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We studied the mechanical properties of living starfish oocytes belonging to two species, Astropecten Auranciacus and Asterina pectinifera, over a wide range of timescales. We monitored the Brownian motion of microspheres injected in the cytoplasm using laser particle-tracking (LPT) and video multiple-particle-tracking (MPT) techniques, to explore high- and low-frequency response ranges, respectively. The analysis of the mean-square-displacements (MSD) allowed us to characterize the samples on different timescales. The MSD behavior is explained by three power-law exponents: for short times (τ < 1 ms) it reflects the semiflexible behavior of the actin network; for intermediate timescales (1 ms < τ < 1 s) it is similar to that of a soft-glass material; finally for long times (τ > 1 s) it behaves mainly like a viscous medium. We computed and compared the viscoelastic moduli using a recently proposed model describing the frequency response of the cell material. The large fluctuations found in the MSD over hundreds of trajectories indicate and confirm the significant cytoplasm heterogeneity., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Training through Research Application Italian iNitiative (TRAIN) is a mobility program financed under the EU action called "Cofinancing of regional, national and international programs" (COFUND) of the European Commission Seventh Framework Program (FP7) - People, and has been designed to encourage the promotion and development of international programs of research through mobility at various stages of research careers. The aim of TRAIN is to improve translational skills in the field of cancer by promoting a three-year international mobility program assigning a total of 51 fellowships subdivided into incoming, outgoing and reintegration fellowships.?The TRAIN proposal has been submitted in February 2009 to the European Commission in reply to the 2008 FP7-PEOPLE-COFUND call and has been successfully evaluated. TRAIN is addressed to postdoctoral scientists or scientists who have at least four years' full-time equivalent research experience and who wish to improve their careers spending one year abroad. The mobility program is open also to non-Italian experienced scientists wishing to spend one year in an Italian research center or private company. Part of the scheme is targeted to experienced Italian scientists who have completed at least three years of research in a foreign country and are interested in returning to Italy.?TRAIN is part of an overall Italian strategy outlined by the International Program of the Italian Cancer Network "Alleanza Contro il Cancro" to promote Italian participation in the building of the European Area for translational cancer research and to enhance the interaction between academy and industry.

Although Iodine-131 (131I) therapy is fully validated for Graves' disease (GD), there is debate about radioiodine amount to be administered (prescribed activity), as well as the use of individualized dosimetry vs fixed 131I activity. The clinical outcome of 119 GD patients treated with 131I from 2003 to 2008 has been evaluated. The prescribed activity was calculated according to a dosimetric protocol taking into account several variables, including thyroid volume reduction during treatment. In addition, we performed a simulation according to other dosimetric protocols, by calculating the corresponding prescribed activities. The patients were followed up for at least 12 months after treatment. In the first period of observation (2003), a 120-200 Gray (Gy) radiation dose to the thyroid was prescribed, according to the guidelines published by the Italian Societies of Endocrinology, Nuclear Medicine and Medical Physics: hyperthyroidism cure with a single radioiodine administration was obtained in 53% of patients. This outcome raised up to 89% when a higher radiation dose to the target (200- 250 Gy) was prescribed, although the administered activities were still lower, as a rule, than the most commonly employed fixed activities (400-600 Mega-Becquerel--MBq). Our method showed a high level of individual dose optimisation, particularly when compared to simplified methods. In conclusion, the protocol adopted in this study ensures a satisfactory rate of hyperthyroidism cure, while administering quite low 131I activities, provided that an adequate committed radiation dose to the thyroid is prescribed. In this context, the dose indication given by the aforementioned guidelines should probably be revised.

In order to minimize the high attrition rate that usually characterizes drug research and development projects, current medicinal chemists aim to characterize both pharmacological and ADME profiles at the beginning of drug R&D initiatives. Thus, the development of ADME High-Throughput Screening in vitro and in silico ADME models has become an important growing research area. Here we present new linear and non-linear predictive QSPR models to predict the human intestinal absorption rate, which are derived from a medium sized, balanced and diverse training set of organic compounds. The structure-property relationships so obtained involve only 4 molecular descriptors, and display an excellent ratio of number of cases to number of descriptors. Their adjustment of the training set data together with the performance achieved during the internal and external validation procedures are comparable to previously reported modeling efforts., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

CTLA-4, initially described as a membranebound molecule, is a costimulatory receptor transducing a potent inhibitory signal. Increasing evidence shows the CTLA-4 gene to be an important susceptibility locus for autoimmune endocrinopathies and other autoimmune disorders. A soluble form of cytotoxic T-lymphocyte-associated antigen-4 (sCTLA-4) has been established and shown to possess CD80/CD86 binding activity and in vitro immunoregulatory functions. sCTLA-4 is generated by alternatively spliced mRNA. Whereas low levels of sCTLA-4 are detected in normal human serum, increased serum levels are observed in several autoimmune diseases (e.g. Graves' disease, myasthenia gravis, systemic lupus erythematosus, type 1 diabetes, systemic sclerosis, coeliac disease, autoimmune pancreatitis and primary biliary cirrhosis). The biological significance of increased sCTLA-4 serum levels is not fully clarified yet. On the one hand, it can be envisaged that sCTLA-4 specifically inhibits early T-cell activation by blocking the interaction of CD80/CD86 with the costimulatory receptor CD28. On the other hand, higher levels of sCTLA-4 could compete for the binding of the membrane form of CTLA-4 with CD80/CD86 in the later phases of T-lymphocyte activation, causing a reduction in inhibitory signalling. This double-edged nature of sCTLA-4 to block the binding of CD28 to CD80/CD86 may result in different outcomes during the clinical course of an autoimmune disease.

Purpose: Specific autoantibodies acting as TSH receptor agonists (TRAb) are responsible for Graves' disease (GD). In the last 30 years three generations of assay methods for the detection of TRAb have become available. The aim of this multicentre study was to evaluate the analytical sensitivity, precision and diagnostic accuracy of TRAb measurement using a new automated assay in comparison with a second-generation standard method., Methods: Serum samples from patients with GD (n=82), autoimmune thyroiditis (AIT, n=57) or hyperthyroidism (HT, n=292), from 106 healthy subjects and from 57 patients with infectious diseases were analysed using a third-generation TRAb immunoassay (anti-TSHR, RAD 120; Radim, Italy) based on the human monoclonal TSH receptor antibody M22., Results: Using a cut-off value of 1.25 mIU/l, established by ROC curve analysis, 80/82 GD patients (97.5%), 68/292 HT patients (23.2%), and 6/57 AIT patients (10.5%) were TRAb-positive with the M22-based automated assay. The percentages of TRAb positivity were lower in the same patients when the measurements were done with the second-generation method (95.1%, 18.9%, 7.0%, respectively)., Conclusion: The M22-based automated immunoassay shows high functional sensitivity (0.4 mIU/l) and high diagnostic specificity, is more sensitive than the standard second-generation method and is less time-consuming and labourintensive, and is therefore the up-to-date technology for TRAb detection in clinical practice.

BT3 is a new family of immunoreceptors belonging to the extended B7 family. BT3 molecules are expressed on the surface of resting and activated monocytes and monocyte-derived dendritic cells (iDC). We show that BT3 cross-linking, in the absence of other survival factors, provides a survival signal for monocytes and iDC and induces up-regulation of costimulatory molecules, such as CD80 and CD86, and HLA-DR. We further analyzed the effects of BT3 cross-linking on various proinflammatory responses on monocytes and iDC. The results obtained showed that BT3 engagement is able to modulate the production of IL8/CXCL8, IL-1β and IL-12/p70. Moreover, we demonstrated a synergistic effect between BT3 and Toll-like receptors ligands on both monocytes and iDC in up-regulating the production of proinflammatory cytokines. Thus, BT3 could be involved in the regulation of the balance between immune activation and suppression. A better understanding of its physiological role of these families of receptors awaits the precise identification of the nature, origin, expression, and distribution of their ligands., (Copyright © 2010. Published by Elsevier Ltd.)

Diffusive mixing in a model polymer blend of limited miscibility (i.e., the pair polydimethylsiloxane/polyisobutene) is investigated. The diffusion process is followed in the actual droplet-based microstructure of the polymer blend, as opposed to the ideal planar geometry used in previous studies (Brochard et al. Macromolecules 1983, 16, 1638; Composto et al. Nature 1987, 328, 234). In our experiments we combine Raman microspectroscopy and video particle-tracking microrheology. The first technique allows us to monitor local concentration of the two polymers with high spatial resolution both inside and outside a micrometer-size droplet of the dispersed phase. In addition, microrheology enables to follow how the local viscosity inside the droplet changes during the diffusion. The polymer viscosity inside the droplet is determined by video tracking the Brownian motion of a polystyrene bead microinjected into the droplet. The microspectroscopic and microrheological data are combined to estimate the concentration dependence of the monomer friction factor of the two species, which is a key parameter to calculate the interdiffusion coefficient D. Numerical calculations based on such concentration-dependent interdiffusion coefficient D and several alternative models of the polymer diffusion are compared to the experimental concentration profiles. A satisfactory agreement is found for the so-called "slow theory" (Brochard et al.). A phenomenological model improving the agreement of the model with the experimental data is also presented.

Cell mechanical properties play an important role in determining many cellular activities. Passive microrheology techniques, such as Multiple-Particle-Tracking (MPT) give an insight into the structural rearrangements and viscoelastic response of a wide range of materials, in particular soft materials and complex fluids like cell cytoplasm in living cells. The technique finds an important field of application in large cells such as oocytes where, during their growth, several organelles and molecules are displaced in specific territories of the cell instrumental for later embryonic development. To measure cell mechanics, cells are usually deformed by many techniques that are slow and often invasive. To overcome these limits, the MPT technique is applied. Probe particles are embedded in the viscoelastic sample and their properties are extracted from the thermal fluctuation spectra measured using digital video-microscopy. The Brownian motion of a probe particle immersed in a network is directly related to the network's mechanical properties. Particles exhibit larger motions when their local environments are less rigid or less viscous. The mean-square-displacement (MSD) of the particle's trajectory is used to quantify its amplitude of motions over different time scales.

This work reports on the application of Raman spectroscopy for the analysis of Xenopus laevis oocytes (stage-I). A two-color home-made microscope has been used for this investigation. In particular, a 785nm Raman probe has been used to acquire the spontaneous Raman scattering from the oocyte cytoplasm, while a 532nm probe has been employed to detect carotenoids through Resonant Raman Scattering. Finally, the distribution of beta-carotene along a diameter of a single oocyte has been investigated.

Background: TSH receptor antibodies (TRAb) play a crucial role in the pathogenesis of Graves' disease (GD). The use of human recombinant TSH-receptor far improved the analytical performance of TRAb assays (2nd-generation assays). The 3rd-generation assay is based on the inhibition of binding of a human biotin-labeled monoclonal thyroid- stimulating antibody (M22) to TSH-receptor by the autoantibodies present in the serum., Aim: We aimed to assess the ability of the 2nd- and 3rd-generation assays to detect serum TRAb following radioiodine therapy for hyperthyroidism., Methods: Sera from 47 hyperthyroid (25 autoimmune, 22 non-autoimmune) patients were tested using the two different assays before and at different time intervals after radioiodine therapy. The modifications of TRAb were evaluated, as well as the correlation between the two methods., Results: The results obtained by the two methods proved to be closely correlated. A rise in TRAb was invariably observed in GD patients following radioiodine, with a median peak at 6 months, irrespective of their initial clinical status, presence of ophthalmopathy, smoking habits or other variables. Such a rise was nearly superimposable using both methods. No TRAb appearance was observed in patients with non-autoimmune hyperthyroidism., Conclusions: The use of methods of higher sensitivity with respect to that formerly used indicate that nearly all GD patients develop TRAb following radioiodine, and that this phenomenon is transient and not related to baseline conditions and clinical outcome/efficacy of treatment.

Background: To report about initial technical and clinical experience in preoperative radiation treatment of rectal cancer with volumetric modulated arcs with the RapidArc (RA) technology., Methods: Twenty-five consecutive patients (pts) were treated with RA. All showed locally advanced rectal adenocarcinoma with stage T2-T4, N0-1. Dose prescription was 44 Gy in 22 fractions (or 45 Gy in 25 fractions). Delivery was performed with single arc with a 6 MV photon beam. Twenty patients were treated preoperatively, five did not receive surgery. Twenty-three patients received concomitant chemotherapy with oral capecitabine. A comparison with a cohort of twenty patients with similar characteristics treated with conformal therapy (3DC) is presented as well., Results: From a dosimetric point of view, RA improved conformality of doses (CI95% = 1.1 vs. 1.4 for RA and 3DC), presented similar target coverage with lower maximum doses, significant sparing of femurs and significant reduction of integral and mean dose to healthy tissue. From the clinical point of view, surgical reports resulted in a down-staging in 41% of cases. Acute toxicity was limited to Grade 1-2 diarrhoea in 40% and Grade 3 in 8% of RA pts, 45% and 5% of 3DC pts, compatible with known effects of concomitant chemotherapy. RA treatments were performed with an average of 2.0 vs. 3.4 min of 3DC., Conclusion: RA proved to be a safe, qualitatively advantageous treatment modality for rectal cancer, showing some improved results in dosimetric aspects.

Optical tweezers have become a powerful tool to explore the viscoelasticity of complex fluids at micrometric scale. In the experiments, the Brownian trajectories of optically confined microparticles are properly analysed to provide the viscous and elastic moduli G' and G'. Nevertheless, the elastic response of the medium is inherently superimposed on the trap stiffness itself. Usually, this drawback is removed by subtracting the elastic trap contribution from the measured medium response. However, it is clear that when trap and medium elasticity become comparable this procedure is no longer reliable. Still, there exists a wide class of complex fluids that exhibit a low elasticity (diluted biopolymers, Boger fluids, etc) for which alternative experimental approaches would be desirable. Herein we propose a new method based on blinking optical tweezers. It makes use of two independent laser beams: the first is used to trap a single bead while the second one, of very weak power, acts as probe to monitor its position with a quadrant photodiode. The trap laser intensity is modulated on-off: when the laser is off the bead follows a free diffusion trajectory that, hence, leads to an estimation of G' and G' free of the influence of the trap. We have successfully applied this technique to highly-diluted hyaluronic acid solutions (c < 0.1 mg/ml) reaching to measure very weak G' modulus ( approximately 0.01 Pa) in a wide range of frequencies.

Celiac disease (CD) is a multifactorial disorder influenced by environmental, genetic and immunological factors. Increasing evidence showed CTLA-4 gene as an important susceptibility locus for autoimmune disorders. A native soluble cytotoxic T-lymphocyte-associated protein-4 (sCTLA-4), lacking of transmembrane sequence, has been described in several autoimmune diseases. We aimed to evaluate the presence of increased sCTLA-4 concentration in the serum of patients with CD and the possible immunoregulatory function. Blood samples were collected from 160 CD patients; sCTLA-4 levels were evaluated by ELISA, western blot and reverse transcription-PCR. The capability of serum sCTLA-4 to modulate T-lymphocyte proliferation in vitro was evaluated by two-way mixed leukocyte reaction assay. We demonstrated high levels of sCTLA-4 in serum of untreated celiac patients. Additionally, we observed that sCTLA-4 concentrations are related to gluten intake and that a correlation between autoantibodies to tissue transglutaminase and sCTLA-4 concentration exists. Moreover, sCTLA-4 levels correlate with the degree of mucosal damage. Conversely, no correlation between sCTLA4 levels and the HLA-related risk was observed. Finally, we show that sCTLA-4 from sera of CD patients displays functional activities. These results strongly suggest a regulation of sCTLA-4 synthesis depending on the presence or absence of dietary gluten and imply a possible immunomodulatory effect on cytotoxic T lymphocyte functions. In gluten-exposed patients, serum sCTLA-4 levels might provide insight about mucosal injury.

Development and maturation of the nascent cardiovascular system requires the recruitment of mural cells (MCs) around the vascular tree in a process called vascular myogenesis. Understanding the origin and development of vascular MCs has been hampered by difficulties in observing these cells in vivo and performing defined genetic and experimental manipulations in available model organisms. Here, we investigate the origin of vascular MCs using molecular and genetic tools in zebrafish. We show that vascular MCs are present around the lateral dorsal aortae (LDA) and anterior mesenteric arteries (AMA) of developing animals, and that they also contribute to the outflow tract of the developing heart and ventral aorta (VA). Genetic data indicate that the vascular MCs of the LDA and AMA do not arise from blood or endothelial progenitors but from other derivatives of the lateral plate mesoderm. We further show that zebrafish vascular MCs share many of the morphological, molecular and functional characteristics of vascular smooth muscle cells and pericytes found in higher vertebrates. These data establish the zebrafish as a useful cellular and genetic model to study vascular myogenesis as well as tumor angiogenesis and other MC-associated diseases.

The aim of surgical treatment of fistula-in-ano is to eradicate the suppurative process permanently without compromising faecal continence. The appearance of a recurrence of fistula-in-ano is often due to non-identification of the internal opening by the surgeon, and to the presence of complex fistulae. We evaluated the clinical course of 214 patients in a randomised. controlled trial with respect to the recurrence rate of anal fistula with or without preoperative endoanal ultrasonography. In this study we demonstrate that endoanal ultrasonography was the most accurate diagnostic modality for detecting internal opening and complex fistulae. Therefore, endoanal ultrasonography is reliable and useful in the preoperative assessment of anal fistula, and particularly for decreasing the recurrence rate of this disease.

This review presents the development of a Raman Tweezers system for detecting hemoglobin-related blood disorders at a single cell level. The study demonstrates that the molecular fingerprint insight provided by Raman analysis holds great promise for distinguishing between healthy and diseased cells in the field of biomedicine. Herein a Raman Tweezers system has been applied to investigate the effects of thalassemia, a blood disease quite diffuse in the Mediterranean Sea region. By resonant excitation of hemoglobin Raman bands, we examined the oxygenation capability of normal, alpha- and beta-thalassemic erythrocytes. A reduction of this fundamental red blood cell function, particularly severe for beta-thalassemia, has been found. Raman spectroscopy was also used to draw hemoglobin distribution inside single erythrocytes; the results confirmed the characteristic anomaly (target shape), occurring in thalassemia and some other blood disorders. The success of resonance Raman spectroscopy for thalassemia detection reported in this review provide an interesting starting point to explore the application of a Raman Tweezers system in the analysis of several blood disorders.

Background: Postsurgical recurrence (PSR) is very common in patients with Crohn's disease (CD) and previous surgery. Endoscopy is crucial for the diagnosis of PSR, also showing high prognostic value. Bowel sonography (BS) with or without oral contrast enhancement (OCBS) is accurate for CD diagnosis but its role in PSR detection and grading is poorly investigated. The aim was to evaluate the diagnostic accuracy of BS and OCBS for PSR compared to the endoscopical Rutgeerts's grading system., Methods: We prospectively performed endoscopy, BS, and OCBS in 40 CD patients with previous bowel resection to provide evidence of possible PSR. Endoscopy, BS, and OCBS were executed 1 year after surgery, with PSR diagnosis and grading made in accordance with Rutgeerts. BS and OCBS were considered suggestive for PSR in the presence of bowel wall thickness (BWT) >3 mm. OCBS was performed after ingestion of 750 mL of polyethylene glycol (PEG). Also, a receiver operating characteristic (ROC) curve was constructed in order to define the best cutoff of BWT to discriminate mild from severe PSR (grade 0-2 versus 3-4 of Rutgeerts) for both BS and OCBS., Results: In all, 22 out of the 40 CD showed an endoscopic evidence of PSR (55%). A severe PSR was present in 14 patients (64%). Sensitivity, specificity, and positive and negative predictive values were 77%, 94%, 93%, and 80% for BS, and 82%, 94%, 93%, and 84% for OCBS. On the ROC curve a BWT >5 mm showed sensitivity, specificity, and positive and negative predictive values of 93%, 96%, 88%, and 97% for the diagnosis of severe PSR at BS, while a BWT >4 mm was the best cutoff differentiating the mild from the severe CD recurrence for OCBS, with a sensitivity, specificity, and positive and negative predictive values of 86%, 96%, 97%, and 79%, respectively., Conclusions: Both BS and OCBS show good sensitivity and high specificity for the diagnosis of PSR in CD, with a BWT >5 mm for BS and BWT >4 mm for OCBS strongly indicative of severe endoscopic PSR. Accordingly, these techniques could replace endoscopy for the diagnosis and grading of PSR in many cases.

Background: In the sera of liver, colorectal and prostate cancer patients, several biomarkers may be detected as IgM immune complexes. To determine whether the presence of immune complexes was correlated to an increase of IgMs, we measured the IgM content in the sera of patients with hepatocellular carcinoma (HCC) and cirrhosis, and evaluated the occurrence of des-gamma-carboxy prothrombin (DCP) as immune complexes (DCP-IgM) compared to the levels of DCP and alpha-fetoprotein (AFP)., Patients and Methods: Serum samples from 31 patients with cirrhosis, 33 untreated HCC patients diagnosed by ultrasound, computed tomography and/or magnetic resonance and confirmed by histopathology, when indicated, and 30 healthy controls were analysed. Concentrations of IgM and DCP-IgM were determined by ELISAs., Results: Circulating IgM in patients with HCC (median level = 1.79 mg mL(-1)) and cirrhosis (1.09 mg mL(-1)) were not significantly different (P = 0.1376) while DCP-IgM were significantly higher in HCC patients (median level = 2171.2 AU mL(-1)) than in those with cirrhosis (1152 AU mL(-1), P = 0.0047). No correlation was found between DCP-IgM and IgM in HCC (r = 0.227) and cirrhosis patients (r = 0.475). DPC-IgM was positive in 55% (18/33) of HCC patients and in 26% (8/31) of cirrhosis patients compared to 39% and 26% for DCP and 48% and 13% for AFP. DCP-IgM, DCP and AFP tests had 100% specificity in healthy controls., Conclusions: DCP-IgM in HCC patients was not associated with an increase in IgM concentration. DCP-IgM was more frequently detected in HCC patients than DCP and AFP, strengthening the diagnostic role of IgM immune complexes for liver cancer.

In this work, the effects of thalassemia, a blood disease quite diffuse in the Mediterranean sea region, have been investigated at single cell level using a Raman Tweezers system. By resonant excitation of hemoglobin Raman bands, we have examined the oxygenation capability of beta-thalassemic erythrocytes. A reduction of this fundamental erythrocyte function has been found. The measurements have been performed on a significant number of red blood cells; the relative statistical analysis is presented. Moreover, the response to photo-induced oxidative stress of diseased cells with respect to the normal ones has been analyzed. Finally, the deformability of thalassemic erythrocytes has been quantified by measuring the membrane shear modulus by using a double-trap system: the measurements have revealed an increase in membrane rigidity of more than 40%, giving evidence that the genetic defect associated to thalassemia, which manly relies on hemoglobin structure, also strongly affects the erythrocyte mechanical properties. Our results demonstrate that the developed set-up may have potential for the monitoring of blood diseases and their response to drug therapies.

Background: A proportion of men with prostate cancer will progress to develop metastatic disease involving the lymph-nodes and bone. To identify novel candidates associated with metastatic progression, we compared the proteomic profiles of LNCaP (lymph-node metastatic, androgen-dependant) and PC-3 (bone metastatic, androgen-independent), human prostate cancer cells., Methods: Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS), was used to identify differentially expressed proteins. Western blotting was used to validate the identity of any candidates. Immunohistochemistry was used to assess tissue expression., Results: 2D-PAGE followed by ESI-MS/MS analyses identified the expression of glutathione S-transferase-pi (GST-pi) and protein gene product 9.5 (PGP 9.5) in PC-3 cells, but absent expression in LNCaP cells. PGP 9.5 expression in PC-3 cells was confirmed by Western blotting, in addition to expression in DU145 cells. Analysis of cell conditioned media showed that PGP 9.5 was secreted. Sequencing of the PGP 9.5 gene promoter region in bisulfite modified DNA, suggested that the regulation of expression involves promoter hypermethylation. RT-PCR analysis for Chromogranin A (ChA) mRNA (a marker of neuroendocrine cells), showed expression in PC-3 and DU145 cells but was undetectable in LNCaP cells. Immunohistochemistry localised PGP 9.5 expression exclusively within neuroendocrine cells and nerve fibres., Conclusions: Our unexpected finding that the neuroendocrine cell markers PGP 9.5 and ChA are expressed by PC-3 and DU145 cells, suggests that these cells may have been derived from metastatic adenocarcinomas which had undergone neuroendocrine differentiation or alternatively the expression occurred ectopically as a result of cell culture.

We analyze the equations governing the evolution of distributions of the work and the heat exchanged with the environment by a manipulated stochastic system, by means of a compact and general derivation. We obtain explicit solutions for these equations for the case of a dragged Brownian particle in a harmonic potential. We successfully compare the resulting predictions with the outcomes of experiments, consisting of dragging a micron-sized colloidal particle through water with a laser trap.

Clinical studies have estimated a 10- to 20-fold increased risk for celiac disease (CD) in patients with selective IgA deficiency (SIgAD). For this reason, screening for CD is mandatory in SIgAD patients, but it represents a special challenge since the specific IgA class antibodies against gliadin (AGA), endomysium (EMA), and tissue-transglutaminase (tTG) are not produced in patients with CD. IgG class counterparts of these antibodies may be informative; in particular IgG EMA has been demonstrated to be a valid marker for diagnosing CD in SIgAD cases, but it is not used much in clinical laboratories, because it is cumbersome and involves some technical difficulties. Even if it was widely used in clinical laboratories, the measuring of IgG AGA has shown a less-than-optimum diagnostic accuracy, so that now it tends to be substituted by tests for anti-tTG IgG, for which the few available studies have shown diagnostic performances superior to AGA. Since it is not known whether various available methods for measuring IgG anti-tTG antibodies offer similar diagnostic performances, we have compared the results obtained from nine second-generation commercial methods (D-tek, Phadia, Immco, Orgentec, Radim, Euroimmun, Inova, Aesku, Generic Assays), measuring IgG anti-tTG antibodies in 20 patients with CD and SIgAD and in 113 controls (9 patients with SIgAD without CD, 54 patients with chronic liver disease, and 50 healthy individuals). Diagnostic sensitivity, calculated by means of ROC plot analysis, ranged between 75% and 95%, and specificity ranged from 94% to 100%. In the same population, the diagnostic sensitivity and specificity of AGA IgG were 40% and 87%, respectively. Even though they perform differently, all IgG anti-tTG methods evaluated are reliable serological assays for the diagnosis of CD in SIgAD patients, with diagnostic accuracy superior to the AGA IgG method. The methods that use a mix of tTG and gliadin peptides as the antigenic preparation have a specificity slightly lower than that of the methods that use only tTG.

Background: To evaluate the diagnostic characteristics of commercially available IgG anti-tTG assays in selective IgA deficiency (SIgAD), we tested different IgG anti-tTG methods and compared the results with those obtained from two other tests: one for IgG anti-gliadin (AGA) and one for IgG to deaminated gliadin peptides (DGP)., Methods: 20 CD patients with SIgAD and 113 controls (9 patients with SIgAD without CD; 54 patients with chronic liver disease; 50 healthy subjects) were tested with 9 IgG anti-tTG assays (2 of which are enriched with gliadin peptides), one IgG AGA assay and one IgG anti-DGP assay., Results: Using optimal cutoffs as determined by ROC curves, the sensitivity of IgG anti-tTG methods ranged from 75% (1 kit) to 95% (7 kits) and the specificity from 94% (1 kit) to 100% (5 kits). Sensitivity and specificity were 40% and 87% for IgG AGA, and 80% and 98% for IgG anti-DGP, respectively., Conclusions: All IgG anti-tTG methods evaluated are reliable serologic assays for the diagnosis of CD in patients with SIgAD and perform better than the gliadin-based assays used in this study. The tests containing both tTG and gliadinic peptides are burdened by a lower specificity than the anti-tTG assays.

The cytoskeleton provides the backbone structure for the cellular organization, determining, in particular, the cellular mechanical properties. These are important factors in many biological processes, as, for instance, the metastatic process of malignant cells. In this paper, we demonstrate the possibility of monitoring the cytoskeleton structural transformations in optically trapped yeast cells (Saccharomyces cerevisiae) by tracking the forward scattered light via a quadrant photodiode. We distinguished normal cells from cells treated with latrunculin A, a drug which is known to induce the actin-cytoskeleton depolymerization. Since the proposed technique relies only on the inherent properties of the optical trap, without requiring external markers or biochemical sensitive spectroscopic techniques, it can be readily combined with existing optical tweezers setups.

Different autoantibodies are often measured simultaneously; this typically occurs when using indirect immunofluorescence on tissue sections or multiplex detection systems and may generate clinically "unexpected" positivities (i.e., without any relation to the disease under investigation). Their number is expected to increase with the development of microarray systems in autoantibody assays. In general, when examining patients with such unexpected findings, it is necessary to take into account that: a) autoantibody positivities are much more frequent than autoimmune diseases; b) the positive predictive value of an autoantibody positivity depends upon the diagnostic accuracy of the test and disease prevalence; c) autoantibodies may be risk factors for autoimmune disease or may also have a pathogenetic role by themselves. In this article we will highlight the possible problems raised by some relatively common situations, related to anti-nuclear, anti-thyroid, anti-phospholipid and anti-tissue transglutaminase autoantibodies. The need for specific strategies is outlined.

Raman spectroscopy has become a powerful tool for microscopic analysis of organic and biological materials. When combined with optical tweezers (Raman tweezers), it allows investigating single, selected micrometric particles in their natural environment, therefore, reducing unwanted interferences from the cover plate. A general problem affecting both Raman spectrometers and Raman tweezers systems is the background caused by the environment surrounding the sample under investigation. In this paper, we report on a novel method that allows acquiring Raman spectra of a single trapped particle (polystyrene microspheres) free from any background contribution. The method is based on the use of two collinear and copropagating laser beams: the first is devoted to trapping (trap laser), while the second one is used to excite the Raman transitions (pump laser). The trap laser moves the trapped particle periodically, by means of a galvomirror, back and forth across the pump laser. The back-scattered photons are analyzed by a spectrometer and detected by a photomultiplier; finally, the resulting signal is sent to a lock-in amplifier for phase-sensitive detection. The purpose of the present work is to give a detailed description of our method and to supply a systematic study concerning the formation of the Raman signal. We trap polystyrene beads and study the dependence of the Raman signal on several parameters, such as height from the coverslip surface, the bead size, the modulation amplitude, and the pump laser intensity. Our results establish a direct and practical approach for background suppression in the spectroscopic analysis of optical trapped microsized samples.

CTLA-4 molecule, expressed by activated T and B lymphocytes, transduces an inhibitory signal. Increasing evidence showed CTLA-4 gene as an important susceptibility locus for autoimmune endocrinopathies and other autoimmune disorders. The aim is to evaluate the augmented sCTLA-4 serum levels in different autoimmune thyroid diseases when compared with normal donors or with non-autoimmune hyperthyroidism and to investigate the functional activities and suggest the possible pathogenetic role of sCTLA-4. We demonstrate the presence of a soluble form of CTLA-4 in 59/90 sera from patients with autoimmune thyroid diseases (both Graves' disease and autoimmune thyroiditis). sCTLA-4 levels were not related to specific clinical manifestations, such as clinical thyroid status (hypo- or hyperthyroidism), circulating thyroid hormones, or other clinical features (ophthalmopathy). sCTLA-4 production does not seem to be affected by disease evolution during time. We showed that sCTLA-4 from sera of patients with thyroid autoimmunity is able to bind its physiological ligands CD80/CD86 and displays functional activities on different in vitro systems (T-cell proliferation induced by specific soluble antigens, bi-directional mixed lymphocyte reaction). In conclusion, we demonstrate an increment of sCTLA-4 in serum of patients with autoimmune thyroid diseases. Its possible pathogenetic role during autoimmune processes can be speculated: sCTLA-4 can specifically inhibit the early T-cell activation by blocking the interaction of CD80/CD86 with the co-stimulatory receptor CD28. Conversely, higher levels of sCTLA-4 could compete with membrane-bound CTLA-4 for CD80/CD86, in later T lymphocytes activation phase, causing a reduction of inhibitory signaling.

Hyaluronic acid (HA) is a polysaccharide widely used in biomedical applications, due to its elevated biocompatibility and the peculiar viscoelastic properties of its solutions. Although the viscoelastic behaviour of HA solutions has been extensively studied in the literature it has been often reported in the range of low frequency (1-100 Hz) and high salt concentration, whereas the main rheological peculiarities of this molecule are expected at high frequency (>100 Hz) and low salt concentration. In this work we studied the viscoelastic properties of low molecular weight HA (155 kDa) in wide range of concentrations (0.01-20 mg/ml) at low ionic strength and over an extended frequency range (0.1-1000 Hz) using both optical tweezers and conventional rheometry. Good agreement between the high frequency dynamic behaviour (optical tweezers) and the viscoelastic properties at low frequency (rheometry) was found. We also found that, in apparent contradiction with polyelectrolyte solution theory, HA solution behaves as liquid-like viscoelastic fluid (G''>G') even at concentrations higher than the entanglement concentration where a weak-gel behavior should be expected.

Objective: A screening program was proposed for the village of Carcare (population 5700), located in a region of Italy with an apparently low prevalence of coeliac disease (CD): only 1 patient diagnosed out of 2557 inhabitants. The study group comprised 1002 individuals (568 F, 434 M, age range 13-90 years) recruited from blood donors, secondary school pupils and people referred to the local outpatient facilities for routine blood chemistry., Material and Methods: Total IgA, IgA anti-tissue transglutaminase (tTG) (ELISA, recombinant human antigen) and IgA antiendomysium (EMA) (IFI, umbilical cord substrate) antibodies were measured in the serum of all participants. All patients with IgA deficiency were investigated for IgG tTG antibodies, and in the case of disagreement between tTG and EMA, they were typed for HLA DQ2-DQ8 haplotypes., Results: Thirteen subjects were positive and 988 negative for autoantibodies (3/988 had IgA deficiency). One serum sample was positive for tTG antibodies but negative for EMA. Ten out of 13 positive subjects consented to undergo duodenal biopsy, which invariably produced evidence of CD despite the absence of clinical signs/symptoms. A post-diagnostic clinical investigation provided evidence showing mild iron deficiency (4 subjects) and osteoporosis (2 subjects). After counselling, all subjects accepted a gluten-free diet., Conclusions: The prevalence of CD in the study group was 1:100 (1.0%; 95% CI: 0.5-1.8%): this indicates that CD is largely underdiagnosed in Carcare. Our results suggest that the low prevalence of CD observed in some regions is likely to be due to underdiagnosis.

We report the case of a female patient in whom gluten-induced entheropathy was revealed at the age of 71 yr by resistance to treatment with levothyroxine (L-T4), calcium carbonate and alfacalcidol. Hypothyroidism and hypoparathyroidism were the consequence of a total thyroidectomy performed at the age of 65 yr for a large multinodular goiter. Six months after thyroid ablation the patient started to complain of abdominal pain, diarrhea and weight loss. Following, anemia and osteopenia were documented. A progressive increase of replacement therapy for hypothyroidism and hypoparathyroidism was necessary. The clinical presentation suggested a malabsorption syndrome: celiac disease (CD) was diagnosed by serological markers and duodenal biopsy. Following gluten-free diet a normalization of clinical and serological findings was observed, bone mass density improved and a reduction of L-T4, calcium and vitamin D requirements was observed.

The present case describes a two-step endoscopic management of hydrocephalus and diagnosis of a single pineal region metastasis arising from a gastric adenocarcinoma. A 62-year-old man presenting with signs of subacute obstructive hydrocephalus from a pineal region mass had at first been treated with an endoscopic third ventriculostomy. As cerebrospinal fluid tumor markers (alpha-fetoprotein, beta-human chorionic gonadotropin) were negative, an endoscopic biopsy of the pineal region tumor was performed through a more anterior frontal burr hole. Pathology showed an adenocarcinoma and primary tumor work-up revealed an unsuspected gastric tumor, the pathology of which matched with the intracranial metastasis. The present report emphasizes the role of neuroendoscopy in pineal region tumors and reports a rare case of a solitary gastric adenocarcinoma metastasis in this location.

Amoebic Dictyostelium discoideum cells were employed in a bioassay to evaluate stress responses after exposures to the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) and two heavy metals (copper and mercury). Furthermore, we developed a recombinant cell line expressing a labile Green Fluorescent Protein (GFP) variant expressed under the control of an actin promoter to monitor stress-related protein degradation. Finally, cell viability was monitored to discriminate lethal exposure concentrations. The results demonstrated that exposure to sub-micromolar concentrations of mercury rendered significant changes in all studied physiological parameters, whereas B[a]P became toxic at low micromolar, and copper at high micromolar concentrations. Exposure to 0.5 microM mercury significantly reduced lysosomal membrane stability (LMS), endocytosis rate, GFP expression, and further resulted in the elevation of cytosolic free Ca(2+) ([Ca(2+)](i)). LMS in mercury-treated cells that had been pre-incubated with a specific Ca(2+)-dependent phospholipase A2 blocking agent was however not affected by the exposure, indicating that the toxic action of mercury is linked to the activation of phospholipase A2 via a Ca(2+)-signaling pathway. Exposure to 20 microM B[a]P significantly reduced LMS, endocytosis rate, and GFP expression, however without affecting [Ca(2+)](i), suggesting a calcium-independent route of toxicity for this compound. None of the physiological parameters were significantly affected by copper exposure at concentrations <400 microM, demonstrating a high resistance to this metal. Our results further showed that neither cell growth nor viability was affected by concentrations altering the studied physiological parameters. LMS, endocytosis rate, and [Ca(2+)](I), therefore, appear sensitive biomarkers of pollutant-related stress in amoebic cells.

Background and Aims: Postsurgical recurrence (PSR) is common in patients with Crohn's disease (CD) who have undergone surgery. Endoscopy is crucial for the diagnosis of PSR, showing also high prognostic value. Bowel sonography (BS) is accurate for CD diagnosis, but its role in PSR detection and grading has been poorly investigated. The aim of this study was to evaluate the diagnostic accuracy of BS compared to endoscopy in the detection of PSR., Materials and Methods: Between March 2002 and October 2005, to gain evidence of possible PSR, we prospectively performed endoscopy and BS in 45 CD patients who had undergone previous bowel resection. Endoscopy and BS were carried out 1 year after surgery, with diagnosis and grading of PSR made in accordance with Rutgeerts. BS was considered suggestive for PSR in the presence of bowel wall thickness (BWT)>3 mm. Also, an ROC curve was constructed to define the best cutoff value for BWT to differentiate mild from severe PSR (grade 1-2 vs 3-4 of Rutgeerts)., Results: Of the 45 patients with CD, 24 showed endoscopic evidence of PSR (53%). Severe endoscopic PSR was present in 16 patients (66%). Sensitivity, specificity, and positive and negative predictive values of BS were 79%, 95%, 95%, and 80%, respectively, with a sensitivity of 93% for severe PSR. On the ROC curve, a BWT>5 mm showed sensitivity, specificity, and positive and negative predictive values of 94%, 100%, 100%, and 96%, respectively, in differentiating mild from severe PSR, in remarkable agreement with endoscopy (kappa=0.90)., Conclusions: BS shows good sensitivity and high specificity for the diagnosis of PSR in CD, with a BWT>5 mm being strongly indicative of severe endoscopic PSR. Hence, BS could replace endoscopy for the diagnosis and grading of PSR in patients who comply poorly with the endoscopic examination.

In this work we discuss on a compact spectrometer based on DFB diode lasers for detection of chloridric and fluoridric acids. HCl and HF concentrations are determined through optical absorption of the P(4) line (lambda=1.7 microm) and the R(3) line (lambda=1.3 microm), respectively. Both lines belong to first overtone vibrational bands and their line strengths are 7.8 x 10(-21)cm/molecule for HCl and 2.8 x 10(-20)cm/molecule for HF. We chose these lines for their relative high intensities and because they are quite far from water vapour lines which represent the main interfering gas for trace-gases analysis. To detect these species we used two different high frequency modulation techniques: two-tone frequency modulation spectroscopy (f(1)=800 MHz and f(2)=804 MHz) was used for HCl while for HF we followed a simpler approach based on wavelength modulation spectroscopy (f=600 kHz). We demonstrate that the two techniques provide comparable detection limit of about 80 ppbV at atmospheric pressure. Positive testing of our spectrometer makes it suitable for in situ measurements of exhaust gases coming from waste incinerators.

Autoimmune thyroid diseases (AITD) are the far most common autoimmune disorders, their prevalence in Western countries exceeding 5% of the general population. In the large majority of individual cases the clinical impact of AITD is not severe, however, their widespread diffusion renders them a significant health problem. AITD are heterogeneous in their clinical presentation: the two main forms are autoimmune thyroiditis (AT) and Graves' disease (GD). Although they probably share, at least in part, a common genetic background and may occur in the same family as well as in the same individual, they are definitely two distinct diseases both in their clinical presentation and their pathophysiology. In fact, AT causes structural thyroid damage (mainly via cell-mediated immune destruction of thyroid follicular cells) which results, as a rule, in functional impairment (hypothyroidism); however, depending on clinical variants, evolution towards hypothyroidism may be very low, or thyroid function impairment occurs after an initial phase of mild thyrotoxicosis due to relatively rapid gland destruction. GD patients have hyperthyroidism, often severe, due to autoantibody-mediated thyrotropin receptor stimulation, with thyroid cell hyperplasia and hyperfunction. Such a functional heterogeneity is a key feature for the clinical management: as a matter of fact, therapy of AITD is mainly therapy of thyroid dysfunction. Moreover, since hyperthyroidism is quite early perceived by the patient as a cause of discomfort, the timing of the natural history of GD is relatively well defined; on the other hand, AT may be asymptomatic for a long time and defining its natural history in a single patient may be difficult. In some AITD patients (mainly, but not exclusively, with GD), clinical features not directly related to thyroid dysfunction, such as orbitopathy, are present. Graves' orbitopathy is probably related, at least in part, to autoantibodies directed to thyrotropin receptor; it may be, in a minority of patients, severe and sight-threatening, and represents an independent clinical problem., (Copyright (c) 2006 S. Karger AG, Basel.)

Background: Presently, sublingual immunotherapy is widely used as an alternative to the injection route for respiratory allergy, but its pharmacokinetics in humans is poorly known, and data are available only for Par j 1 allergen. We aimed at assessing the biodistribution of iodine-123-radiolabelled Der p 2 in allergic volunteers., Methods: Purified Der p 2 and its monomeric allergoid were radiolabelled with iodine-123 and administered sublingually to 7 allergic volunteers. The subjects were allowed to swallow 6 min after administration. Dynamic (up to 10 min) and static scintigraphic images (30 min, 1, 2, 3 and 20 h) were recorded, and blood samples were obtained at different time points to measure the plasma radioactivity and to assess the presence of circulating radiolabelled species by gel chromatography., Results: The local pharmacokinetics did not differ between allergen and allergoid. Plasma radioactivity began to increase only after swallowing and peaked at 1-2 h. Both the allergen and the allergoid persisted in the mouth for several hours, and traces could be detectable up to 20 h. At radioactivity plasma peak, gel chromatography showed that a fraction of the allergoid, but not the allergen, was absorbed as an intact molecule., Conclusions: These results indicate that the pharmacokinetics of sublingual administration is independent of the allergen used and characterized by the long persistence in the mouth. The contribution of enteric absorption of the allergoid in the mechanism of action of sublingual immunotherapy remains to be defined., (Copyright (c) 2005 S. Karger AG, Basel.)

Background: The aim of this study was to assess the efficacy of new agents in patients with malignant glioma in a neoadjuvant setting not confounded by surgery. The first study of neoadjuvant temozolomide aimed to provide a benchmark for future evaluation of new treatments., Patients and Methods: This was a multicentre phase II study of chemotherapy in patients with histologically verified glioblastoma multiforme (GBM) and anaplastic astrocytoma (AA) who had undergone biopsy alone. Patients were planned to receive two cycles of temozolomide at 200 mg/m(2) orally daily for 5 days at a 28-day interval prior to radiotherapy. Response was assessed by two central observers on pre- and post-chemotherapy enhanced scans using bi-dimensional criteria and as progression-free survival (PFS) at the time of second assessment prior to radiotherapy. Withdrawal from the study due to worsening clinical condition was, in the absence of second imaging, assessed as progressive disease. Survival and quality of life (QOL) were secondary endpoints., Results: Between August 1999 and June 2002, 188 patients from 15 UK and two Italian centres were entered into the study and 187 were analysed. Overall, 162 patients were assessable for response; seven had partial and 25 had minimal response. The objective response rate was 20% [95% confidence interval (CI) 14-26%] and PFS prior to commencing radiotherapy was 64% (95% CI 57-72%). The median survival was 10 months, and 1-year survival 41%. The median survival of responders was 16 months compared to 3 months in patients with progressive disease (P <0.001 on multivariate analysis)., Conclusion: The phase II study design of primary chemotherapy in patients with malignant glioma following biopsy alone is feasible and provides as objective a method of assessment of efficacy as is currently available. The baseline data on temozolomide provide a benchmark for assessment of efficacy of other agents and combinations.

Introduction: The aim of the study was to define the therapeutic approach used for obstructing colon cancer., Patients and Methods: In the period 1990-2002 in the Emergency Surgery Department of the University of Naples Federico II, 81 patients underwent colon surgery for obstructing colon cancer (25 M, 57 F, age range: 55-88 years, mean: 72). Were performed subtotal colectomies, left or right hemicolectomies, sigmoid colectomies, anterior resections, temporary or permanent enterostomies. The authors put morbidity and mortality in touch with the type of surgical operation and with results of the literature., Results: Mortality was 32%: 2.4% as a result of an anastomotic dehiscence; 4.9% for cardiovascular pathologies; 1.2% for sepsis; 23.4% for metastasis. Morbidity for anastomotic leakage was 4.9%: 2.4% after left emicolectomy and colo-colonic anastomosis; 1.2% after right emicolectomy and ileo-colonic anastomosis; 1.2% after defunctioning colostomy and left emicolectomy after 20 days., Conclusions: This experience suggests that a subtotal colectomy with primary anastomosis (one stage) can be performed more safely, by an experienced, skilled surgeon, in patients in good clinical conditions with acute obstruction of the colon. Healing remains a process depending more on the patient than on any aspect of the surgical technique. A defunctioning colostomy may be idealfor surgeons with little experience in colorectal surgery and in high risk patients with a very poor prognosis (unresectable lesions).

We detect acetylene and water-vapor lines by using a difference-frequency generator in the spectral region around 3 mm. Both C2H2 and H2O lines belong to fundamental vibrational bands and exhibit a line strength of the order of 10-20 cm/mol. Acetylene molecules were detected either by pure absorption or by first-derivative wavelength-modulation spectroscopy. The minimum detection sensitivity achieved for C2H2 in nitrogen was 4 ppb (parts in 109). Moreover, we discuss the effects of C2H2 pressure reduction in the presence of nitrogen in order to estimate systematic errors in the concentration measurements. Finally, we tested the accuracy of our spectrometer by detecting water vapor present as an impurity in a nitrogen cylinder at a nominal concentration of approximately 5 ppm.

First-level in vitro diagnostic tests for specific IgE against common inhalant or food allergens have been used to identify allergic patients. We evaluated the performance of Phadiatop and Fx5 (mixed food allergens) serological tests (Pharmacia Diagnostics AB, Uppsala, Sweden) in different groups of pediatric patients. We studied two groups of pediatric patients: 61 children recruited from an Allergy and Clinical Immunology Unit (Group 1); 136 children from a Pediatric Unit not specifically devoted to allergic diseases (Group 2); the two groups comprised patients with (A) or without (B) clinical suspicion of allergic disease. Sera were collected from routine blood analysis. Frequencies of positivities for Phadiatop and/or Fx5 were very high (68.8%) in Group 1, however, as many as 35.5% of Group 2 children were positive, as well. All the patients of Group 1 with clinical suspicion of allergic disease (1A), confirmed by allergologic diagnostic tests, had a positive first-level test; 42.8% only of the patients in Group 2 with suspicion of allergic disease (2A) had a positive first-level test. None of the Phadiatop/Fx5-negative children of Group 2A had specific-IgE with conventional tests. In 30% of children not suspected for allergic diseases (1B and 2B), positive first-level tests were observed. Such unexpected positivities were confirmed by single specific-IgE assays in 94.7% (for inhalants) or 71% (for foods) of cases. In conclusion, altogether the sensitivity and overall performance of first-level tests in pediatric populations (especially for inhalant allergens) may suggest their use, under appropriate circumstances, both as a first diagnostic approach (to rule out negative patients) and for screening purposes.

We report the realisation of a laser spectrometer in the mid-infrared spectral region based on difference-frequency generation in a periodically poled LiNbO3 crystal. Tunable coherent radiation around 3 microm was produced by mixing a diode-pumped monolithic cw Nd-YAG laser and an injection-locked diode laser at 0.785 microm. High sensitivity N2O detection was demonstrated by observing pure absorption spectra of lines in the v1 + v3 combination band. We estimate a minimum detectable pressure of pure N2O of 1 x 10(-2) Pa with 0.9 m absorption path-length, corresponding to an absorbance of 3 x 10(-4). Nitrous oxide was also detected in presence of O2, N2 and air. Collisional broadening coefficients for the P(33) line at 3447.678 per cm are reported for N2O-N2 and N2O-O2 mixtures.

Dysregulation of apoptosis is associated with the pathogenesis of organ-specific autoimmune diseases, through altered target organ susceptibility. Apoptosis signaling pathways can be initiated through activation of death receptors such as Fas. A comparative analysis of the expression of Fas and FasL, the antiapoptotic molecule Bcl-2, and apoptosis in both thyrocytes and thyroid-infiltrating lymphocytes (TILs) from patients with either Graves' disease (GD) or Hashimoto's thyroiditis (HT) was performed. GD thyrocytes expressed less Fas than HT thyrocytes, whereas GD TILs had higher levels of Fas and FasL than HT TILs. GD thyrocytes expressed higher levels of Bcl-2 compared with HT thyrocytes. The opposite pattern was observed in GD (low Bcl-2) and HT (high Bcl-2) TILs. Consistently, thyrocyte apoptosis was marked in HT and poor in GD thyroids, and TIL apoptosis was marked in GD and poor in HT. Our findings suggest that in GD thyroid the regulation of Fas/FasL/Bcl-2 favors apoptosis of infiltrating lymphocytes. Moreover, the reduced levels of Fas/FasL and increased levels of Bcl-2 should favor thyrocyte survival and hypertrophy associated with stimulatory thyroid-stimulating hormone receptor antibodies. In contrast, the regulation of Fas/FasL/Bcl-2 expression in HT can promote thyrocyte apoptosis via homophylic Fas-FasL interactions, and a gradual reduction in thyrocyte numbers leading to hypothyroidism. Fas-mediated apoptosis may be a general mechanism of cell damage in destructive organ-specific autoimmunity.

At least two signals for proliferation and cytokine secretion by T-cells are required. The first signal is delivered through the interaction of the T-cell receptor with major histocompatibility complex (MHC) molecules expressed on the surface of antigen-presenting cells (APC). The second or costimulatory signal is delivered by cell surface molecules expressed by APC. The interaction of B7.1/B7.2 with CD28 provide the most potent costimulatory signal for T-cell activation. CD40 antigen and its ligand (CD40L) have been shown to play a major role in regulating both humoral and cellular immune responses. In autoimmune thyroid diseases autoantigen presentation could be provided by "professional" APC, such as dendritic cells, as well as "nonprofessional" APC, such as thyroid follicular cells (TFC). In fact, these cells aberrantly express MHC class II molecules in Graves' disease (GD) and Hashimoto's thyroiditis (HT), together with large amounts of MHC class I antigens: moreover, the expression of CD40 on TFC, has been demonstrated. On the other hand B7.1 has been demonstrated in HT, but not in GD TFC. This could provide in HT a local costimulatory signal for T-cell differentiation towards a type 1 cytokine secretion pattern and also result in rescue from apoptosis of infiltrating lymphocytes. The presence of ICAM-1 on the surface of HT TFC may further strengthen contact and facilitate cross-signaling between T-cells and TFC. In contrast, the absence of B7 and ICAM-1 antigens in most GD TFC may more easily be associated with anergy and apoptosis of infiltrating T-cells, preventing the perpetuation and expansion of a "destructive" autoimmune reaction.

Cultured thyroid epithelial cells can be induced to express intercellular adhesion molecule-1 (ICAM-1, or CD54). However, constitutive follicular expression of ICAM-1 has been reported only in thyroid autoimmunity. We evaluated the expression of ICAM-1 mRNA and protein on thyroid tissue from different autoimmune thyroid diseases, and its relationship with other immunologically relevant surface markers, namely costimulatory molecules of B7 family. Thyroid tissue sections were obtained by surgically removed thyroid glands from 6 patients with Hashimoto's thyroiditis (HT), 6 with Graves' disease (GD) and 3 with multinodular nontoxic goiter. We used in situ hybridization to localize ICAM-1 mRNA, and immunohistochemical analysis by alkaline phosphatase anti-alkaline phosphatase (APAAP) method. We showed a clear hybridization pattern, localized in follicular cells, in sections of glands with HT. The hybridization pattern was far less pronounced in GD: no staining was apparent on follicular cells. These results were strictly consistent with those obtained by means of immunohistochemistry. Moreover, double-staining experiments demonstrated colocalization of ICAM-1 and B7.1 molecules in HT, whereas no B7.1 expression was observed in Graves' or in non-autoimmune thyroid diseases. These data agree with the hypothesis of distinct immunoregulatory phenomena and effector mechanisms in the 2 main autoimmune thyroid diseases.

Background: We have previously shown aberrant expression of the 'leukocyte' integrin LFA-1 on epithelial cells in chronic autoimmune thyroiditis. In the present study we investigated whether conjunctival epithelial cells, which bear the adhesion molecule ICAM-1 on their surface during allergic inflammation, may also aberrantly express its natural ligand, the 'leukocyte' integrin LFA-1., Methods: We studied 13 patients with rhinoconjunctivitis allergic to mites, chronically exposed to the allergen, 11 patients allergic to pollen tested out of the pollen season and 8 normal volunteers. Single and double immunocytochemical staining of conjunctival smears was employed., Results: LFA-1 staining on epithelial cells was demonstrated in 12/13 patients allergic to mites and not in normal controls or in patients allergic to pollen tested out of the pollen season. The epithelial localization of LFA-1 was confirmed by double staining with anti-LFA-1 and anti-cytokeratin antibodies (both immunocytochemical and immunofluorescence)., Conclusions: Coexpression of LFA-1 and ICAM-1 during persistent allergen stimulation may be relevant for interaction between epithelial cells and activated effector cells, such as eosinophils, which bear on their surface both ICAM-1 and its beta2 integrin ligands., (Copyright 2001 S. Karger AG, Basel)

Background: Tissue transglutaminase (t-TG) is the main autoantigen recognized by the endomysium antibodies (EMA) observed in patients with celiac disease (CD). The aim of the study was to assess an ELISA method for t-TG antibodies (t-TGA) with respect to EMA IF assay in pediatric and adult patients., Methods: t-TGA were analyzed by ELISA in 220 sera samples: 82 patients with biopsy-proven untreated CD (23 adults and 59 children), 14 CD children on gluten-free diet, 18 asymptomatic relatives of CD patients, and 106 age-matched control patients with gluten-unrelated gastrointestinal diseases (58 adults and 48 children). Serum IgA EMA were tested on umbilical cord sections in all patients., Results: The great majority (92.7%) of untreated CD patients (both adults and children) were t-TGA positive (values ranging from 20.1 to > 300 AU). None of the child control patients and only two out of 58 (3.4%) of the adults with unrelated gastrointestinal diseases had serum t-TGA positivity; two out of 18 first-degree relatives with biopsy-proved silent CD were t-TGA (as well as EMA) positive. Finally, two out of 14 CD children, assuming a gluten-free diet, had serum t-TGA (as well as EMA). A highly significant correlation (P < 0.001) was observed between t-TGA concentrations and EMA. t-TGA showed a sensitivity of 87% and 95%, a specificity of 97% and 100% for adults and children, respectively., Conclusion: The method is highly sensitive and specific in the diagnosis of CD and is promising as a tool for routine diagnostic use and population screening, especially in children.