Carotid plaque instability is not related to quantity but to elemental composition of calcification.

BACKGROUND AND AIMS: Recent studies highlighted the role of calcification processes in the clinical progression of chronic cardiovascular diseases. In this study we investigated the relationship between the chemical composition of calcification and atherosclerotic plaque stability in carotid arteries. METHODS AND RESULTS: To this end, we characterized the calcification on 229 carotid plaques, by morphology, immunohistochemistry, transmission electron microscopy and energy dispersive X-ray microanalysis. Plaques were classified into two categories: unstable and stable. No significant differences were found in the incidence of the various risk factors between patients with and without carotid calcification, with the exception of diabetes. The energy dispersive X-ray microanalysis allowed us to identify two types of calcium salts in the atheromatous plaques, hydroxyapatite (HA) and calcium oxalate (CO). Our results showed that calcification is a common finding in carotid plaques, being present in 77.3% of cases, and the amount of calcium is not a factor of vulnerability. Noteworthy, we observed an association between HA calcification and unstable plaques. On the contrary, CO calcifications were mainly detected in stable plaques. CONCLUSIONS: The presence of different types of calcification in atheromatous plaques may open new perspectives in understanding the molecular mechanisms of atheroma formation and plaque instability.

Elemental analysis of histological specimens: a method to unmask nano asbestos fibers.

There is recent mounting evidence that nanoparticles may have enhanced toxicological potential in comparison to the same material in the bulk form. The aim of this study was to develop a new method for unmask asbestos nanofibers from Formalin-Fixed, Paraffin-Embedded tissue. There is an increasing amount of evidence that nanoparticles may enhance toxicological potential in comparison to the same material in the bulk form. The aim of this study was to develop a new method to unmask asbestos nanofibers from Formalin-Fixed Paraffin-Embedded (FFPE) tissue. For the first time, in this study we applied Energy Dispersive X-ray (EDX) microanalysis through transmission electron microscopy to demonstrate the presence of asbestos nanofibers in histological specimens of patients with possible occupational exposure to asbestos. The diagnostic protocol was applied to 10 randomly selected lung cancer patients with no history of previous asbestos exposure. We detected asbestos nanofibers in close contact with lung cancer cells in two lung cancer patients with previous possible occupational exposure to asbestos. We were also able to identify the specific asbestos iso-type, which in one of the cases was the same rare variety used in the workplace of the affected patient. By contrast, asbestos nanofibers were not detected in lung cancer patients with no history of occupational asbestos exposure. The proposed technique can represent a potential useful tool for linking the disease to previous workplace exposure in uncertain cases. Furthermore, Formalin-Fixed Paraffin-Embedded (FFPE) tissues stored in the pathology departments might be re-evaluated for possible etiological attribution to asbestos in the case of plausible exposure. Since diseases acquired through occupational exposure to asbestos are generally covered by workers' insurance in most countries, the application of the protocol used in this study may have also relevant social and economic implications.

Differentiation of human neuroblastoma cells toward the osteogenic lineage by mTOR inhibitor.

Current hypothesis suggest that tumors can originate from adult cells after a process of 'reprogramming' driven by genetic and epigenetic alterations. These cancer cells, called cancer stem cells (CSCs), are responsible for the tumor growth and metastases. To date, the research effort has been directed to the identification, isolation and manipulation of this cell population. Independently of whether tumors were triggered by a reprogramming of gene expression or seeded by stem cells, their energetic metabolism is altered compared with a normal cell, resulting in a high aerobic glycolytic 'Warburg' phenotype and dysregulation of mitochondrial activity. This metabolic alteration is intricately linked to cancer progression.The aim of this work has been to demonstrate the possibility of differentiating a neoplastic cell toward different germ layer lineages, by evaluating the morphological, metabolic and functional changes occurring in this process. The cellular differentiation reported in this study brings to different conclusions from those present in the current literature. We demonstrate that 'in vitro' neuroblastoma cancer cells (chosen as experimental model) are able to differentiate directly into osteoblastic (by rapamycin, an mTOR inhibitor) and hepatic lineage without an intermediate 'stem' cell step. This process seems owing to a synergy among few master molecules, metabolic changes and scaffold presence acting in a concerted way to control the cell fate.

A chondrocyte infiltrated collagen type I/III membrane (MACI® implant) improves cartilage healing in the equine patellofemoral joint model.

UNLABELLED: Autologous chondrocyte implantation (ACI) has improved outcome in long-term studies of joint repair in man. However, ACI requires sutured periosteal flaps to secure the cells, which precludes minimally-invasive implantation, and introduces complications with arthrofibrosis and graft hypertrophy. This study evaluated ACI on a collagen type I/III scaffold (matrix-induced autologous chondrocyte implantation; MACI(®)) in critical sized defects in the equine model. METHODS: Chondrocytes were isolated from horses, expanded and seeded onto a collagen I/III membrane (ACI-Maix™) and implanted into one of two 15-mm defects in the femoral trochlear ridge of six horses. Control defects remained empty as ungrafted debrided defects. The animals were examined daily, scored by second look arthroscopy at 12 weeks, and necropsy examination 6 months after implantation. Reaction to the implant was determined by lameness, and synovial fluid constituents and synovial membrane histology. Cartilage healing was assessed by arthroscopic scores, gross assessment, repair tissue histology and immunohistochemistry, cartilage glycosaminoglycan (GAG) and DNA assay, and mechanical testing. RESULTS: MACI(®) implanted defects had improved arthroscopic second-look, gross healing, and composite histologic scores, compared to spontaneously healing empty defects. Cartilage GAG and DNA content in the defects repaired by MACI implant were significantly improved compared to controls. Mechanical properties were improved but remained inferior to normal cartilage. There was minimal evidence of reaction to the implant in the synovial fluid, synovial membrane, subchondral bone, or cartilage. CONCLUSIONS: The MACI(®) implant appeared to improve cartilage healing in a critical sized defect in the equine model evaluated over 6 months.

Assessment of metal contaminants in non-small cell lung cancer by EDX microanalysis.

Human cardio-respiratory diseases are strongly correlated to concentrations of atmospheric elements. Bioaccumulation of heavy metals is strictly monitored, because of its possible toxic effects. In this work, we utilized the EDX microanalysis in order to identify the potential heavy metal accumulation in the lung tissue.  To this aim, we enrolled 45 human lung biopsies: 15 non-small cell lung cancers, 15 lung benign lesions and 15 control biopsies. Lung samples were both paraffin embedded for light microscopy study and eponepoxid embedded for transmission electron microscopy. EDX microanalysis was performed on 100 nm thick unstained ultrathin-sections placed on specific copper grids. Our results demonstrated that the EDX technology was particularly efficient in the study of elemental composition of lung tissues, where we found heavy metals, such as Cobalt (Co), Chromium (Cr), Manganese (Mn) and Lead (Pb). Furthermore, in malignant lesions we demonstrated the presence of multiple bio-accumulated elements. In fact, a high rate of lung cancers was associated with the presence of 3 or more bio-accumulated elements compared to benign lesions and control tissue (91.7%, 0%, 8.3%, respectively). The environmental impact on pulmonary carcinogenesis could be better clarified by demonstrating the presence of polluting agents in lung tissues. The application of EDX microanalysis on biological tissuescould shed new light in the study of the possible bioaccumulation of polluting agents in different human organs and systems.

Tetracycline accumulates in Iberis sempervirens L. through apoplastic transport inducing oxidative stress and growth inhibition.

Environmental antibiotic contamination is due mainly to improper and illegal disposal of these molecules that, yet pharmacologically active, are excreted by humans and animals. These compounds contaminate soil, water and plants. Many studies have reported the bioaccumulation of antibiotics in plants and their negative effects on photosynthesis, cell growth and oxidative balance. Therefore, the principal objective of this paper was the study of antibiotic accumulation sites in plants and its uptake modality. Iberis sempervirens L., grown in soil and in agar in the presence or absence of tetracycline, were used as a model system. Using confocal and transmission electron microscopy, we demonstrated that tetracycline was absorbed and propagated in plants through apoplastic transport and also accumulated in intercellular spaces. Tetracycline was rarely detected inside cells (in cytoplasm and mitochondria where, coherent to its pharmacological activity, it probably affected ribosomes), except in stomata. Moreover, we verified and clarified further the phytotoxic effects of tetracycline on plants. We observed that the antibiotic induced a large reduction in plant growth and development and inhibition of photosynthetic activity. As tetracycline may lead to oxidative stress in plants, plant cells tried to balance this disequilibrium by increasing the amount and activity of some endogenous enzyme antioxidant agents (superoxide dismutase 1 and catalase) and levels of antiradical secondary metabolites.

A role for the Tubby-like protein 1 in rhodopsin transport.

PURPOSE: To test the hypothesis that a lack of Tubby-like protein 1 (TULP1) function causes aberrant transport of nascent rhodopsin and to examine the functional relationship between the homologous proteins TULP1 and Tubby by studying mice carrying combined mutations. METHODS: Subcellular localization of TULP1 and rhodopsin in photoreceptors was determined by immunofluorescence and by postembedding immunoelectron microscopy. Mice carrying different tulp1/tubby allele combinations were examined by histology, electroretinograms (ERGs), and immunofluorescence microscopy. RESULTS: TULP1 is distributed throughout the photoreceptor cytoplasm but is excluded from the outer segments and the nuclei. In the tulp1-/- mice, ectopic accumulation of rhodopsin occurs at an early age. Both the vesicular profiles in the interphotoreceptor space and the inner segment plasma membranes are immunoreactive for rhodopsin. Mice doubly homozygous for null mutations in the tulp1 and tubby genes initially develop photoreceptors and express a battery of photoreceptor markers at age 14 days. Thereafter their photoreceptors undergo a fulminant degeneration that reaches completion by postnatal day 17. The disease phenotype in the double homozygote is much more severe than either single homozygote. Double heterozygotes are phenotypically normal. CONCLUSIONS: A lack of TULP1 function results in misrouting of nascent rhodopsin. TULP1 may be a component of the cellular machinery that targets nascent rhodopsin to the outer segments. Comparison of disease phenotypes in the single and double mutants suggests that TULP1 and Tubby are not functionally interchangeable in photoreceptors nor do they form an obligate functional complex.

Treatment of pain in methadone-maintained patients.

Patients with opioid dependency experience trauma, acute medical illness and chronic diseases, and may have to undergo surgery to the same extent as other individuals. They need to be treated for relief of symptoms, including pain. Undertreatment or inadequate treatment of pain for these individuals is a particular problem because of opioid dependency and/or methadone maintenance treatment. The guiding principles governing treatment of these patients are to maintain the methadone treatment and to use short-acting narcotics administered at higher doses, and to do so as often as necessary, preferably on a fixed schedule, to relieve the pain. Supplemental analgesic medication may also be employed, except that opiate antagonists must be avoided.

Safer sex strategies for women: the hierarchical model in methadone treatment clinics.

Women clients of a methadone maintenance treatment clinic were targeted for an intervention aimed to reduce unsafe sex. The hierarchical model was the basis of the single intervention session, tested among 63 volunteers. This model requires the educator to discuss and demonstrate a full range of barriers that women might use for protection, ranking these in the order of their known efficacy. The model stresses that no one should go without protection. Two objections, both untested, have been voiced against the model. One is that, because of its complexity, women will have difficulty comprehending the message. The second is that, by demonstrating alternative strategies to the male condom, the educator is offering women a way out from persisting with the male condom, so that instead they will use an easier, but less effective, method of protection. The present research aimed at testing both objections in a high-risk and disadvantaged group of women. By comparing before and after performance on a knowledge test, it was established that, at least among these women, the complex message was well understood. By comparing baseline and follow-up reports of barriers used by sexually active women before and after intervention, a reduction in reports of unsafe sexual encounters was demonstrated. The reduction could be attributed directly to adoption of the female condom. Although some women who had used male condoms previously adopted the female condom, most of those who did so had not used the male condom previously. Since neither theoretical objection to the hierarchical model is sustained in this population, fresh weight is given to emphasizing choice of barriers, especially to women who are at high risk and relatively disempowered. As experience with the female condom grows and its unfamiliarity decreases, it would seem appropriate to encourage women who do not succeed with the male condom to try to use the female condom, over which they have more control.