Estrogen regulates cytokine production and apoptosis in PMA-differentiated,macrophage-like U937 cells

We have investigated the effects of sex steroids, estradiol (E2), and testosterone (T) on the synthesis of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) in phorbol-myristate-acetate (PMA)-differentiated human monoblastic U937 cells. The ability of both hormones to modulate the viability and programmed cell death of macrophage-like PMA-differentiated U937 cells was also inspected. E2 increased TNF-alpha synthesis, whereas T had no effect on the production of this cytokine. The combination of E2 and its antagonist tamoxifen or ICI-182,789 completely abolished the induction of TNF-alpha, while combination of T and its antagonist Casodex (CSDX) did not significantly affect TNF-alpha production by U937 cells. Exposure of cells to E2 resulted in a dose-dependent decrease of IL-10 synthesis, while again T did not show any detectable effect. In addition, E2 induced a significant increase of apoptosis in macrophage-like U937 cells and this increase was inhibited by the simultaneous addition of either tamoxifen or ICI-182. In contrast, T alone or in combination with CSDX did not modify apoptotic rates of U937 cells. This evidence, taken together, suggests that estrogens, but not androgens, exert a pro-inflammatory action through the modulation of TNF-alpha and IL-10, and regulate the immune effector cells by the induction of programmed cell death.     

Mtp-40 and alpha antigen gene fragment amplification for the detection of Mycobacterium tuberculosis in Colombian clinical specimens

In this study, the use of Mtp-40 and alpha antigen polymerase chain reaction (PCR) amplification fragments for the precise tuberculosis (TB) diagnosis was evaluated. One hundred and ninety two different samples were obtained from 113 patients with suspected TB. Mtp-40 and alpha antigen protein genes were amplified by the PCR technique and compared to both the "gold standard" (culture) test, as well as the clinical parameters (including a clinical record and X-ray film exam in 113 patients). Thirty-eight of the 113 patients had a presumptive clinical diagnosis of TB; 74% being detected by PCR technique, 58% by culture and 44% by direct microscopic visualization. Weconclude that it is possible to use PCR as a suitable technique for the detection of any mycobacteria by means of the alpha antigen product, or the specific infection of Mycobacterium tuberculosis by means of the mtp-40 gene. This might be a good supporting tool in difficult clinical TB diagnosis and pauci-bacillary cases.     

Characterization of nitroproteome in neuron-like PC12 cells differentiated with nerve growth factor: identification of two nitration sites in alpha-tubulin

Nitric oxide (NO) is a precursor of reactive nitrating species, peroxynitrite and nitrogen dioxide, which modify proteins to generate oxidized species such as 3-nitrotyrosine that has been used as a hallmark of peroxynitrite-mediated oxidative stress on proteins. In the last few years however, a growing body of evidence indicates that NO also regulates a myriad of physiologic responses by modifying tyrosine residues. Looking for the molecular event triggered by NO in nerve growth factor (NGF)-induced neuronal differentiation, we recently reported that in differentiating PC12 cells, the cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins, and alpha-tubulin is the major target. In the present work, we focus on the investigation of the sites of tyrosine nitration in alpha-tubulin purified by two-dimensional gel electrophoresis following anti-alpha-tubulin immunoprecipitation of protein extract from NGF-treated PC12 cells. Using Western blotting and matrix-assisted laser desorption/ionization-time of flight analysis, we show for the first time, both in vivo and in vitro, that nitration can occur on alpha-tubulin at sites other than the C-terminus and we positively identify Tyr 161 and Tyr 357 as two specific amino acids endogenously nitrated.     

Legionella Contamination in Hot Water of Italian Hotels

A cross-sectional multicenter survey of Italian hotels was conducted to investigate Legionella spp. contamination of hot water. Chemical parameters (hardness, free chlorine concentration, and trace element concentrations), water systems, and building characteristics were evaluated to study risk factors for colonization. The hot water systems of Italian hotels were strongly colonized by Legionella; 75% of the buildings examined and 60% of the water samples were contaminated, mainly at levels of ≥10³ CFU liter⁻¹, and Legionella pneumophila was the most frequently isolated species (87%). L. pneumophila serogroup 1 was isolated from 45.8% of the contaminated sites and from 32.5% of the hotels examined. When a multivariate logistic model was used, only hotel age was associated with contamination, but the risk factors differed depending on the contaminating species and serogroup. Soft water with higher chlorine levels and higher temperatures were associated with L.pneumophila serogroup 1 colonization, whereas the opposite was observed for serogroups 2 to 14. In conclusion, Italian hotels, particularly those located in old buildings, represent a major source of risk for Legionnaires' disease due to the high frequency of Legionella contamination, high germ concentration, and major L. pneumophila serogroup 1 colonization. The possible role of chlorine in favoring the survival of Legionella species is discussed.     

Turner syndrome, insulin sensitivity and growth hormone treatment

Mild insulin resistance appears to be an early metabolic defect in girls with Turner syndrome (TS). Impaired glucose tolerance has been reported in 10-34% of patients with TS, and type 2 diabetes mellitus is 2-4 times more common and occurs at a younger age in girls with TS than in the general population. In a mixed longitudinal and cross-sectional study, we analysed carbohydrate tolerance and insulin sensitivity in 46 children and adolescents with TS who reached their final height after long-term treatment (mean 6.3 +/- 2.5 years) with growth hormone (GH: 0.33 mg/kg/week [0.05 mg/kg/day]), and in 36 of these patients who were followed-up after the cessation of GH therapy (mean follow-up, 2.6 +/- 2.5 years; range, 1-9.5 years). Patients with TS were compared with an age-matched female control group. Insulin sensitivity appeared to be lower in patients with TS than in controls, even before the start of GH therapy. As in controls, insulin sensitivity decreased with age in patients with TS, and levels were lower in those aged >12 years than in those aged <12 years. GH therapy resulted in good catch-up growth in patients with TS, with final height significantly higher than projected height evaluated before the initiation of GH therapy. Insulin sensitivity increased after 7-8 years of therapy and, on the cessation of GH therapy, returned to pre-treatment levels. The increase in insulin sensitivity seen on the cessation of GH therapy appeared to be influenced negatively by body mass index and triglyceride levels, and correlated positively with the number of years since cessation of GH therapy. As in the general population, excess weight and an abnormal lipid profile, in particular excess triglyceride levels, worsened insulin sensitivity. In conclusion, our study confirms that GH therapy reduces insulin sensitivity, but at its cessation there is a return to pre-therapy values. We therefore report a progressive improvement in carbohydrate tolerance and insulin function in patients with TS, despite an increase in age.     

JNK/p53 mediated cell death response in K562 exposed to etoposide-ionizing radiation combined treatment

The study of the ability of chemotherapeutic agents and/or ionizing radiation (IR) to induce cell death in tumor cells is essential for setting up new and more efficient therapies against human cancer. Since drug and ionizing radiation resistance is an impediment to successful chemotherapy against cancer, we wanted to check if etoposide/ionizing radiation combined treatment could have a synergic effect to improve cell death in K562, a well-known human erythroleukemia ionizing radiation resistant cell line. In this study, we examined the role played by JNK/SAPK, p53, and mitochondrial pathways in cell death response of K562 cells to etoposide and IR treatment. Our results let us suppose that the induction of cell death, already evident in 15 Gy exposed cells, mainly in 15 Gy plus etoposide, may be mediated by JNK/SAPK pathway. Moreover, p53 is a potential substrate for JNK and may act as a JNK target for etoposide and ionizing radiation. Thus further investigation on these and other molecular mechanisms underlying the cell death response following etoposide and ionizing radiation exposure could be useful to overcome resistance mechanisms in tumor cells.