High doses of hydroxytyrosol induce apoptosis in papillary and follicular thyroid cancer cells.

PURPOSE: Recent evidences indicates that hydroxytyrosol, one of the main olive oil phenols, possess antitumor effects because of its pro-oxidant properties and the capacity to inhibit proliferation and to promote apoptosis in several tumor cell lines, although most of the results were obtained for breast and digestive systems cancers. METHODS: In this study, we evaluated the activities of hydroxytyrosol against papillary (TPC-1, FB-2) and follicular (WRO) thyroid cancer cell lines. RESULTS: Cellular viability revealed that high doses of hydroxytyrosol reduced cancer cells viability concomitantly with a reduction of cyclin D1 expression and an up-regulation of cell cycle key modulator p21 levels. In the same experimental conditions, Annexin V-PI staining and DNA laddering revealed that hydroxytyrosol exerts proapoptotic effects on papillary and follicular cancer cells. Furthermore, by Western blot analysis, we observed that hydroxytyrosol treatment reduced thyroid cancer cells viability by promoting apoptotic cell death via intrinsic pathway. CONCLUSIONS: Collectively, our results demonstrated for the first time that in thyroid cancer cells hydroxytyrosol promoted apoptosis at higher doses with respect to other cancer cells lines. Therefore, further studies will reveal the mechanisms by which thyroid cancer cells are more resistant to the proapoptotic effect exerted by hydroxytyrosol as well as the potential application as novel target therapeutic in thyroid cancer.

Early cytomegalovirus-specific T-cell response and estimated glomerular filtration rate identify patients at high risk of infection after renal transplantation.

BACKGROUND: Assessing the risk of cytomegalovirus (CMV) viremia in kidney transplant recipients (KTR) may be helpful to indicate in which patient it is worth starting antiviral treatment during preemptive strategy. METHODS: In 40 CMV-seropositive KTR preemptively treated with ganciclovir, we used interferon (IFN)-γ ELISpot test to evaluate whether monitoring T cells directed against phosphoprotein (pp) 65 and immediate early (IE)-1 antigens could predict the onset of viremia. RESULTS: CMV viremia occurred in 24 patients (60%) within 120 days after transplantation. Non-viremic patients had higher anti-pp65, anti-IE-1 T cells, and estimated glomerular filtration rate (eGFR) in the first 90 days after transplantation. At logistic regression, anti-pp65, anti-IE-1 T cells, and eGFR measured at day 30 were significantly associated with CMV infection. Cutoff values of 15 spot-forming cells (SFCs)/200,000 peripheral blood mononuclear cells (PBMCs) for anti-IE, 40 SFCs/200,000 PBMCs for anti-pp65, and 46.6 mL/min/1.73 m(2) for eGFR, respectively, predicted the risk of CMV infection with high sensitivity and specificity (area under the receiver operating characteristic curve >0.75). Using a classification tree model, we identified as high-risk patients those showing anti-pp65 <42 SFCs/200,000 PBMCs and eGFR <62 mL/min/1.73 m(2) , as well as anti-pp65 ≥42 and anti-IE-1 <6.5 SFCs/200,000 PBMCs. CONCLUSION: Monitoring CMV-specific T-cell responses and eGFR in the first month post transplant can identify patients at high risk of CMV infection, for whom preemptive antiviral therapy is recommended.

Monitoring chronic hemodialysis patients: a questionnaire-based survey.

BACKGROUND: The monitoring program for patients on regular hemodialysis treatment (RDT) is not well defined yet by current international guidelines (CIG). METHODS: To evaluate the extent to which CIG are implemented, we sent a questionnaire to 100 Italian hemodialysis units (DU) with questions concerning: (a) the frequency with which routine tests were performed for the follow-up of patients on RDT; (b) which other non-routine tests were performed. We analyzed the response data and compared them with the CIG. RESULTS: We received 37 replies. We found several differences between the monitoring program of our respondents and the CIG. CONCLUSION: Because of the small number of responses, this survey is only preliminary; however, it shows the difficulty nephrologists have in using the CIG to create a correct monitoring program in patients on RDT. Although our analysis is limited to 37 DUs, it suggests that specific guidelines are necessary to optimize the management of patients on RDT.

Rapamycin-induced autophagy protects proximal tubular renal cells against proteinuric damage through the transcriptional activation of the nerve growth factor receptor NGFR.

Experimental evidence demonstrated that macroautophagy/autophagy exerts a crucial role in maintain renal cellular homeostasis and represents a protective mechanism against renal injuries. Interestingly, it has been demonstrated that in the human proximal tubular renal cell line, HK-2, the MTOR inhibitor rapamycin enhanced autophagy and mitigated the apoptosis damage induced by urinary protein overload. However, the underlying molecular mechanism has not yet been elucidated. In our study we demonstrated, for the first time, that in HK-2 cells, the exposure to low doses of rapamycin transactivated the NGFR promoter, leading to autophagic activation. Indeed, we observed that in HK-2 cells silenced for the NGFR gene, the rapamycin-induced autophagic process was prevented, as the upregulation of the proautophagic markers, BECN1, as well as LC3-II, and the autophagic vacuoles evaluated by transmission electron microscopy, were not found. Concomitantly, using a series of deletion constructs of the NGFR promoter we found that the EGR1 transcription factor was responsible for the rapamycin-mediated transactivation of the NGFR promoter. Finally, our results provided evidence that the cotreatment with rapamycin plus albumin further enhanced autophagy via NGFR activation, reducing the proapoptotic events promoted by albumin alone. This effect was prevented in HK-2 cells silenced for the NGFR gene or pretreated with the MTOR activator, MHY1485. Taken together, our results describe a novel molecular mechanism by which rapamycin-induced autophagy, mitigates the tubular renal damage caused by proteinuria, suggesting that the use of low doses of rapamycin could represent a new therapeutic strategy to counteract the tubule-interstitial injury observed in patients affected by proteinuric nephropathies, avoiding the side effects of high doses of rapamycin.

Olive leaf extract counteracts cell proliferation and cyst growth in an in vitro model of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive enlargement of kidney cysts, leading to chronic kidney disease. Since the available treatment for ADPKD is limited, there is emerging interest for natural compounds as potential therapeutic candidates. The aim of our study was to investigate whether an olive leaf extract may be able to counteract the cyst growth in an in vitro model of ADPKD. We treated WT9-12 cells with an olive leaf extract (OLE). In monolayer culture we evaluated cell viability by the MTT assay, protein expression by western-blot analysis and apoptosis by DNA laddering and TUNEL assays. For functional studies we used transient transfection and ChIP assays. Intracellular calcium measurement was performed with a spectrofluorimeter using a fluorescent probe. 3D-cell-culture was used for cyst growth studies. OLE reduced the WT9-12 cell growth rate and affected intracellular signaling due to high c-AMP levels, as OLE reduced PKA levels, enhanced p-AKT, restored B-Raf-inactivation and down-regulated p-ERK. We elucidated the molecular mechanism by which OLE, via Sp1, transactivates the p21WAF1/Cip1 promoter, whose levels are down-regulated by mutated PKD1. We demonstrated that p-AKT up-regulation also played a crucial role in the OLE-induced anti-apoptotic effect and that OLE ameliorated intracellular calcium levels, the primary cause of ADPKD. Finally, using a 3D-cell-culture model we observed that OLE reduced the cyst size. Therefore, multifaceted OLE may be considered a new therapeutic approach for ADPKD treatment.

Active compounds extracted from extra virgin olive oil counteract mesothelial-to-mesenchymal transition of peritoneal mesothelium cells exposed to conventional peritoneal dialysate: in vitro and in vivo evidences.

During peritoneal dialysis (PD), peritoneal mesothelial cells undergo a transition from an epithelial phenotype to a mesenchymal phenotype that, together with the inflammatory process, promotes tissue fibrosis and a failure of peritoneal membrane function. To date, there is no definitive treatment for the progressive thickening and angiogenesis of the peritoneal membrane associated with PD. In this study we tested, in vitro and in vivo, the ability of active compounds extracted from extra virgin olive oil (AC-EVOO) to counteract the mesothelial-to-mesenchymal transition process (MMT) observed in mesothelial cells chronically exposed to the conventional peritoneal dialysate (DL). In particular, we used a cultivar from southern Italy known to have a high polyphenol content. Our results showed that, in mesothelial cells exposed to DL, the combined treatment with AC-EVOO prevented the genic and protein upregulation of key mesenchymal and inflammatory markers, as well as the MCs' migratory capacity. Concomitantly, we tested the antifibrotic efficacy of AC-EVOO in mesothelial cells obtained from effluents of patients undergoing PD, whose "fibroblast-like" phenotype was defined by flow-cytometry assay. We observed that in these cells AC-EVOO significantly mitigated, but did not reverse, the MMT process. In conclusion, our preliminary results suggest that AC-EVOO can interfere with critical factors in the process of differentiation, preventing myofibroblast formation, but once fibrosis has already progressed it is unable to promote the redifferentiation to the epithelial phenotype. Further studies are needed to establish whether AC-EVOO could represent a new therapeutic target to prevent peritoneal fibrosis.

Relationships between plasma CoQ10 levels and thyroid hormones in chronic obstructive pulmonary disease.

In previous works we demonstrated an inverse correlation between plasma Coenzyme Q 10 (CoQ10) and thyroid hormones; in fact, CoQ10 levels in hyperthyroid patients were found among the lowest detected in human diseases. On the contrary, CoQ10 is elevated in hypothyroid subjects, also in subclinical conditions, suggesting the usefulness of this index in assessing metabolic status in thyroid disorders. On the other hand, a low-T3 syndrome, due to reduced peripheral conversion from the prohormone T4, is observed in different chronic diseases: this condition is considered an adaptation mechanism, usually not to be corrected by replacement therapy. In order to perform a metabolic evaluation, we have studied a group of 15 patients, aged 69-82 ys, affected by chronic obstructive pulmonary disease (COPD), comparing respiratory indexes, thyroid hormones and CoQ10 levels (also normalized with cholesterol levels) in patients with low (group A) or normal (group B) free-T3 (FT3) concentrations. We found that CoQ10 levels were significantly higher in patients of group A than in B (0.91+/- 0.03 vs 0.7 +/- 0.04 microg/ml respectively); the same difference was observed when comparing the ratios between CoQ10/cholesterol in the two groups (200.16 +/- 8.96 vs 161.08 +/- 7.03 nmol/mmol respectively). These preliminary data seem to indicate that low T3 levels are accompanied by metabolic indexes of a true hypothyroidism in COPD patients. Whether this datum supports the need to perform a replacement therapy in such a condition requires further studies.

Postoperative Autologous Reinfusion in Total Knee Replacement.

Surgeries for total knee replacement (TKR) are increasing and in this context there is a need to develop new protocols for management and use of blood transfusion therapy. Autologous blood reduces the need for allogeneic blood transfusion and the aim of the present study was to verify the safety and the clinical efficacy. An observational retrospective study has been conducted on 124 patients, undergoing cemented total knee prosthesis replacement. Observed population was stratified into two groups: the first group received reinfusion of autologous blood collected in the postoperative surgery and the second group did not receive autologous blood reinfusion. Analysis of data shows that patients undergoing autologous blood reinfusion received less homologous blood bags (10.6% versus 30%; p = 0.08) and reduced days of hospitalization (7.88 ± 0.7 days versus 8.96 ± 2.47 days for the control group; p = 0.03). Microbiological tests were negative in all postoperatively salvaged and reinfused units. Our results emphasize the effectiveness of this procedure and have the characteristics of simplicity, low cost (€97.53 versus €103.79; p < 0.01), and easy reproducibility. Use of autologous drainage system postoperatively is a procedure that allows reducing transfusion of homologous blood bags in patients undergoing TKR.

Standardization of a method for the routine analysis of polychlorinated biphenyl congeners and selected pesticides in human serum and milk.

A methodology is presented for the routine determination of specific polychlorinated biphenyl (PCB) congeners in serum and milk samples. The procedures include standardized extraction, cleanup and quantitation by high-resolution gas chromatography (GC) and comprehensive quality assurance program to minimize systematic and erratic errors. The analyses of 68 PCB congeners and three pesticides, p,p1-dichloro diphenyl dichloro ethylene (DDE), hexachlorobenzene (HCB), and Mirex, at part-per-billion levels include the addition of surrogate congener standards (IUPAC isomers #46 and #142), extraction with hexane after protein precipitation, cleanup with Florisil, and analysis by GC with capillary column and electron capture detection. Quantitation is based on calibration standards and response factors using isomers #30 and #204 as internal standards. The quality control activities consist of analyses of samples in batches of 6 to 10 simultaneously with quality control (QC) samples. The quality assurance program checks that the procedures are under control by the use of control charts and set the criteria for data acceptability. The detection limits for the congeners and pesticides associated with the analyses of 500 serum samples and of 100 milk samples are reported. In addition, typical profiles of congener distribution in both matrices are illustrated.

High nerve growth factor blood concentration in renal transplantation: a new prognostic marker?

BACKGROUND: Nerve growth factor (NGF) belongs to the family of neurotropic proteins NGF is markedly expressed in proteinuric renal diseases and in end-stage renal disease; it might be involved in kidney physiopathology. To date, little is known about NGF concentrations in kidney transplant recipients (KTRs). Because NGF exerts its action on cell survival and differentiation, tissue repair, and inflammatory responses, it may also be implicated in the pathogenesis of chronic allograft nephropathy. The aim of this study was to determine circulating NGF concentrations in KTRs and to ascertain their use as a prognostic marker for kidney transplant outcomes. METHODS: Using enzyme-linked immunosorbent assay, we performed quantification of NGF in the serum of 40 prevalent KTRs at baseline and at 6 months. RESULTS: NGF concentrations in KTRs averaged 1.16 ± 0.67 ng/mL. They negative-linearly correlated with recipient age. Logistic multivariate regression analysis showed NGF to be independently associated with increased proteinuria over the 6-month follow-up. CONCLUSIONS: Our data demonstrated that serum concentrations of NGF in KTRs were elevated and that they could be considered to be a prognostic marker in kidney transplantation.

Relationship between plasma antioxidants and thyroid hormones in chronic obstructive pulmonary disease.

BACKGROUND: A low-T3 syndrome is observed in chronic diseases, but its treatment is still debated. Chronic obstructive pulmonary disease (COPD) has not been conclusively studied under this aspect. COPD is a complex condition, which cannot be considered a lung-related disorder, but rather a systemic disease also associated to increased oxidative stress. We evaluated thyroid hormones and antioxidant systems, the lipophilic Coenzyme Q10 (CoQ10) and total antioxidant capacity (TAC) in COPD patients to reveal the presence of a low-T3 syndrome in COPD and investigate the correlation between thyroid hormones, lung function parameters and antioxidants. METHODS: We studied: 32 COPD patients and 45 controls, evaluating thyrotropin (TSH), free-triiodotyronine (fT3), free-tetraiodotyronine (fT4), CoQ10 (also corrected for cholesterol) and TAC. CoQ10 was assayed by HPLC; TAC by the metmyoglobin-ABTS method and expressed as latency time (LAG) in radical species appearance. RESULTS: We found significantly lower LAG values, fT3 and fT4 levels and significantly higher TSH in COPD patients vs. controls. LAG values significantly correlated with fT3 concentration. 12 out of 32 patients exhibited fT3 levels lower than normal range. So we divided COPD patients in 2 groups on the basis of the fT3 concentration (normal fT3 COPD and low fT3 COPD). We observed lower LAG values in normal fT3-COPD, compared to healthy subjects, with a further significant reduction in low fT3-COPD patients. Moreover higher TSH concentration was present in normal fT3-COPD, compared to healthy subjects, with a further significant increase in low fT3-COPD patients. CoQ10/cholesterol ratio was higher in low fT3-COPD vs. normal fT3-COPD, with a nearly significant difference. CONCLUSIONS: These data seem to indicate an increased oxidative stress in low fT3-COPD and a role of fT3 in modulating antioxidant systems. However low fT3 levels are joined to metabolic indexes of true hypothyroidism, suggesting that elevated CoQ10 expresses a reduced tissue utilization. These data might suggest the need of thyroid replacement therapy in such a condition.

The effect of bis(p-chlorophenyl) acetic acid on the renal function of the rat.

The principle water-soluble metabolite of DDT in mammals has been shown to be DDA (bis(p-chlorophenyl)acetic acid). Previous studies suggested that DDA was secreted by the renal proximal tubule and was reabsorbed at an unspecified site in the nephron. Since DDA has been known to produce alterations in cellular functions, the present study examined the possibility that the renal transport of DDA was capable of causing acute nephrotoxicity. When 100 mg/kg of DDA was infused iv into the rat during a 90 min period, there was a significant decrease (congruent to 20%) in the glomerular filtration rate (GFR) after 110 min from the start of administration. During these experiments, there was no change in the mean arterial blood pressure (MABP), urine flow rate (V), renal clearance of tetraethylammonium (CTEA) or fractional reabsorption of Na (FRNa). After 200 mg/kg of DDA was infused iv into the rat during a 90 min period, there was a 60% decrease in the GFR, CTEA and V. However, the decrease in renal function was accompanied by a dramatic reduction in MABP (125 to 60 mmHg). To determine whether DDA could have produced acute renal failure when the perfusion pressure was kept constant, isolated kidney experiments were performed. In these experiments, DDA (1.0 mM) was present in a dextran perfusate and the perfusion pressure was kept constant at 90 mmHg. During these experiments, the GFR, V and FRNa were decreased significantly. The results indicated that a high perfusate concentration of DDA caused acute renal failure in the isolated kidney which was produced even when the perfusion pressure was kept constant. In conclusion, DDA produced renal failure in vivo which was associated with a reduction in renal perfusion pressure; however, perfused kidney experiments indicated that DDA could have caused a direct effect on nephron function.

Hepatic uptake and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran.

The pharmacokinetics of TCDD and related compounds is congener, dose, and species specific, with urinary and biliary excretion being dependent on the metabolism of these compounds. Isolated hepatocytes and liver slices in suspension culture and hepatic microsomes were used as in vitro models to assess the hepatic uptake and metabolism of [3H]- and [14C]TCDD and [3H]TCDF (0.01-1.0 microM) in control and induced (5 micrograms TCDD/kg, 3 days earlier) male Sprague-Dawley rats. TCDD pretreatment, with an increase in cytochromes P450 1A1 and 1A2 (CYP1A1, CYP1A2), produced an increase in the hepatic uptake of TCDD, while no increase in the hepatic uptake of TCDF was observed. The results are consistent with CYP1A2 serving as a hepatic binding protein for TCDD but not for TCDF. The rates of metabolism of TCDD and TCDF were directly proportional to their concentrations, indicating that the reaction follows first order kinetics at concentrations from 0.01 to 1.0 microM. Very limited metabolism of TCDD and TCDF was observed in control rat liver (0.45 and 3.2 pmol/hr/g hepatocyte wet wt at 0.1 microM, respectively). TCDD induced its own rate of metabolism about two- to fivefold at 1.0 microM but no induction was observed at 0.01 and 0.1 microM. In contrast, TCDD markedly induced the rate of TCDF metabolism at all substrate concentrations. While the results support the role of rat CYP1A1 in TCDF metabolism, the data suggest that CYP1A1 or CYP1A2 may not metabolize TCDD. These results also support the hypothesis that the more rapid metabolism and excretion of TCDF accounts for the relative resistance of the rat to the acute toxicity of TCDF. Comparative studies in rat and human liver microsomes found that TCDF metabolism exhibited first order kinetics in both species. Furthermore, the rate of TCDF metabolism in human liver microsomes was similar to that of control rat liver microsomes. Together the results suggest that TCDF will be far more persistent in rats, and possibly humans, following exposure at low doses which do not significantly induce cytochrome P450 1A1 and/or 1A2.

Two consecutive thunderstorm associated epidemics of asthma in the city of Melbourne. The possible role of rye grass pollen.

OBJECTIVE: To document the clinical impact and identify the meteorological and environmental circumstances surrounding two epidemics of asthma exacerbations associated with thunderstorms in the city of Melbourne and to find a possible aetiology for these events. DESIGN: Collection of meteorological and environmental data from the Victorian Bureau of Meteorology and the Environment Protection Authority; collection of clinical data from metropolitan emergency departments and the Victorian Ambulance Service; and study of a cohort of affected patients with asthma and a control group of asthmatics who were not affected by the storms. SETTING: Tertiary institution. PATIENTS: Twelve storm-affected patients with asthma and 16 asthmatics not affected by the storms. INTERVENTION: Administration of a questionnaire, medical interview, pulmonary function tests and skin prick tests with common allergens. MAIN OUTCOME AND RESULTS: Both epidemics caused a major increase in the number of hospital attendances and admissions because of asthma exacerbation (five to ten fold rise). These events could not be related to atmospheric pollution or specific meteorological features of the storms. Patients affected by the second storm were significantly more likely to suffer from hay fever (P less than 0.05), rye grass pollen allergy (P less than 0.05) and allergy to rainfall released rye grass starch granules (P less than 0.025). CONCLUSIONS: Late spring thunderstorms in the city of Melbourne can trigger epidemics of asthma attacks. The seasonal nature of the phenomenon and the pattern of allergic responses found in affected patients suggest a possible aetiological role for rye grass pollen.