Separation of hemodynamic signals from GCaMP fluorescence measured with wide-field imaging.

Wide-field calcium imaging is often used to measure brain dynamics in behaving mice. With a large field of view and a high sampling rate, wide-field imaging can monitor activity from several distant cortical areas simultaneously, revealing cortical interactions. Interpretation of wide-field images is complicated, however, by the absorption of light by hemoglobin, which can substantially affect the measured fluorescence. One approach to separating hemodynamics and calcium signals is to use multiwavelength backscatter recordings to measure light absorption by hemoglobin. Following this approach, we develop a spatially detailed regression-based method to estimate hemodynamics. This Spatial Model is based on a linear form of the Beer-Lambert relationship but is fit at every pixel in the image and does not rely on the estimation of physical parameters. In awake mice of three transgenic lines, the Spatial Model offers improved separation of hemodynamics and changes in GCaMP fluorescence. The improvement is pronounced near blood vessels and, in contrast with the Beer-Lambert equations, can remove vascular artifacts along the sagittal midline and in general permits more accurate fluorescence-based determination of neuronal activity across the cortex.NEW & NOTEWORTHY This paper addresses a well-known and strong source of contamination in wide-field calcium-imaging data: hemodynamics. To guide researchers toward the best method to separate calcium signals from hemodynamics, we compare the performance of several methods in three commonly used mouse lines and present a novel regression model that outperforms the other techniques we consider.

The release of Doxorubicin from liposomes monitored by MRI and triggered by a combination of US stimuli led to a complete tumor regression in a breast cancer mouse model.

The work aimed at developing a novel MRI-based theranostic protocol for improving the anticancer efficacy of a Doxil-like liposomal formulation. The goal was achieved stimulating the intratumor release of the drug from the nanocarrier and favoring its diffusion in the lesion by the sequential application of low-intensity pulsed ultrasound. The protocol was tested on mice bearing a syngeneic breast cancer model. The combination of acoustic waves with different characteristics allowed for: i) the release of the drug and the co-encapsulated MRI agent (Gadoteridol) from the liposomes in the vessels of the tumor region, and ii) the extravasation of the released material, as well as intact liposomes, in the tumor stroma. The MR-T1 contrast enhancement measured in the tumor reported on the delivery and US-triggered release of Doxorubicin. The developed protocol resulted in a marked increase in the intratumor drug concentration that, in turn, led to the complete regression of the lesion. The protocol has a good clinical translatability because all the components of the theranostic agent (Doxorubicin, liposomes, Gadoteridol) are approved for human use.

Sonosensitive theranostic liposomes for preclinical in vivo MRI-guided visualization of doxorubicin release stimulated by pulsed low intensity non-focused ultrasound.

The main goal of this study was to assess the theranostic performance of a nanomedicine able to generate MRI contrast as a response to the release from liposomes of the antitumor drug Doxorubicin triggered by the local exposure to pulsed low intensity non focused ultrasounds (pLINFU). In vitro experiments showed that Gadoteridol was an excellent imaging agent for probing the release of Doxorubicin following pLINFU stimulation. On this basis, the theranostic system was investigated in vivo on a syngeneic murine model of TS/A breast cancer. MRI offered an excellent guidance for monitoring the pLINFU-stimulated release of the drug. Moreover, it provided: i) an in vivo proof of the effective release of the liposomal content, and ii) a confirmation of the therapeutic benefits of the overall protocol. Ex vivo fluorescence microscopy indicated that the good therapeutic outcome was originated from a better diffusion of the drug in the tumor following the pLINFU stimulus. Very interestingly, the broad diffusion of the drug in the tumor stroma appeared to be mediated by the presence of the liposomes themselves. The results of this study highlighted either the great potential of US-based stimuli to safely trigger the release of a drug from its nanocarrier or the associated significant therapeutic improvement. Finally, MRI demonstrated to be a valuable technique to support chemotherapy and monitoring the outcome. Furthermore, in this specific case, the theranostic agent developed has a high clinical translatability because the MRI agent utilized is already approved for human use.

In vivo maps of extracellular pH in murine melanoma by CEST-MRI.

PURPOSE: A novel method based on the use of Yb-HPDO3A as MRI Para-CEST agent for in vivo pH mapping of the tumor region in a melanoma murine model is reported. This method does not require the knowledge of the concentration of the imaging agent. METHODS: C57BL/6-mice were inoculated with B16-F10 cells. CEST-MR images of tumor and bladder were acquired upon the i.v. administration of Yb-HPDO3A (1.2 mmol/Kg). pH was assessed by the use of a ratiometric method. RESULTS: Yb-HPDO3A distributes well in the extracellular space of the tumor allowing the detection of good levels of saturation transfer (ST). It is excreted throughout kidneys and accumulated in the bladder thus yielding a strong CEST signal from urine. By comparing the ST% obtained upon selective irradiation of the two OH resonances belonging to the two isomeric forms of Yb-HPDO3A, it has been possible to measure the extracellular pH for each voxel (0.22 mm(3) ). The obtained pH-maps of tumors show a great heterogeneity. Marked differences are associated to tumor staging. CONCLUSION: The application of Yb-HPDO3A to measure extracellular tumor pH provides a good spatio-temporal resolution and it does not require the prior knowledge of the contrast agent concentration. The herein reported data support the potential clinical translation of Yb-HPDO3A.

Advances in metal-based probes for MR molecular imaging applications.

The role of MRI in the armory of diagnostic modalities for the medicine of the forthcoming years largely depends on how chemistry will provide advanced tools to meet the medical needs. This review aims at outlining the most innovative approaches that have been undertaken in the recent history of MRI contrast agents for tackling the challenges of sensitivity and specificity required by the new generation of contrast agents that should allow the visualization of pathological processes occurring on cellular and molecular scale (the so-called Molecular Imaging). Most of the classes of MRI agents clinically approved or currently under investigation in a preclinical phase exploit peculiar magnetic properties of metals. The conventional agents acting as T(1) or T(2)/T(2)* relaxation enhancers are primarily based on the paramagnetic or the superparamagnetic properties of Gd(III)-, Mn(II)- and iron oxides systems. Recently, there has been a renewed interest towards paramagnetic lanthanide complexes with an anisotropic electronic configuration thanks to their ability to induce strong effect on the resonance frequency of the spins dipolarly coupled with them. Such systems, formerly mainly used as shift reagents, have now attracted much attention in the emerging field of Chemical Exchange Saturation Transfer (CEST) MRI agents.

The effect of acute treadmill walking on cognitive control and academic achievement in preadolescent children.

The effect of an acute bout of moderate treadmill walking on behavioral and neuroelectric indexes of the cognitive control of attention and applied aspects of cognition involved in school-based academic performance were assessed. A within-subjects design included 20 preadolescent participants (age=9.5+/-0.5 years; eight female) to assess exercise-induced changes in performance during a modified flanker task and the Wide Range Achievement Test 3. The resting session consisted of cognitive testing followed by a cardiorespiratory fitness assessment to determine aerobic fitness. The exercise session consisted of 20 min of walking on a motor-driven treadmill at 60% of estimated maximum heart rate followed by cognitive testing once heart rate returned to within 10% of pre-exercise levels. Results indicated an improvement in response accuracy, larger P3 amplitude, and better performance on the academic achievement test following aerobic exercise relative to the resting session. Collectively, these findings indicate that single, acute bouts of moderately-intense aerobic exercise (i.e. walking) may improve the cognitive control of attention in preadolescent children, and further support the use of moderate acute exercise as a contributing factor for increasing attention and academic performance. These data suggest that single bouts of exercise affect specific underlying processes that support cognitive health and may be necessary for effective functioning across the lifespan.

Lichens on asbestos-cement roofs: bioweathering and biocovering effects.

Asbestos-cement roofs, the most widespread sources of airborne, toxic and carcinogenic asbestos fibres, are often colonized by lichens. Since these latter are physical and chemical weathering agents, they have been often considered as significant responsible of disaggregation processes increasing fibre dispersion. Consequently, official guidelines for the management of asbestos often suggest their removal. Weathering and/or covering effects of lichens on asbestos-cement, however, have never been deeply investigated and available procedures to evaluate asbestos-cement aging do not take the biological colonization into account. In this study we show that a 25% lichen cover modifies physical and chemical properties of asbestos-cement sheets containing chrysotile and crocidolite fibres. By innovatively coupling pull up tests and image analysis of linear structures, we show that fibre loss is significantly lower ( approximately 30%) where lichens develop and offer a physical barrier to the fibre detachment. Below the most covering lichens (Acarospora cervina, Candelariella ssp.), chrysotile and crocidolite undergo a partial incongruent dissolution, which in laboratory assays generally determined a reduction of their surface reactivity. Because of their biocovering and bioweathering effects, lichens on asbestos-cement play a role which differs from the current public opinion and the assumptions of some official regulations, acting as effective spontaneous bioattenuation agents.

Development and validation of a smoothing-splines-based correction method for improving the analysis of CEST-MR images.

Chemical exchange saturation transfer (CEST) imaging is an emerging MRI technique relying on the use of endogenous or exogenous molecules containing exchangeable proton pools. The heterogeneity of the water resonance frequency offset plays a key role in the occurrence of artifacts in CEST-MR images. To limit this drawback, a new smoothing-splines-based method for fitting and correcting Z-spectra in order to compensate for low signal-to-noise ratio (SNR) without any a priori model was developed. Global and local voxel-by-voxel Z-spectra were interpolated by smoothing splines with smoothing terms aimed at suppressing noise. Thus, a map of the water frequency offset ('zero' map) was used to correctly calculate the saturation transfer (ST) for each voxel. Simulations were performed to compare the method to polynomials and zero-only-corrected splines on the basis of SNR improvement. In vitro acquisitions of capillaries containing solutions of LIPOCEST agents at different concentrations were performed to experimentally validate the results from simulations. Additionally, ex vivo investigations of bovine muscle mass injected with LIPOCEST agents were performed as a function of increasing pulse power. The results from simulations and experiments highlighted the importance of a proper 'zero' correction (15% decrease of fictitious CEST signal in phantoms and ex vivo preparations) and proved the method to be more accurate compared with the previously published ones, often providing a SNR higher than 5 in different simulated and experimentally noisy conditions. In conclusion, the proposed method offers an accurate tool in CEST investigation.

Effect of a topical treatment in organotypic culture of human breast skin after exposure to gamma-rays.

The early radiation of epidermal reactions can lead to healing of the lesion or radiation necrosis. There is no general agreement for either the prevention and/or treatment of radiation skin response, also as little is known about the immediate phases of this phenomenon. We investigated the early effects exerted by Healing and Wound Emulsion (HWE) on human skin response after ionizing radiation. Epidermal morphology, Heat Shock Protein (HSP) 70, and Transforming Growth Factor-beta1 (TGF-beta1) gene expression were investigated in organotypic human skin cultures undergoing a double dose of gamma-rays (2 Gy). HSP70 gene expression tended to be induced in the HWE group 6 hours after cream administration and was significantly up-regulated after 48 hours, when epidermal morphological alterations were evident. TGF-beta1 seems not affected in cream treated samples. HWE may stimulate skin to mount an early defensive response against damage induced by gamma rays.

An in vivo method to assess the photostability of UV filters in a sunscreen.

An in vivo method was developed in order to study the photostability of UV filters in a sunscreen. This method assesses sunscreen photostability in the emulsion after direct interaction with the skin. For this purpose UV filters were recovered from volunteers' forearms by using the stripping technique, then extracted from tapes and quantified by high-performance liquid chromatography (HPLC). The photostability of the filters tested was evaluated by comparing the amount of filters recovered from the strippings of UV-irradiated skin (40 minimal erythema dose, or MED) versus non-irradiated skin. Sequential analysis of several successive tapes reflected the distribution profile of the filters in the stratum corneum. Photochemical change was observed for one filter: it was shown to undergo a photochemical modification with the appearance of an additional HPLC peak. Moreover, UV filters tested displayed a high affinity for the stratum corneum but presented different distribution profiles. This in vivo method takes into account the interaction of the sunscreen agents with the stratum corneum. Furthermore, unlike spectrometric methods usually used for photostability assessment, it gives quantitative data for each individual filter of a finished product by using an HPLC technique.

Expression studies of key adipogenic transcriptional factors reveal that the anti-adipogenic properties of retinol in primary cultured human preadipocytes are due to retinol per se.

The aim of this study was to investigate the mechanism(s) underlying the antiadipogenic effect of retinol that we recently reported in primary cultured human preadipocytes. Exposure of human preadipocytes to the potent alcohol dehydrogenase inhibitor, 4-methyl-pyrazole, failed to alter the antiadipogenic effect of retinol (3.5 microm), suggesting that the latter effect is due to retinol per se rather than to its oxidation product, retinoic acid (RA). Moreover, retinol, in contrast to what is generally observed with RA, did not alter the expression of the major adipogenic transcriptional factors PPARgamma and C/EBPalpha but, like RA, reduced transcription of an adipospecific gene controlled in part by C/EBP, the ob gene. These results indicate that retinol per se inhibits the adipo-conversion of human preadipocytes and suggest that the mechanisms of this antiadipogenic action implies at least in part inhibition of C/EBP transcriptional activity.

Prevalence of Helicobacter pylori and hepatitis C virus infections among non-Hodgkin's lymphoma patients in Southern Switzerland.

BACKGROUND AND OBJECTIVE: Several recent studies have reported a high rate of previous hepatitis C virus (HCV) infection in patients with non-Hodgkin's lymphoma (NHL). However, it appears that there are marked geographical differences in the prevalence of HCV among NHL patients. There is further controversy concerning a possible pathogenetic link between HCV and certain histologic lymphoma subtypes, in particular MALT lymphomas, and it has recently been speculated that HCV might be involved in the multistep process of gastric lymphoma genesis, in addition to the well established role of chronic Helicobacter pylori infection. The aim of this study was to investigate the prevalence of HCV and H. pylori infections in patients with B-cell NHL in Southern Switzerland. DESIGN AND METHODS: One hundred and eighty newly diagnosed HIV-negative B-cell NHL patients, consecutively seen at a referral oncology center in Southern Switzerland between 1990 and 1995 were prospectively studied. A microparticle enzyme immunoassay was used to detect antibodies to HCV. Serologic determination of HCV genotype was done by the Murex method. The quantitative detection of IgG anti-H. pylori was performed by the Biorad GAP test. RESULTS: Infection with HCV was detected in 17/180 patients (9.4%; 95% C.I., 6%-15%). This prevalence is significantly higher than that observed in a large survey of 5424 new blood donors from the same area tested in 1992-97 (0.9%; 95% C.I., 0.7-1.2). Neither histologic subtypes nor specific extranodal presentations of NHL were associated with a higher prevalence of HCV. HCV serotype 2 (corresponding to genotypes 2a-c) was the most common. HCV infection was significantly associated with a shorter progression-free survival at both univariate and multivariate analysis. Anti-Helicobacter antibodies were detected in 81/180 patients (45%; 95% C.I., 38%-53%) and H. pylori infection was significantly associated with the development of primary lymphomas of the stomach. INTERPRETATION AND CONCLUSIONS: A high prevalence of HCV infection was detected in NHL lymphoma patients and was associated with a shorter time to lymphoma progression. HCV infection was not correlated with primary gastric presentation or with MALT-type histology. Our findings further support the key role of H.pylori infection in the pathogenesis of primary gastric lymphoma of MALT-type. The possible role of HCV in the pathogenesis of NHL should be further investigated.

Antiadipogenic properties of retinol in primary cultured differentiating human adipocyte precursor cells.

The aim of this study was to investigate the effect of retinol on the human adipose conversion process using primary cultured human adipocyte precursor cells. When these cells were seeded in a medium containing retinol (concentrations ranging from 3.5 nM to 3.5 muM), cell proliferation was slightly inhibited by high concentrations of retinol, as demonstrated by cell counting and [(3)H]-thymidine incorporation. Moreover, the differentiation capacities of these cells were markedly and dose-dependently inhibited by retinol, as shown by the reduced expression of the lipogenic enzyme glycerol-3-phosphate dehydrogenase and by microscopic morphological analysis. These results strongly suggest that retinol, by inhibiting the ability of human preadipocytes to convert into mature adipocytes, could be of potential interest in the prevention of human adipose tissue development in general and of cellulitis in particular.

[Usefulness of atrial vulnerability in predicting atrial fibrillation in patients with sick sinus syndrome treated with DDD pacemaker]. / Utilità della vulnerabilità atriale nel prevedere l'insorgenza di fibrillazione atriale in pazienti con malattia del nodo del seno trattata con pacemaker DDD.

BACKGROUND: Atrial fibrillation (AF) is common in patients with sick sinus syndrome (SSS) treated with dual-chamber pacing and it may complicate their management. This study was undertaken to establish the usefulness of atrial vulnerability (AV), determined by means of transesophageal electrophysiological study (TES), in predicting the risk of developing AF and in deciding the type and program of pacemaker (PM) to be implanted in patients with SSS. METHOD: AV was assessed preoperatively using TES in 81 consecutive patients with SSS. AV (AF > 1 min) was divided into "low threshold" (induction with burst < or = 300/min) and "high threshold" (induction with burst > or = 350 min or with incremental ramp). The PM was programmed to ensure constant atrial capture in all patients. Follow-up lasted three years. No patients received antiarrhythmic drugs. RESULT: AV was positive in 52% of patients (Group A) and negative in 48% of patients (Group B). A history of paroxysmal AF was present in 52% of patients in Group A and in 12% of patients in Group B. At follow-up, 38% of Group A and 2% of Group B patients had chronic AF. AV had sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of 94, 59, 38 and 97%, respectively. Thirty-eight percent of patients showed low threshold vulnerability, with sensitivity, specificity, ppv and npv of 87, 92, 87 and 93%, respectively. Sensitivity, specificity, ppv and pnv for history of AF were 93, 100, 98 and 84%, respectively. When the vulnerability threshold and the history of paroxysmal AF were considered together, the sensitivity, specificity, ppv and npv was 94, 100, 100 and 83%, respectively. Multivariate analysis was shown to be an independent predictive value only for history of AF (p = 0.0002). CONCLUSION: AV determined by means of TES, especially with a low induction threshold, shows excellent sensitivity and specificity in evaluating the risk of chronic AF. It could be useful in patients with SSS undergoing cardiac pacing who have never had AF, thus allowing a more accurate choice of the type and program of PM to be implanted. Case histories of paroxysmal AF could represent useful criteria for selecting patients at a high risk of developing chronic AF.

Relevance and reliability of the PREDISAFE assay in the COLIPA eye irritation validation program (phase 1).

The 6th Amendment of the European Directive on Cosmetics induces a potential ban on animal testing for cosmetic ingredients and finished products. In this new context, COLIPA (The European Cosmetic Toiletry and Perfumery Association) has initiated an international multicentric study with the main goal of validating available alternatives to in vitro methods for assessing the eye irritation potential of cosmetic raw materials and formulations. In order to test undiluted and hydrophobic ingredients and formulations, a cytotoxicity test named PREDISAFE was incorporated into our internal battery of in vitro tests for 3 years. This cell culture test based on the neutral red release procedure was prevalidated with several cosmetic formulations and used systematically by comparison with internal benchmarks. In this article, the defined prediction model and the protocol used in the COLIPA eye irritation program are described, and furthermore the PREDISAFE assay results obtained during Phase I of the above mentioned study are presented and discussed in detail. The statistical analysis proves clearly a great interest in the PREDISAFE test for the prediction of eye irritation potential of cosmetic formulations. Its strong compatibility for a wide category of finished products associated with its ease of use offer relevant advantages for a routine use in the ocular irritancy screening in the cosmetics industry. This paper also explains the reasons for false negative and false positive in vitro tests results and describes possible technical modifications to avoid these wrong predictions. At the end, some recommendations for the Phase II of the COLIPA study are considered with the main objective to prove that a multivariable analysis could be useful to find the best battery of in vitro assays for acceptance by the regulators for the replacement of the Draize eye irritation test.

Intrapatient comparison of an intermittent and a continuous flow cell separator for the collection of progenitor and stem cells from the blood.

BACKGROUND AND OBJECTIVES: Continuous-flow and intermittent-flow blood cell separators (CFCS and IFCS) are both used to collect stem cells from the blood to rescue patients undergoing myeloablative treatment for cancer. MATERIALS AND METHODS: We designed a study to compare the collection efficiency of the two systems. The continuous-flow Cobe Spectra and the intermittent-flow Haemonetics MCS-3P were used to collect cells on consecutive days from 9 patients mobilised with G-CSF with or without chemotherapy. Blood obtained before leukapheresis and the leukapheresis product were analysed for their content of red and white cells, platelets, CD34-positive cells, GM-CFC, CFC-E, and BFU-E. An extraction ratio was calculated. RESULTS: We found that the CFCS extracted about 4 times more mononuclear cells per unit time, 3 times more CD34-positive, and 4 times more clonogenic cells than the IFCS. The subject acceptability of the two systems was similar. CONCLUSION: The CFCS is a more efficient system for stem cell collection. IFCS requires a longer harvesting time for the same result.

Ageing and rheological properties of facial skin in women.

Ageing affects the mechanical properties of skin. Studies using objective measurements on facial skin have been rare and yielded contradictory information. In the present study, the age-related changes occurring in the mechanical properties of facial skin were reviewed using a computerized suction method. A total of 200 healthy women, aged from 17 to 68 years, were enrolled. Data show a significant increase in skin extensibility and a significant decline in elasticity with ageing. These changes become obvious in women approaching 40 years.

Pretreatment of skin with a Ginkgo biloba extract/sodium carboxymethyl-beta-1,3-glucan formulation appears to inhibit the elicitation of allergic contact dermatitis in man.

The clinical efficiency of mitigating contact dermatitis with a Ginkgo biloba extract and carboxymethyl-beta-1,3-glucan formulation was investigated in a double-blind versus placebo study using 22 subjects (Caucasian women aged 22-55 years) with allergic contact dermatitis from various substances in the European standard series. The formulation was applied to intact skin 2X a day for 2 weeks ("in use" application) prior to a single application of a selected contact allergen under a Finn Chamber for 24 h. Readings were carried out in a blind study by a dermatologist 2 and 3 days after patch removal. Representative photographs were taken of treated, placebo and untreated test areas. 68.2% of the panelists showed significantly reduced skin reactivity (p = 0.037*) on the treated site 2 days after patch removal, versus untreated and/or placebo sites. This finding indicates that the Ginkgo biloba/carboxymethyl-beta-1,3-glucan formulation can mitigate against allergic contact dermatitis.

Industrial pollutants in ground waters from northern Milan.

Ground water samples from an industrialised area near Milan were analysed by gas chromatography-mass spectrometry (GC-MS) to identify the main pollutants and to quantify two classes of chemicals: polychloro-1,3-butadienes (PCBD) and some aromatic amines. The water contained several halogenated aromatic and aliphatic compounds and heavy contamination due to PCBD, probably arising from contaminated land where a disused chemical plant is located. All the samples contained low levels of aromatic amines indicating a diffuse contamination probably arising from different sources.