Evaluation of Kisspeptin levels in prepubertal obese and overweight children: sexual dimorphism and modulation of antioxidant levels.

OBJECTIVE: Kisspeptin, neuropeptide involved in puberty beginning and regulation of pituitary-gonadal axis, has been shown to stimulate antioxidant defenses in murine models. Its levels are greater in females than males and also in obese prepubertal girls. Therefore, our aim was to evaluate sex-related differences in prepubertal obese patients and the relationships of Kisspeptin with metabolic/hormonal parameters. PATIENTS AND METHODS: We studied Kisspeptin concentrations in 54 children (22 males and 32 females, Tanner stage 1), 5-12 ys, classified according to Cole's criteria into 17 overweight and 37 obese; 25 normal-weight children, aged 6-12 years, were studied as controls. We evaluated metabolic (glucose and insulin levels after oral glucose load, total- LDL- HDL-cholesterol, triglycerides, uric acid) and hormonal (fT3, fT4, TSH, IGF-1, leptin) parameters. Moreover, total antioxidant capacity (TAC) was evaluated by spectrophotometric method, using the system H202-metmyoglobin-ABTS. Kisspeptin levels were measured by RIA. RESULTS: We did not find significant differences between obese and normal-weight children, but obese males presented significantly lower levels than females. Kisspeptin did not correlate with BMI, HOMA-IR, Insulin peak levels and TAC; a significant correlation was found between Kisspeptin and fT3 (r2=0.25; p=0.003) in the obese group; leptin levels, significantly greater in obese vs. overweight and control children, significantly correlated with TAC (r2=0.39; p=0.03). CONCLUSIONS: These data suggest that both hormones could modulate antioxidants, Kisspeptin indirectly via influence on thyroid hormones, and Leptin by a direct effect. This mechanism seems to be sex-related, not attributable to peripheral steroid levels. Further studies can clarify the complex interrelationship between central and peripheral Kisspeptin secretion and oxidative stress in children obesity.

Analysis of mammographic diagnostic errors in breast clinic.

Mammography is the gold standard for detection of early breast cancer and it is still the only diagnostic tool which shows reduction of the mortality from that. Despite that, there is a high chance of false negatives that can lead to diagnostic errors resulting in delays of treatment and worsening of prognosis. The aim of this study is to analyze the rate of false negative in mammography and assess the source of diagnostic errors. Two radiologists have retrospectively evaluated 500 mammograms performed between January 2008 and December 2011 in Breast Imaging Clinic. 250 patients (Group A) had been operated for breast cancer and 250 patients (Group B) were healthy woman submitted to mammography according to the guideline for early detection of breast cancer. In Group A, 138 patients (55.2 %) were true missed cancer, 61 had minimal sign (24.4 %) and 53 were false negative (FN) (20.4 %). The source of errors amongst the FN were in 42 % of cases due to perception, in 15 % to interpretation, in 10 % to subtle/unusual lesion characteristics, in 9 % error for satisfaction of search, in 7 % to inherent limitations of mammography, in 4 % to poor technique and 13 % for inadequate clinical management. The diagnostic errors in breast clinic services are not negligible. The largest number of FN results from perception errors, misinterpretation and inadequate clinical management. These can be related to factors such as inattention, fatigue or lack of experience. To reduce it, it is necessary to have a dedicated multidisciplinary staff and adequate equipment and workloads.

A real-time chirp-coded imaging system with tissue attenuation compensation.

In ultrasound imaging, pulse compression methods based on the transmission (TX) of long coded pulses and matched receive filtering can be used to improve the penetration depth while preserving the axial resolution (coded-imaging). The performance of most of these methods is affected by the frequency dependent attenuation of tissue, which causes mismatch of the receiver filter. This, together with the involved additional computational load, has probably so far limited the implementation of pulse compression methods in real-time imaging systems. In this paper, a real-time low-computational-cost coded-imaging system operating on the beamformed and demodulated data received by a linear array probe is presented. The system has been implemented by extending the firmware and the software of the ULA-OP research platform. In particular, pulse compression is performed by exploiting the computational resources of a single digital signal processor. Each image line is produced in less than 20 µs, so that, e.g., 192-line frames can be generated at up to 200 fps. Although the system may work with a large class of codes, this paper has been focused on the test of linear frequency modulated chirps. The new system has been used to experimentally investigate the effects of tissue attenuation so that the design of the receive compression filter can be accordingly guided. Tests made with different chirp signals confirm that, although the attainable compression gain in attenuating media is lower than the theoretical value expected for a given TX Time-Bandwidth product (BT), good SNR gains can be obtained. For example, by using a chirp signal having BT=19, a 13 dB compression gain has been measured. By adapting the frequency band of the receiver to the band of the received echo, the signal-to-noise ratio and the penetration depth have been further increased, as shown by real-time tests conducted on phantoms and in vivo. In particular, a 2.7 dB SNR increase has been measured through a novel attenuation compensation scheme, which only requires to shift the demodulation frequency by 1 MHz. The proposed method characterizes for its simplicity and easy implementation.

Prognostic role of glutathione S-transferase polymorphisms in acute myeloid leukemia.

Glutathione S-transferases (GSTs) are phase II detoxification enzymes involved in the metabolism of carcinogens and anticancer drugs, known also to interact with kinase complexes during oxidative or chemical stress-induced apoptosis. We were interested whether their polymorphic variants may account for differences in outcome of patients with acute myeloid leukemia (AML) following chemotherapy. We studied the prognostic role of polymorphisms in three GST genes (GSTP1/M1/T1) in a large patient cohort of the German Austrian Acute Myeloid Leukemia Study Group, treated according to prospective multicenter clinical trials (AML HD98A: 254 patients; AML HD98-B: 100 patients), with a median follow-up of 46 months. Looking at short-term adverse drug reactions, homozygous carriers of the GSTP1*105 Val allele had a faster neutrophil and platelet recovery (P=0.002 and 0.02, respectively) and a reduced need of red cell and platelet transfusions (P=0.01 and 0.03, respectively). Response to induction chemotherapy did not vary according to GST polymorphisms. Multivariable Cox regression models revealed a significant better relapse-free (RFS) and overall survival for the GSTP1(*)105 Val (P=0.003 and 0.03, respectively), whereas GSTT1 and GSTM1 genotypes had no significant impact. The favorable impact of GSTP1(*)105 Val on RFS seems to be restricted to the subgroup of patients exhibiting a normal karyotype.

Increased risk of acute myeloid leukaemia due to polymorphisms in detoxification and DNA repair enzymes.

BACKGROUND: Polymorphisms in genes involved in detoxification and DNA-repair pathways may modify the individual's risk for genomic damage, and, as a consequence, the risk of developing malignant diseases. PATIENTS AND METHODS: We performed a case-control study including 160 cases of acute myeloid leukaemia (AML) and 162 matched controls to test the impact of six genomic polymorphisms on the risk to develop AML and/or therapy-related AML. RESULTS: We found a significantly higher prevalence of the polymorphic variants RAD51-G135C and CYP3A4-A-290G genes in AML cases, when compared with controls (P = 0.02 and P = 0.04), increasing the risk of AML 2.1-folds (95% CI: 1.1-4.0) and 3.2-fold (95% CI: 1.1-11.5), respectively. Carriers of both the RAD51-G135C and CYP3A4-A-290G variants were at highest AML risk (P = 0.003; OR:13,6; 95% CI: 2.0-585.5), suggesting a synergistic effect between these polymorphisms. CONCLUSIONS: These results suggest that polymorphic variants in DNA-repair and detoxification enzymes may co-operate in modulating the individual's risk of AML.

Polymorphism in cytokine genes as prognostic markers in Hodgkin's lymphoma.

BACKGROUND: In Hodgkin's lymphoma (HL), the production of cytokines by Reed-Sternberg cells and the surrounding tissue is thought to contribute to the biology of the disease. Cytokine expression can be altered by common single nucleotide polymorphisms (SNPs) in the 5'-promoter regions. PATIENTS AND METHODS: We studied polymorphic allele variants of the cytokine genes interleukin (IL)-10 (T-3575A, G-2849A, C-2763A, A-1082G and C-592A), IL-6 (G-174C) and tumor necrosis factor-alpha (C-863A and G-308A) in 184 patients with HL, and analyzed for associations with treatment outcome. RESULTS: Carriers of the IL-10-592AA and the IL-6-174GG genotypes had a significantly lower probability of freedom from treatment failure (FFTF) with adjusted hazard ratios (HRs) for failure of 2.92 [95% CI (confidence interval) 1.58-5.41, P = 0.001] and of 1.75 (95% CI 1.04-2.92, P = 0.03), respectively. Reconstructing haplotypes from the five SNPs in the IL-10 promoter revealed that homozygous carriers of the IL-10.4 haplotype (T-G-C-A-A) had a worse FFTF (HR, 2.35; 95% CI 1.2-4.6, P = 0.01). In the Cox multivariate analysis, the IL-10-592AA, the IL-6-174GG genotypes and stage were independent prognostic factors. CONCLUSIONS: Our study indicates that cytokine genotypes predict clinical outcome in patients with HL and points to the importance of the genetic background of the host for treatment response.

Reduced BRCA1 expression due to promoter hypermethylation in therapy-related acute myeloid leukaemia.

BRCA1 plays a pivotal role in the repair of DNA damage, especially following chemotherapy and ionising radiation. We were interested in the regulation of BRCA1 expression in acute myeloid leukaemia (AML), in particular in therapy-related forms (t-AML). Using real-time PCR and Western blot, we found that BRCA1 mRNA was expressed at barely detectable levels by normal peripheral blood granulocytes, monocytes and lymphocytes, whereas control BM-mononuclear cells and selected CD34+ progenitor cells displayed significantly higher BRCA1 expression (P=0.0003). Acute myeloid leukaemia samples showed heterogeneous BRCA1 mRNA levels, which were lower than those of normal bone marrows (P=0.0001). We found a high frequency of hypermethylation of the BRCA1 promoter region in AML (51/133 samples, 38%), in particular in patients with karyotypic aberrations (P=0.026), and in t-AML, as compared to de novo AML (76 vs 31%, P=0.0002). Examining eight primary tumour samples from hypermethylated t-AML patients, BRCA1 was hypermethylated in three of four breast cancer samples, whereas it was unmethylated in the other four tumours. BRCA1 hypermethylation correlated to reduced BRCA1 mRNA (P=0.0004), and to increased DNA methyltransferase DNMT3A (P=0.003) expression. Our data show that reduced BRCA1 expression owing to promoter hypermethylation is frequent in t-AML and that this could contribute to secondary leukaemogenesis.

Effect of vessel curvature on Doppler derived velocity profiles and fluid flow.

Side-branches and curvatures in the arterial tree yield deviations from the axial oriented velocity. Velocity or volume flow estimates based on the assumption that flow is axially oriented are of limited value at these sites. This article evaluates information obtainable by using a multigate Doppler ultrasound (US) instrument used with curved phantoms, which resemble the human coronary arteries. The comparison of experimental velocity data with data provided by an accurate computational fluid dynamics (CFD) method shows differences in the range of 4 to 11% for four curvatures with different radii. Multigate data are also used to estimate the volume flow in the curved segments at different experimental conditions. An error lower than 15% is obtained, to be compared with a 24% error obtained by assuming a parabolic velocity profile. In particular, it is shown that the residual error is not related to the small deviation of the velocity vectors from the axial direction due to the presence of secondary velocity components, which are found to be of magnitude less than 10% with respect to the axial velocity component.

A novel ultrasound instrument for investigation of arterial mechanics.

The study of arterial mechanics concerns functional characteristics depending on wall elasticity and flow profile. Wall elasticity can be investigated through the estimation of parameters like the arterial distensibility, which is of high clinical interest because of its known correlation not only with the advanced atherosclerotic disease, but also with aging and major risk factors for cardiovascular disease. The flow velocity profile is also clinically relevant, because it modulates endothelial function and can be responsible for the development and distribution of atherosclerotic plaques. A clinically relevant variable extracted from the blood velocity profile is the wall shear rate (WSR), which represents the spatial velocity gradient near the vessel wall. This paper describes an integrated ultrasound system, capable of detecting both the velocity profile and the wall movements in human arteries. It basically consists of a PC add-on board including a single high-speed digital signal processor. This is dedicated to the analysis of echo-signals backscattered from 128 range cells located along the axis of the interrogating ultrasound (US) beam. Echoes generated from the walls (characterized by high amplitudes and low Doppler frequencies) and from red blood cells (characterized by low amplitudes and relatively high Doppler frequencies) are independently processed in real-time. Wall velocity is detected through the autocorrelation algorithm, while blood velocity is investigated through a complete spectral analysis of all signals backscattered by erythrocytes and WSR is extracted from the estimated velocity profile. Preliminary applications of the new system, including the simultaneous analysis of blood flow and arterial wall movement in healthy volunteers and in a diseased patient, are discussed, and first results are presented.

Transforming growth factor-beta1 transcriptionally activates CD34 and prevents induced differentiation of TF-1 cells in the absence of any cell-cycle effects.

A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among these is transforming growth factor beta1 (TGF-beta1), which regulates cell cycle and differentiation of hematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has been previously shown by us and other authors that TGF-beta1 maintains human CD34(+) hematopoietic progenitors in an undifferentiated state, independently of any cell cycle effects, and that depletion of TGF-beta1 triggers differentiation accompanied by a decrease in CD34 antigen expression. In the present work, we show that exogenous TGF-beta1 upregulates the human CD34 antigen in the CD34(+) cell lines TF-1 and KG-1a, but not in the more differentiated CD34(-) cell lines HL-60 and K-562. We further studied this effect in the pluripotent erythroleukemia cell line TF-1. Here, TGF-beta1 did not effect cell growth, but induced transcriptional activation of full-length CD34 and prevented differentiation induced by differentiating agents. This effect was associated with nuclear translocation of Smad-2, activation of TAK-1, and with a dramatic decrease in p38 phosphorylation. In other systems TGF-beta1 has been shown to activate a TGF-beta-activated kinase 1 (TAK1), which in turn, activates p38. The specific inhibitor of p38 phosphorylation, SB202190, also increased CD34 RNA expression, indicating the existence of a link between p-38 inhibition by TGF-beta1 and CD34 overexpression. Our data demonstrate that TGF-beta1 transcriptionally activates CD34 and prevents differentiation of TF-1 cells by acting independently through the Smad, TAK1 and p38 pathways, and thus provide important clues for the understanding of hematopoietic development and a potential tool to modify response of hematopoietic cells to mitogens or differentiating agents.

A simplified approach for real-time detection of arterial wall velocity and distension.

Arterial stiffness is known to increase with age and with many vascular diseases, but its noninvasive assessment in patients still represents a difficult task. The measurement of diameter change during the cardiac cycle (distension) has been proposed as a means to estimate arterial compliance and stiffness. Therefore, we have developed a simple PC-based device and algorithm for noninvasive quantification of vessel wall motion and diameter change in humans. This goal is achieved in real-time by processing the base-band signals from a commercial ultrasound Doppler system. Real-time operation is of crucial importance, because it allows a rapid achievement of optimal measurement conditions. The system was evaluated in a laboratory using a string phantom and was tested on the carotid arteries of 10 volunteers. Wall velocities from 0.05 to 600 mm/s and displacements lower than 2 microns were detected with phantoms. The measured carotid diameter change in the volunteers ranged from 7.5 to 11.8% (mean = 9.8%) and agrees closely with values reported in the literature. The difference between values taken one hour apart ranged from 0.2 to 0.5%. We conclude that the new system provides rapid, accurate, and repeatable measurements of vessel distension in humans.

Adoptive immunotherapy by avidin-driven cytotoxic T lymphocyte-tumor bridging.

We have shown previously that T cells, tagged with biotinylated anti-CD3 antibody fragments, can exert avidin-dependent cytolytic activity on suitably biotinylated tumor cells in vitro. In this study, we demonstrate that avidin-driven CTL-tumor bridging in vivo leads to growth inhibition of murine tumors WEHI-164 fibrosarcoma and RMA lymphoma. The biodistribution of biotin-tagged 111In-labeled T cells demonstrated a selective avidin-dependent and time-dependent accumulation of radioactivity at tumor sites. The specificity of lymphocyte tumor localization was demonstrated by the concurrent time-dependent decrease of radioactivity in the blood and in all other organs. Furthermore, we documented a therapeutic effect of the adoptively transferred T cells, i.e., a significant delay of tumor growth at early stages. All of the experiments included a control group of mice, which received all of the reagents, except avidin. These avidin-minus mice showed no specific localization and no delay in tumor growth, indicating that avidin bridging was essential for T-cell activity at tumor sites.

The N-terminal portion of the main cytosolic loop mediates K+ sensitivity in the retinal rod Na+/Ca2+-K+-exchanger.

Two types of Na+/Ca2+-exchangers have been characterized in the literature: The first is the cardiac, skeletal muscle and brain type, which exchanges 1 Ca2+ for 3 Na+, the second, found in retinal photosensor cells, transports 1 Ca2+ and 1 K+ in exchange for 4 Na+. The present work describes the properties of chimeric constructs of the two exchanger types. Ca2+ gel overlay experiments have identified a high affinity (Kd in the 1 microM range) Ca2+-binding domain between Glu601 and Asp733 in the main cytosolic loop of the retinal protein, just after transmembrane domain 5. Insertion of the retinal Ca2+-binding domain in the cytosolic loop of the cardiac exchanger conferred K+-dependence to the Ca2+ uptake activity of the chimeric constructs expressed in HeLa cells. The apparent Km of the K+ effect was about 1 mM. Experiments with C-terminally truncated versions of the retinal insert indicated that the sequence between Leu643 and Asp733 was critical in mediating K+ sensitivity of the recombinant chimeras. Thus, the high affinity Ca2+-binding domain in the main cytosolic loop of the retinal exchanger may regulate the activity of the retinal protein by binding Ca2+, and by conferring to it K+ sensitivity.

Hematopoietic reconstitution after lethal irradiation and bone marrow transplantation: effects of different hematopoietic cytokines on the recovery of thymus, spleen and blood cells.

Lethally irradiated mice were grafted with syngeneic bone marrow cells or left ungrafted. Mice of each group were injected with different hematopoietic cytokines for 5 consecutive days starting immediately after irradiation or left uninjected. The recovery of lymphoid tissues induced by hematopoietic cytokines 7 days after irradiation and bone marrow cell transplantation was comparable to that observed at days 21-28 in irradiated, bone marrow-grafted, but cytokine-uninjected mice. IL-11 or IL-6, in combination with IL-3, was able to hasten thymus, spleen and blood cell numbers and functions. SCF also displayed a detectable effect when used with IL-3. Conversely, the IL-6 superagonist K-7/D-6 was able, when injected alone, to induce significant recovery of thymus, spleen and blood cells. Thus, K-7/D-6 appears to be a most efficient cytokine for fast reconstitution of lymphoid tissues after irradiation and bone marrow transplantation.

Modulation of X-ray-induced damage recognition and repair in ageing human peripheral blood mononuclear cells by an interleukin-6-type cytokine.

We have investigated the effects of an interleukin (IL)-6-type cytokine on the DNA-binding activity of ku and on unscheduled DNA repair in X-ray-treated peripheral blood mononuclear cells (PBMC) from human subjects of different ages. The cytokine used, called K-7/D-6, is an IL-6 variant with increased in vivo and in vitro biological activity compared to the wild type molecule. Ku is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK). It binds the ends of various types of DNA discontinuity and is involved in the repair of DNA breaks caused by V(D)J recombination, isotype switching, physiological oxidation reactions, ionizing radiation and some chemotherapeutic drugs. The ku-dependent repair process, called non-homologous end joining, is the main DNA double strand break repair mechanism in irradiated mammalian cells. Results show that K-7/D-6 significantly increases DNA-binding activity of ku in irradiated PBMC from young but not from elderly subjects. However, K-7/D-6 is able to induce unscheduled DNA repair in irradiated PBMC from both young and elderly subjects. These effects of K-7/D-6 are relevant to the mechanisms of the cellular response to DNA damage.

Haemopoietic reconstitution after sublethal irradiation: comparison of the effects of different haemopoietic cytokines on murine lymphocytes and bone marrow cells.

Sublethally irradiated mice were injected with recombinant cytokines to stimulate haemopoietic reconstitution. Interleukin (IL)-11 and IL-6 were able to significantly accelerate the recovery of thymus, spleen and bone marrow cells when used in combination with IL-3, but not alone. Stem cell factor (SCF) also displayed detectable effects when used with IL-3. Conversely, the IL-6 superagonist K-7/D-6 was able, even when used alone, to induce recovery of thymus, spleen and bone marrow cells up to the level of unirradiated controls. Together, these results indicate that it is possible to attain complete recovery of lymphoid organs and tissues as early as 7 d after irradiation by use of haemopoietic cytokines.

Activation of gp130 signaling in vivo by the IL-6 super-agonist K-7/D-6 accelerates repopulation of lymphoid organs after irradiation.

Stimulation of the gp130 signaling pathway by IL-6 is known to contribute significantly to hematopoietic expansion in vitro, mostly in combination with other cytokines. In the present study we have investigated whether a similar effect can be observed also in vivo using short-term assays in which irradiated mice were analyzed for repopulation of lymphoid organs. Mice were injected with a combination of soluble IL-6Ralpha either with wild-type (wt) human IL-6 or with an IL-6 variant, called K-7/D-6, that shows a 70-fold higher IL-6Ralpha affinity. We observed that while wt IL-6 was able to induce a partial effect only in combination with IL-3, K-7/D-6 bypassed the need for IL-3 and yielded complete recovery. In lethally irradiated mice reconstituted with syngeneic bone marrow cells K-7/D-6 strongly accelerated the repopulation of thymus and spleen and hastened blood neutrophil recovery. These results underscore the potential of the gp130 signaling pathway in hematopoietic reconstitution after myeloablative regimens and open the possibility to fully exploit it with a super-active IL-6 variant.

Detection of vascular haemodynamics through a high-speed velocity profiler.

OBJECTIVE: This paper aims at demonstrating that ultrasound Doppler multigate spectral analysis performed with advanced equipment may provide detailed and significant haemodynamic information. METHODS: A novel multigate system was recently introduced and shown capable of performing real-time spectral analysis of Doppler data from 64 resolution cells located at different depths from the transducer. The system extends the typical capabilities of conventional Pulsed Wave (PW) equipment by displaying the full spectral content of Doppler signals over an ultrasound scan line rather than in a single resolution cell. In cases where it is appropriate to display the available information in a simpler form, parameters such as the maximum frequency can be extracted from each spectrum, by using conventional or advanced image processing methods. RESULTS: In-vitro experiments show that the multigate system can perform velocity measurements with good accuracy and precision. Examples of in vivo profiles detected from carotid, femoral and radial arteries are presented. In particular, the first results obtained from the aorta are shown. CONCLUSIONS: Blood flow behavior can be accurately investigated using a real-time multigate system which extends Doppler spectral analysis to a whole scan line.