Stochastic epigenetic mutations as possible explanation for phenotypical discordance among twins with congenital hypothyroidism.

PURPOSE: The elevated frequency of discordance for congenital hypothyroidism (CH) phenotype between monozygotic twins suggests the involvement of non-mendelian mechanisms. The aim of the study was to investigate the role of epigenetics in CH pathogenesis. METHODS: A genome-wide DNA methylation analysis was performed on the peripheral blood of 23 twin pairs (10 monozygotic and 13 dizygotic), 4 concordant and 19 discordant pairs for CH at birth. RESULTS: Differential methylation analysis did not show significant differences in methylation levels between CH cases and controls, but a different methylation status of several genes may explain the CH discordance of a monozygotic twin couple carrying a monoallelic nonsense mutation of DUOX2. In addition, the median number of hypo-methylated Stochastic Epigenetic Mutations (SEMs) resulted significantly increased in cases compared to controls. The prioritization analysis for CH performed on the genes epimutated exclusively in the cases identified SLC26A4, FOXI1, NKX2-5 and TSHB as the genes with the highest score. The analysis of significantly SEMs-enriched regions led to the identification of two genes (FAM50B and MEG8) that resulted epigenetically dysregulated in cases. CONCLUSION: Epigenetic modifications may potentially account for CH pathogenesis and explain discordance among monozygotic twins.

Oxidative stress in myelin sheath: The other face of the extramitochondrial oxidative phosphorylation ability.

Oxidative phosphorylation (OXPHOS) is not only the main source of ATP for the cell, but also a major source of reactive oxygen species (ROS), which lead to oxidative stress. At present, mitochondria are considered the organelles responsible for the OXPHOS, but in the last years we have demonstrated that it can also occur outside the mitochondrion. Myelin sheath is able to conduct an aerobic metabolism, producing ATP that we have hypothesized is transferred to the axon, to support its energetic demand. In this work, spectrophotometric, cytofluorimetric, and luminometric analyses were employed to investigate the oxidative stress production in isolated myelin, as far as its respiratory activity is concerned. We have evaluated the levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), markers of lipid peroxidation, as well as of hydrogen peroxide (H2O2), marker of ROS production. To assess the presence of endogenous antioxidant systems, superoxide dismutase, catalase, and glutathione peroxidase activities were assayed. The effect of certain uncoupling or antioxidant molecules on oxidative stress in myelin was also investigated. We report that isolated myelin produces high levels of MDA, 4-HNE, and H2O2, likely through the pathway composed by Complex I-III-IV, but it also contains active superoxide dismutase, catalase, and glutathione peroxidase, as antioxidant defense. Uncoupling compounds or Complex I inhibitors increase oxidative stress, while antioxidant compounds limit ROS generation. Data may shed new light on the role of myelin sheath in physiology and pathology. In particular, it can be presumed that the axonal degeneration associated with myelin loss in demyelinating diseases is related to oxidative stress caused by impaired OXPHOS.

Whole-lung volume and density in spirometrically-gated inspiratory and expiratory CT in systemic sclerosis: correlation with static volumes at pulmonary function tests.

BACKGROUND: Spiral low-dose computed tomography (LDCT) permits to measure whole-lung volume and density in a single breath-hold. OBJECTIVE: To evaluate the agreement between static lung volumes measured with LDCT and pulmonary function test (PFT) and the correlation between the LDCT volumes and lung density in restrictive lung disease. DESIGN: Patients with Systemic Sclerosis (SSc) with (n = 24) and without (n = 16) pulmonary involvement on sequential thin-section CT and patients with chronic obstructive pulmonary disease (COPD)(n = 29) underwent spirometrically-gated LDCT at 90% and 10% of vital capacity to measure inspiratory and expiratory lung volumes and mean lung attenuation (MLA). Total lung capacity and residual volume were measured the same day of CT. RESULTS: Inspiratory [95% limits of agreement (95% LoA)--43.8% and 39.2%] and expiratory (95% LoA -45.8% and 37.1%) lung volumes measured on LDCT and PFT showed poor agreement in SSc patients with pulmonary involvement, whereas they were in substantial agreement (inspiratory 95% LoA -14.1% and 16.1%; expiratory 95% LoA -13.5% and 23%) in SSc patients without pulmonary involvement and in inspiratory scans only (95% LoA -23.1% and 20.9%) of COPD patients. Inspiratory and expiratory LDCT volumes, MLA and their deltas differentiated both SSc patients with or without pulmonary involvement from COPD patients. LDCT lung volumes and density were not correlated in SSc patients with pulmonary involvement, whereas they did correlate in SSc without pulmonary involvement and in COPD patients. CONCLUSIONS: In restrictive lung disease due to SSc there is poor agreement between static lung volumes measured using LDCT and PFT and the relationship between volume and density values on CT is altered.

Effects of experimental occlusal interference on body posture: an optoelectronic stereophotogrammetric analysis.

In recent years, there has been increasing interest in the relationship between dental occlusion and body posture both among people and in scientific literature. The aim of the present longitudinal study is to investigate the effects of an experimental occlusal interference on body posture by means of a force platform and an optoelectronic stereophotogrammetric analysis. An occlusal interference of a 0- to 2-mm-thick glass composite was prepared to disturb the intercuspal position while not creating interference during lateral or protrusive mandibular excursions. Frontal and sagittal kinematic parameters, dynamic gait measurements and superficial electromyographic (SEMG) activity of head and neck muscles were performed on 12 healthy subjects. Measurements were taken 10 days before the application of the occlusal interference, and then immediately before the application, the day after it, and at a distance of 7 and 14 days under four different exteroceptive conditions. The outcomes of this study show that an occlusal interference does not modify significantly over time static and dynamic parameters of body posture under different exteroceptive conditions. It has a minimal influence only on the frontal kinematic parameters related to mandibular position, and it induces a transient increase of the activity of masticatory muscles. In this study, the experimental occlusal interference did not significantly influence the body posture during a 14-day follow-up period.

[Societal cost of pre-lingual deafness]. / Costi sociali della sordità pre-linguale.

Congenital hearing loss still remain an important medical and social problem for the delayed language development. Object of this study is to provide an updated and close estimate of the economic burden involved in pre-lingual hearing loss. Data were provided by the Ministry of Health data bank, the Ministry of Education national data bank, the National Institute of Social Insurance national data bank and the Italian Central Statistics Institute. The information was collected by means of a specially provided Societal Cost Questionnaire (SCQ). Direct medical costs, direct non-medical costs and indirect welfare costs involved in deafness were included in the cost estimate. Was enrolled in the study a sample of subjects with pre-lingual deafness, with a mean bilateral neuro-sensorial hearing impairment equal to 60 dB or more for 500, 1,000 and 2,000 Hz frequency tones in the better ear detected in neonatal age, had prevented speech from developing. The statistical assessment was performed according to an actuarial approach, considering the estimated life expectancy at birth, based on updated population data from census 2001. Based on life expectancy, the lifetime mean cost assessed for a subject affected by profound pre-lingual deafness turned out to be equal to Euro 737,994.76 for a male and Euro 755,404.02 for a female. Unlike other disabling affections, deafness weighs significantly more on the social system than on the health system. As a matter of fact, the direct medical costs, such as audiological diagnosis, hearing aids, etc., only account for 3.8% of the societal cost, whereas education, rehabilitation and welfare costs reach 96.2% of the total. Finally, our results suggest that societal costs can only be reduced by zeroing in on promotion and broadening of effective prevention strategies. The appropriate public health measures (such as the universal newborn hearing screening) set up and implemented in several European and non-European countries proved effective and reduced the handicap degree resulting from deafness. The investment in prevention will be widely paid.

Screening of lung cancer with low dose spiral CT: results of a three year pilot study and design of the randomised controlled trial ''Italung-CT''.

PURPOSE: To report the results of a three-year observational pilot study of lung cancer screening with low dose computed tomography (CT) and to present the study design of a randomised clinical trial named as ''Italung-CT''. MATERIALS AND METHODS: Sixty (47 males and 13 females, mean age 64+/-4.5 years) heavy smokers (at least 20 packs-year) underwent three low-dose spiral CT screening tests one year apart on a single slice or multislice CT scanner. Indeterminate nodules were managed according to the recommendations of the Early Lung Cancer Action Project. RESULTS: and Indeterminate nodules were observed in 33 (55%) of the subjects (60% at the baseline screening test, 24% at the first annual test and 16% at the second annual test). The size of the largest indeterminate nodule was <5 mm in diameter in 20 subjects, 10 of whom showed the nodule at the baseline test. Forty-five subjects (75%) completed the first annual test and 42 (70%) the second annual test. One (1.6%) prevalent lung cancer (adenosquamous carcinoma) and one (2.2%) incident lung cancer (small cell cancer at the first annual examination) were observed, as well as a pulmonary localisation of Hodgkin's lymphoma (at the second annual test). In addition, one subject underwent lung surgery for a chondromatous hamartoma. CONCLUSIONS: The results of the pilot study are substantially in line with those of other observational studies of greater sample size. This justifies optimism about the reliability of the results in the screened arm of the ''Italung CT'' trial which has just begun.

[High resolution and spirometric synchronization computerized tomography in chronic obstructive bronchopneumopathy]. / Tomografia Computerizzata con alta risoluzione e sincronizzazione spirometrica nella broncopneumopatia cronica ostruttiva.

PURPOSE: To assess the feasibility of spirometrically gated HRCT in patients with chronic obstructive pulmonary disease and to correlate the density measures with the results of pulmonary function tests. MATERIAL AND METHODS: Pulmonary function tests and spirometrically gated HRCT were performed, on the same day, in 20 patients with chronic obstructive pulmonary disease. Spirometrically gated HRCT scans at 90 and 10% of the patient's vital capacity were acquired, at three anatomical levels (carena, carena plus 5 cm, carena minus 5 cm). In each section, lung parenchyma was isolated from the other thoracic structures and from the main bronchi and a densitometric evaluation was applied to the regions of interest. A histogram showing the distribution of the pixel frequencies versus lung densities, the mean CT number and the pixel index at -910 HU (for scans acquired at 10% of the patient's vital capacity) and at -950 HU (for scans obtained at 90% of the patient's vital capacity) were obtained. Average acquisition time for the spirometrically gated HRCt was 15-20 minutes, average data processing time was 20 minutes. RESULTS: The mean CT number and the pixel index at -910 HU obtained at 10% of the patient's vital capacity correlated with the Tiffeneau index (r = 0.85; R = 0.78 respectively). The pixel index at -910 HU correlated also with the ratio of residual volume to total lung capacity (r = 0.65). The mean CT number and the pixel index obtained at 90% of the vital capacity correlated with the lung diffusion of carbon monoxide (r = 0.64 and r = 0.79 respectively). CONCLUSIONS: In our study, we found good correlation between the densitometric values obtained with spirometrically gated HRCT and the respiratory function tests routinely employed to assess the presence and the gravity of obstructive pulmonary disease. A good reproducibility of the data is guaranteed by the spirometical gating which enables standardization of the lung acquisition volume, and by the semiautomatic contour tracing program combined with an automated densitometric evaluation. The main disadvantages of the technique are the relatively long acquisition and evaluation times. Spirometrically gated HRCT densitometry is a promising tool for longitudinal studies of diffuse lung disease (pulmonary emphysema and fibrosis).

S-phase fraction can predict event free survival in patients with pT2-T3N0M0 colorectal carcinoma: implications for adjuvant chemotherapy.

BACKGROUND: Adjuvant chemotherapy for colorectal carcinoma was found to improve survival of patients with American Joint Committee on Cancer/International Union Against Cancer Stage III disease. The usefulness of chemotherapy in patients with Stage II disease continues to be debated, and it is likely that only those patients with a poor prognosis should receive adjuvant chemotherapy. Biologic prognostic factors may allow further insight into the optimal treatment strategy for patients with Stage II or earlier disease. In this study the prognostic role of S-phase fraction (SPF) determined by flow cytometry was assessed in patients with Stage I-II colorectal carcinoma. METHODS: Specimens of surgically resected colorectal carcinoma were examined for SPF by flow cytometric DNA analysis. Consecutive patients referred to the study institution were considered eligible for this study. The main inclusion criteria were a Stage I-II tumor together with sufficient tumor material and adequate follow-up information. For each stage of disease, SPF data were associated with the recurrence rate and the disease free survival (DFS). RESULTS: Analysis was performed on 167 patients (65 with Stage I disease and 102 with Stage II disease). Among Stage I patients, SPF was high in 20 patients and low in 45 patients. In Stage II patients, there were 36 patients with low SPF and 66 patients with high SPF. In both stages, the recurrence rate and DFS were significantly worse for the subgroups of patients with high SPF. CONCLUSIONS: SPF has revealed prognostic differences among patients with surgically resected Stage I-II colorectal carcinoma. These data should be considered for planning future trials in the adjuvant setting because patients with high SPF may benefit from adjuvant chemotherapy.

A three-year prospective study of specific immunotherapy to inhalant allergens: evidence of safety and efficacy in 300 children with allergic asthma.

The effectiveness and safety of specific immunotherapy (SIT) in allergic diseases such as asthma have increasingly come under question. Some authors advocate eliminating SIT as a therapeutic option for allergic patients, since the risks associated with this form of asthma may outweigh its positive effects. However, in a review of twenty-nine controlled studies in 2077 children and an equal number of controls, 27 (93.1%) have shown the effectiveness of SIT in pediatric age for the treatment of asthma due to inhalant allergens (p < 0.0001). The scope of this study was to ascertain whether this form of therapy is safe and effective in pediatrics. 300 children (median age 4.4 years) with asthma due to pollen or house dust mite were prospectively followed for three years. They were randomly divided into two groups: the study group and control group, being almost equal in number of children and clinical characteristics such as sex and age. No child suffered severe reactions due to SIT. Children receiving SIT had significantly greater reductions in days (p = 0.0001) and nights (p = 0.0005) without asthma and drug usage (p = 0.0003), compared with drug-treated children. In addition, the number of asthma attacks (p = 0.0001), and the quality of life were significantly improved in the study group (p = 0.0001). These findings suggest that if suitable allergen extracts are used with close observation of therapeutic indications, and children are followed by their doctors as frequently as required, SIT is effective in the treatment of pediatric asthma, with few adverse effects.

CD40 ligand expression on the surface of colostral T cells.

The proportion of T lymphocytes, mainly CD4 positive, co-expressing the CD40 ligand (CD40-L) was significantly greater (P = 0.001) in the colostrum of 10 breast-feeding mothers than in either autologous or heterologous blood. This surface glycoprotein is a T cell molecule involved in B cell isotype switching and immunoglobulin production with its natural counter-receptor, CD40, expressed by both adult and infant B lymphocytes. As the T cells of newborn infants fail to express the CD40-L when stimulated in vitro, the in vivo upregulation on milk T lymphocytes may be one of the mechanisms through which the mother transfers immune protection to the suckling infant.

Gamma delta T lymphocytes in mumps meningitis patients.

Flowcytometric analysis on T-cell subpopulations in the blood and cerebrospinal fluid (CSF) of 10 children with mumps meningitis (MM) revealed that the proportion of lymphocytes which bore the gamma delta T-cell receptor for antigen was significantly higher in CSF than in autologous and heterologous blood samples. gamma delta T-cell values in CSF of MM patients were also significantly higher than those calculated in CSF specimens from nine children with non-inflammatory neurological disorders. Whether and how the expanded CSF gamma delta T-lymphocyte subset, either CD8+ or double-negative (CD4-/CD8-), contributes to the eradication of mumps virus infection from the central nervous system represents a central focus of our ongoing research.

Seroprevalence of anti-HTLV I/II and anti-HIV-1 antibodies in a sample population in the Marche Region, Italy.

A total of 1292 sera, provided by different USLs (Local Health Centers) in the Marche Region (Italy), was tested by ELISA and Western Blot for the detection of anti-HTLV I/II ad HIV-1 antibodies. It was decided not to follow any particular criterion when collecting the sera, so no protocol of admission was established in advance. Of the 1292 sera tested, 12 were found antibody positive to HTLV I/II, 18 to HIV-1 and 4 to both viruses. Of the seropositives, for HTLV I/II and HIV-1, respectively, 2 and 3 were prisoners; 2 and 4 were IVDAs, 3 and 7 were hematological patients, 3 and 2 were hospitalized subjects (not for hematological disorders), and 2 and 2 were positives belonging to a healthy population group. These data suggest that the viruses may also be spreading among groups other than the high-risk ones (homosexuals, IVDAs, prisoners, blood-transfusion recipients, promiscuous heterosexuals).

The blockage of the human transferrin receptor by a monoclonal antibody, EA.3, induces growth inhibition in leukemia cell lines.

In our program to produce monoclonal antibodies against hemopoietic functional antigens we have obtained an hybridoma cell line EA.3 reacting with human transferrin receptor. This monoclonal antibody is an IgM that displays "in vitro" antiproliferative activity against leukemic cells due to its competition with transferrin on the receptor. Cell cytotoxicity was demonstrated either with the blockage of BrdU incorporation in HL-60 cells and with leukemic CFU inhibition. In contrast, K562 cells were insensitive to the blockage of the transferrin receptor induced by this MAb.

Inhibition of NK binding to K562 cells induced by MAb saturation of adhesion molecules on target membrane.

Cell to cell interaction play a major role in the induction of a immune response. NK cells represent a special lymphoid subset which displays its cytotoxic function without the engagement of MHC system. In order to investigate the role of different adhesion molecules in the mechanism of binding of the NK cell to the classic tumor target K562 cell, we have employed different unclustered mAbs of the Adhesion session (5th "CD" Workshop, Boston 1993) mostly of the CAM (cell adhesion molecule) subpanel. After their reactivity characterization both on lymphocytes and K562 cells, those that demonstrate reactivity against the tumor target were tested in the binding assay. The target was pretreated with the monoclonal in order to block a possible reacting molecule for the effector. Then, after the incubation of lymphocytes with PE-labelled anti CD16 mAb, their ability to bind to the target was tested in flow. While the majority of the mAbs did not induce any significant change in the binding capacity of the NK subset, few of them did, and precisely anti-CD58 (LFA-3) and anti-CD54 (ICAM-1) which showed different level of inhibition, particularly drastic with S002, S083 and S100. Other mAbs, such as the S011 (anti-CD59), due to the presence of the PI-linked glycoprotein recognized on both target and effector membranes, and to its capacity to stimulate NK activity, produced a total binding of the NK population. The coincubation of targets with anti-CD54 and anti-CD58 allowed to reduce at the lowest level this function. This data seem to support the hypothesis of specific surface molecules involved in the binding process of the NK cell, after recognition of the target.

Fate of iron stores in thalassaemia after bone-marrow transplantation.

After successful bone-marrow transplantation (BMT) in thalassaemia, the individual acquires the pattern of globin synthesis of the donor. We call such an individual "ex-thalassaemic after BMT", a term that underscores the cure of the genetic defect but maintenance of residual signs of organ damage due to iron overload and dysfunction acquired during the pretransplant years. We have analysed the extent and fate of tissue iron overload in 151 ex-thalassaemic patients after BMT, according to the risk factors of hepatomegaly, hepatic portal fibrosis, and inadequate chelation therapy. Serum ferritin concentrations decreased and unbound iron binding capacity (UIBC) increased slowly during the years after the transplant. When analysed according to risk group (assigned at the time of the transplant), ferritin and UIBC returned within the normal ranges in only the low-risk group (without hepatomegaly or portal fibrosis, and with adequate chelation pre-BMT). Ferritin and UIBC were still abnormal 7 years after the transplant in the moderate-risk group (those with one or two risk factors) and highly abnormal in the high-risk group (all three risk factors) indicating persistence of, respectively, moderate and severe iron overload at the time of transplant. In ex-thalassaemic patients who were studied before and yearly after the transplant the extent of haemosiderosis, as judged by staining of liver biopsy samples, decreased during the years after transplant. The degree of iron deposition and rate of post-BMT linear growth seem to influence rate of post-BMT decrease in tissue iron overload in different risk groups at the time of BMT.

Cytokinetic effects of interferon in colorectal cancer tumors: implications in the design of the interferon/5-fluorouracil combinations.

Interferon (IFN) has been shown to enhance the cytotoxic effects of 5-fluorouracil (5FUra) in colorectal cancer, and clinical trials with this combination resulted in higher response rate with respect to 5FUra alone. IFN is generally administered s.c. three times a week. This prolonged exposure could determine a block of tumor cells in the G0-G1 phase of the cell cycle, thus rendering tumor cells insensitive to 5FUra, an S-phase specific agent. In order to verify the presence of this block, 21 operable colorectal cancer patients were treated with IFN-alpha 2b at the dose of 3 megaunits every other day in the week before operation, while another 22 represented the control group. Samples of tumor tissue were taken at endoscopy and operation. [3H]Thymidine labeling index and flow cytometry were used to assess the S-phase fraction. In IFN treated patients, we found a significant statistical difference between the mean percentage of S-phase fractions evaluated either by labeling index (P = 0.00001) or by flow cytometry (P < 0.001). On the contrary, this difference was not present in the control group: labeling index, P = 0.06; flow cytometry, P = 0.08. Furthermore a significant increase in the G0-G1 phase of the cell cycle was found after IFN administration (P < 0.001) but not in the control group. Our results suggest that IFN reduces the S-phase fraction in colorectal cancer tumors. This action should be considered in the design of the 5FUra/IFN combination because it could decrease 5FUra activity, leading to a loss or a decrease in the advantage of 5FUra modulation by IFN.