 on proliferation and telomerase activity of human melanoma cells in vitro.

Previous studies have shown that cyclopentenone prostaglandins are endowed with antitumour activity in various murine and human tumour models. In the present investigation four human melanoma cell lines were treated with graded concentrations (4-16microg/ml) of prostaglandin A1 (PGA1) for 24 or 48 h in vitro. At the end of the treatment, cell proliferation (measured in terms of DNA synthesis) and telomerase activity were determined. The results showed that PGA1 induced concentration-dependent inhibition of DNA synthesis at 48 h but not at 24 h in SK-MEL-28 cells. In contrast, marked inhibition of telomerase activity was detected after only 24 h of PGA1 treatment. Moreover, after 48h of treatment with the agent, inhibition of telomerase was more pronounced than inhibition of cell proliferation. Additional studies performed with three freshly generated melanoma cell lines confirmed that PGA1 produced early inhibition of cell growth accompanied by marked impairment of telomerase activity. These results suggest that PGA1 could be of potential value as antitumour agent, on the basis of two distinct mechanisms: direct cytostatic/cytotoxic effects on melanoma cells, and inhibitory activity on a tumour-associated enzymatic function (i.e. telomerase) that is responsible for cancer cell immortality.

The basic residues of placenta growth factor type 2 retrieve sequestered angiogenic factors into a soluble form: implications for tumor angiogenesis.

Placenta growth factor type 1 (PIGF-1) can be synthesized by neoplastic cells in an alternative form (PIGF-2) by the addition of basic amino acids to its classic sequence. Here we show that the basic residues of PIGF-2 compete for the binding of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) to heparan sulfate proteoglycans of the cell surface and extracellular matrix. In doing so, PIGF-2 basic sequences inhibit the sequestering of VEGF and bFGF and maintain them in a highly diffusible form, thus enhancing their angiogenic effect. In agreement with these in vitro data, the presence of PIGF-2 transcripts in tumors correlates with their blood vessel number. These results suggest a mechanism by which growth factor isoforms produced by neoplastic cells enhance the formation of new blood vessels supporting tumor growth and progression.

Functional antagonism between IL-2 and PGA1 or PGJ2 in the control of proliferation of human cord blood-derived mononuclear cells.

Cyclopentenone prostaglandins PGA1 and PGJ2 induce growth arrest at the G1/S interphase of the cell cycle in tumour cell lines. Notably, PGE, the precursor molecule of PGA, downregulates the interleukin (IL)-2-dependent proliferation of lymphocytes. Therefore the IL-2/IL-2 receptor system and relative signal transduction is a possible target of the antiproliferative effect of PGA/PGJ. In the present study the PGA1/PGJ2-dependent growth inhibition of IL-2-stimulated primary human cord blood mononuclear cells (CBMCs) was found to be mediated by interference with the IL-2 proliferative signal. Both prostaglandins (PGs) inhibited the synthesis of total RNA and protein in IL-2 stimulated cells. PGA1 and even more PGJ2 downregulated the expression of IL-2 receptor alpha (CD25 phenotype). IL-2 partly reversed this effect. Moreover, suppression of IL-2-stimulated cells was not the result of PG-mediated activation of apoptosis. On the contrary, PGs reduced both apoptosis and the high expression of c-Jun detectable in CBMCs spontaneously. Cyclin A/Cdk2 complexes regulate G1/S transition during the cell cycle. In IL-2-stimulated cells, the levels of Cdk2 were found to be lower in PG-treated cells than those detected in controls. In conclusion, cyclopentenone PGs inhibit CBMCs spontaneous or IL-2-dependent proliferation in part by interfering with the IL-2 pathway.

Prostaglandin E2 induces apoptosis in resting immature and mature human lymphocytes: a c-Myc-dependent and Bcl-2-independent associated pathway.

Prostaglandin E2 (PGE2) is a known negative regulator of T lymphocyte proliferation. Previously we have indirectly evidentiated the involvement of PGE2 in apoptosis of lymphocytes both in vitro and in vivo. We have evaluated a possible direct effect of PGE2 on apoptosis. To this end we have investigated the in vitro effects of PGE2 on cell death, and its possible correlation with c-Myc and Bcl-2 proteins. We used freshly isolated unstimulated human lymphocytes from neonatal thymus, cord blood and adult peripheral blood. PGE2 induced DNA fragmentation in both peripheral and cord blood at 10(-7) to 10(-5) M concentrations, even though this induction was delayed in peripheral blood with respect to cord blood. Apoptosis induced by PGE2 was always associated with a dose-dependent increase of cellular steady state c-Myc protein levels, whereas Bcl-2 protein levels were not substantially affected. Unstimulated thymocytes showed spontaneous DNA fragmentation that occurred earlier and at higher levels in PGE2-(10(-5) M) treated cells with respect to untreated controls. Also in these cells, PGE2 produced an early increase of c-Myc protein expression, although Bcl-2 protein levels remained unchanged. In conclusion, PGE2 induces apoptosis with different kinetics on immature and mature T cells: this induction is associated with the increase of c-Myc protein expression and seems to be independent from Bcl-2 regulation.