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Multipotent stem cells persist within the stromal vascular fraction (SVF) of adipose tissue during adulthood. These cells, commonly referred to as adipose-derived stromal cells (ASC), have been extensively investigated over the past years as a promising therapeutic tool based on their regenerative and immunomodulatory properties. However, how ASC might mirror the age-related alteration of the fat they reside in remains unclear. Herein, we show that inguinal adipose tissue in mice turns from brown/beige- to white-like with age and resident ASC readily mirror these changes both at mRNA and microRNA transcriptional level. Mechanistically, our data suggest that these brown/age-related changes in ASC transcription rely on changes in the activity of E2F1 and NFkB transcription factors.

Rhabdoid colorectal carcinoma (RC) is a rare lesion localized to the proximal colon of patients with a mean age at diagnosis of around 70 years. This tumor shows an aggressive behavior with an overall survival period shorter than 12 months. The diagnostic hallmark is the presence of rhabdoid cells. Alterations in chromatin remodeling (SMARCB1) and in the centrosome structure (CROCC) are reported in RC usually BRAFmut and MSI-H. RKO intestinal neoplastic cells culture (BRAFmut, SMARCB1wt, MSI-H) with CROCC knockdown exhibit rhabdoid features and develop prominent projections from the edge of the cell. Here, we investigated two cases of CROCCmutSMARCB1wt RC by scanning and transmission electron microscopy (SEM, TEM). TEM confirmed the diagnostic presence of intermediate cytoplasmic filaments and nucleolar margination. SEM showed cellular protrusions (lamellipodia) in the intercellular spaces not evident at light microscopy. These protrusions CROCC-related might represent the pathogenetic mechanism underlying the rhabdoid aggressive behavior, independently of tumor staging. To our knowledge, the SEM technique was applied in the study of this neoplasm for the first time.

Dysregulated immunity and widespread metabolic dysfunctions are the most relevant hallmarks of the passing of time over the course of adult life, and their combination at midlife is strongly related to increased vulnerability to diseases; however, the causal connection between them remains largely unclear. By combining multi-omics and functional analyses of adipose-derived stromal cells established from young (1 month) and midlife (12 months) mice, we show that an increase in expression of interferon regulatory factor 7 (IRF7) during adult life drives major metabolic changes, which include impaired mitochondrial function, altered amino acid biogenesis and reduced expression of genes involved in branched-chain amino acid (BCAA) degradation. Our results draw a new paradigm of aging as the ‘sterile’ activation of a cell-autonomous pathway of self-defense and identify a crucial mediator of this pathway, IRF7, as driver of metabolic dysfunction with age.

Background The major intrinsic cause of facial skin degeneration is age, associated with extrinsic factors such as exposure to sun. Its major pathologic causes are degeneration of the elastin matrix, with loss of oxytalan and elaunin fibers in the subepidermal region, and actinic degeneration of elastin fibers that lose their functional properties in the deep dermis. Therapy using autologous adipose mesenchymal stem cells for regeneration of extracellular matrix in patients with solar elastosis was addressed in qualitative and quantitative analyses of the dermal elastic fiber system and the associated cells. Methods Mesenchymal stem cells were obtained from lipoaspirates, expanded in vitro, and introduced into the facial skin of patients submitted after 3 to 4 months to a face-lift operation. In the retrieved skin, immunocytochemical analyses quantified elastic matrix components; cathepsin K; matrix metalloproteinase 12 (macrophage metalloelastase); and the macrophage M2 markers CD68, CD206, and hemeoxygenase-1. Results A full de novo formation of oxytalan and elaunin fibers was observed in the subepidermal region, with reconstitution of the papillary structure of the dermal-epidermal junction. Elastotic deposits in the deep dermis were substituted by a normal elastin fiber network. The coordinated removal of the pathologic deposits and their substitution by the normal ones was concomitant with activation of cathepsin K and matrix metalloproteinase 12, and with expansion of the M2 macrophage infiltration. Conclusion The full regeneration of solar elastosis was obtained by injection of in vitro expanded autologous adipose mesenchymal stem cells, which are appropriate, competent, and sufficient to elicit the full structural regeneration of the sun-aged skin. Clinical question/level of evidence Therapeutic, IV.

The implantation of breast prostheses for both aesthetic and reconstructive purposes has been growing exponentially in the last 20 years. Safety and prosthesis lifespan are majorly debated issues in relation to the correlated long-term complications. Mainly the periprosthetic capsule that develops around the implant is often the cause of complications and particularly for macrotextured silicone breast implants. Some reports have tried to elucidate the mechanism by which macrotextured silicone implants undergo damage and cause double capsule formation. In this study, we investigated the morphological characteristics of double capsule of macrotextured implants surgically removed from patients. With the use of microscopy techniques, this work analyzed the newly formed tissue observed in the interaction between synthetic and biological surfaces.

Background. Stem cells from adipose tissue (ADSCs) and platelet-rich plasma (PRP) are innovative modalities that arise due to their regenerative potential. Objective. The aim of this study was to characterize possible histological changes induced by PRP and ADSC therapies in photoaged skin. Methods. A prospective randomized study involving 20 healthy individuals, showing skin aging. They underwent two therapeutic protocols (protocol 1: PRP; protocol 2: ADSCs). Biopsies were obtained before and after treatment (4 months). Results. PRP protocol showed unwanted changes in the reticular dermis, mainly due to the deposition of a horizontal layer of collagen (fibrosis) and elastic fibers tightly linked. Structural analyses revealed infiltration of mononuclear cells and depot of fibrotic material in the reticular dermis. The ADSC protocol leads to neoelastogenesis with increase of tropoelastin and fibrillin. There was an improvement of solar elastosis inducing an increment of macrophage polarization and matrix proteinases. These last effects are probably related to the increase of elastinolysis and the remodeling of the dermis. Conclusions. The PRP promoted an inflammatory process with an increase of reticular dermis thickness with a fibrotic aspect. On the other hand, ADSC therapy is a promising modality with an important antiaging effect on photoaged human skin.

Malignant gliomas, the most frequent primary brain tumors, are characterized by a dismal prognosis. Reliable biomarkers complementary to neuroradiology in the differential diagnosis of gliomas and monitoring for post-surgical progression are unmet needs. Altered expression of several microRNAs in tumour tissues from patients with gliomas compared to normal brain tissue have been described, thus supporting the rationale of using microRNA-based biomarkers. Although different circulating microRNAs were proposed in association with gliomas, they have not been introduced into clinical practice so far. Blood samples were collected from patients with high and low grade gliomas, both before and after surgical resection, and the expression of miR-21, miR-222 and miR-124-3p was measured in exosomes isolated from serum. The expression levels of miR-21, miR-222 and miR-124-3p in serum exosomes of patients with high grade gliomas were significantly higher than those of low grade gliomas and healthy controls and were sharply decreased in samples obtained after surgery. The analysis of miR-21, miR-222 and miR-124-3p in serum exosomes of patients affected by gliomas can provide a minimally invasive and innovative tool to help the differential diagnosis of gliomas at their onset in the brain and predict glioma grading and non glial metastases before surgery.

Background In previous papers, we demonstrated that the treatment of human photoaged skin with stromal-vascular fraction-enriched fat or expanded adipose-derived stem cells showed a decrease of elastosis and the appearance of new oxytalan elastic fibers in dermis and an increase in the vascular network. The utilization of fat plus platelet-rich plasma (PRP) led to an increase in the vascular permeability and reactivity of the nervous component. Objectives The purpose of this study was to analyze the histologic and ultrastructural changes of human skin after the injection of only PRP in the retroauricular area that was not exposed to sun and did not present the photoaging process, in comparison with our previous results. Methods This study was performed in 13 patients who were candidates for facelift and whose ages ranged between 45 and 65 years. The PRP injection was performed in the mastoidea area. Fragments of skin were removed before and 3 months after treatment and analyzed by optical and electron microscopy. Results After the injection of PRP, we observed an increase of reticular dermis thickness because of the deposition of elastic fibers and collagen, with a fibrotic aspect. A modified pattern of adipose tissue was also found at the dermohypodermal junction. Significative regenerative aspects were not found at histologic and ultrastructural analysis. The presence of foci of moderate inflammation and microangiopathy were observed. Conclusions Treatment with PRP increased reticular dermis thickness with a fibrotic aspect. In the long term, the presence of inflammation and microangiopathy caused by PRP injection could lead to trophic alteration of the skin and the precocious aging process. Level of Evidence 4

For targeted brain delivery, nanoparticles (NPs) should bypass the blood-brain barrier (BBB). Novel functionalization strategies, based on low-density lipoprotein receptor (LDLR) binding domain, have been here tested to increase the brain targeting efficacy of poly d,l-lactic-co-glycolic acid (PLGA) NPs, biodegradable and suited for biomedical applications. Custom-made PLGA NPs were functionalized with an apolipoprotein E modified peptide (pep-apoE) responsible for LDLR binding, or with lipocalin-type prostaglandin-d-synthase (L-PGDS), highly expressed in the brain. At the comparison of pep-apoE and L-PGDS sequences, a highly homologs region was here identified, indicating that also L-PGDS could bind LDLR. Non-functionalized and functionalized NPs did not affect the viability of cultured human dendritic cells, protagonists of the immune response, and did not activate them to a proinflammatory profile. At 2 h after intravenous injection in mice, functionalized, but not the non-functionalized ones, fluorescent-tagged NPs were observed in the cerebral cortex parenchyma. The NPs were mostly internalized by neurons and microglia; glial cells showed a weak activation. The findings indicate that the tested functionalization strategies do not elicit adverse immune responses and that the peptidic moieties enable BBB traversal of the NPs, thus providing potential brain drug carriers. These could be especially effective for brain diseases in which LDLR is involved. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 847-858, 2017.

Background In a previous study, the authors demonstrated that treatment with expanded adipose-derived stem cells or stromal vascular fraction (SVF)-enriched fat modify the pattern of the dermis in human beings, representing a skin rejuvenation effect. Considering that expanded stem cells require a cell factor, the authors wanted to assess similar results by replacing them with platelet-rich plasma (PRP), which is easier to obtain and for which an empirical regenerative effect has been already described. Objectives To determine if PRP injection could replace the cutaneous regenerative effect of adipose-derived stem cells. Methods This study was performed in 13 patients who were candidates for facelift. The patients underwent sampling of fat by liposuction from the abdomen and submitted to one of three protocols: injection of SVF-enriched fat or expanded adipose-derived stem cells or fat plus PRP in the preauricular areas. Fragments of skin were removed before and 3 months after treatment and analyzed by optical and electron microscopy. Results The use of fat plus PRP led to the presence of more pronounced inflammatory infiltrates and a greater vascular reactivity, increasing in vascular permeability and a certain reactivity of the nervous component. The addition of PRP did not improve the regenerative effect. Conclusion The use of PRP did not have significant advantages in skin rejuvenation over the use of expanded adipose-derived stem cells or SVF-enriched fat. The effect of increased vascular reactivity may be useful in pathological situations in which an intense angiogenesis is desirable, such as tissular ischemia. Level of Evidence: 4 ![Graphic][1] Therapeutic [10.1093/asj/sjv239][2] [1]: /embed/inline-graphic-1.gif [2]: /lookup/doi/10.1093/asj/sjv239

Therapeutic strategies for the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) have not yet provided satisfactory results. Interest in stem cells for the treatment of neurodegenerative diseases is increasing and their beneficial action seems to be due to a paracrine effect via the release of exosomes, main mediators of cell-cell communication. Here we wished to assess, in vitro, the efficacy of a novel non-cell therapeutic approach based on the use of exosomes derived from murine adipose-derived stromal cells on motoneuron-like NSC-34 cells expressing ALS mutations, and used as in vitro models of disease. In particular, we set out to investigate the effect of exosomes on NSC-34 naïve cells and NSC-34 cells overexpressing human SOD1(G93A) or SOD1(G37R) or SOD1(A4V) mutants, exposed to oxidative stress. The data presented here indicate for the first time that exosomes (0.2 µg/ml) are able to protect NSC-34 cells from oxidative damage, which is one of the main mechanism of damage in ALS, increasing cell viability. These data highlight a promising role of exosomes derived from stem cells for potential therapeutic applications in motoneuron disease.

Porous silicon (pSi) is a sponge-like material obtained by electrochemical etching of a crystalline silicon wafer. Due to quantum confinement effects, this material is photoluminescent and this is a fundamental property from the perspective of bioimaging applications. Limitations in nanomedicine to the use of photoluminescent pSi structures are mainly due to optical quenching in an aqueous environment and to the adverse effects of reactive groups introduced by etching procedures. In this work, we exploited an inorganic TiO2 coating of pSi microparticles by Atomic Layer Deposition (ALD) that resulted in optical stability of pSi particles in a biological buffer (e.g. PBS). The use of a rotary reactor allows deposition of a uniform coating on the particles and enables a fine tuning of its thickness. The ALD parameters were optimized and the photoluminescence (PL) of pSi–TiO2 microparticles was stabilized for more than three months without any significant effect on their morphology. The biocompatibility of the coated microparticles was evaluated by analyzing the release of cytokines and superoxide anion (O2−) by human dendritic cells, which play an essential role in the regulation of inflammatory and immune responses. We demonstrated that the microparticles per se are unable to significantly damage or stimulate human dendritic cells and therefore are suitable candidates for nanomedicine applications. However, a synergistic effect of the microparticles with bacterial products, which are known to stimulate immune-response, was observed, indicating that a condition unfavorable to the use of inorganic nanomaterials in biological systems is the presence of infection diseases. These results, combined with the proved PL stability in biological buffers, open the way for the use of pSi–TiO2 microparticles as promising materials in nanomedicine, but their ability to increase immune cell activation by other agonists should be considered and even exploited.

Loss-of-function mutations of the the ATP-binding cassette-1 (ABCA1) gene are the cause of Tangier disease (TD) in homozygous subjects and familial HDL deficiency (FHD) in heterozygous subjects. These disorders are characterized by reduced plasma HDL-cholesterol (HDL-C) and altered efflux of cholesterol from cells. Previous studies in TD patients and ABCA1−/− murine models reported defects in platelet count, morphology, and function, but the issue is still controversial. We analyzed three subjects with low to very low HDL-C levels due to the loss-of-function mutations of the ABCA1 gene. Two related patients with FHD were heterozygous carriers of two mutations on the same ABCA1 allele; one, with TD, was homozygous for a different mutation. Mild to moderate thrombocytopenia was observed in all the patients. No morphological platelet abnormalities were detected under optical or EM. History of moderate bleeding tendency was recorded only in one of the FHD patients. Only limited alterations in platelet aggregation and activation of the integrin αIIbβ3 were observed in one FHD patient. While α-granule secretion (P-selectin), content, and secretion of platelet δ-granules (serotonin, ATP, and ADP) and thromboxane (TX) A2 synthesis were normal in all the patients, the expression of lysosomal CD63, in response to some agonists, was reduced in TD patients. In conclusion, three patients carrying ABCA1 genetic variants had low platelet count, with the lowest values observed in TD, not associated with major alterations in platelet morphology and response to agonists or bleeding.

Introduction: Cellulite is a complex pathology that affects about 85% of women. The origin of cellulite is poorly understood, even if some studies describe different hypotheses based on the observation of subcutaneous architecture, vascular and lymphatic patterns. Material and methods: in this work an ultra-structural description of cellulite specimens, harvested from fifteen healthy women affected by first degree cellulite, was provided. Specimens were harvested from trochanter, after local anaesthesia using punch biopsy. Samples were fixated, immediately after biopsy, in glutaraldehyde 2% and processed for electron microscopy. Results: with scanning and transmission electron microscopy it was possible to describe the hypothetical cellulite starting unit, consisting of mature unilocular adipocytes strictly connected with sweat glands. The peculiar structure of this complex was not described in other adipose depots, even if this formations resembled the mammary glands during the pubertal phase in which there is a hormonal influence, leading to breast augmentation. With the ultra-structural and morphological analysis of cellulite specimens it was possible to define the possible role of sweat glands associated with mature adipocytes in the dermis of patients. Sweat glands probably stimulate mature adipocytes and mesenchymal stem cells to cause an increase in adipose tissue. In the case of cellulite the increase in adipose tissue was associated also with the stimulation of fibroblasts with the effect of collagen fibres deposition and with the consequent formation of the typical cellulite morphology. Discussion: understanding cellulite origin might be conducive to developing more specific and accurate treatments and to hypothesize novel prevention strategies.

Chorea-acanthocytosis is one of the hereditary neurodegenerative disorders known as the neuroacanthocytoses. Chorea-acanthocytosis is characterized by circulating acanthocytes deficient in chorein, a protein of unknown function. We report here for the first time that chorea-acanthocytosis red cells are characterized by impaired autophagy, with cytoplasmic accumulation of active Lyn and of autophagy-related proteins Ulk1 and Atg7. In chorea-acanthocytosis erythrocytes, active Lyn is sequestered by HSP90-70 to form high-molecular-weight complexes that stabilize and protect Lyn from its proteasomal degradation, contributing to toxic Lyn accumulation. An interplay between accumulation of active Lyn and autophagy was found in chorea-acanthocytosis based on Lyn coimmunoprecipitation with Ulk1 and Atg7 and on the presence of Ulk1 in Lyn-containing high-molecular-weight complexes. In addition, chorein associated with Atg7 in healthy but not in chorea-acanthocytosis erythrocytes. Electron microscopy detected multivesicular bodies and membrane remnants only in circulating chorea-acanthocytosis red cells. In addition, reticulocyte-enriched chorea-acanthocytosis red cell fractions exhibited delayed clearance of mitochondria and lysosomes, further supporting the impairment of authophagic flux. Because autophagy is also important in erythropoiesis, we studied in vitro CD34+-derived erythroid precursors. In chorea-acanthocytosis, we found (1) dyserythropoiesis; (2) increased active Lyn; (3) accumulation of a marker of autophagic flux and autolysososme degradation; (4) accumlation of Lamp1, a lysosmal membrane protein, and LAMP1-positive aggregates; and (5) reduced clearance of lysosomes and mitochondria. Our results uncover in chorea-acanthocytosis erythroid cells an association between accumulation of active Lyn and impaired autophagy, suggesting a link between chorein and autophagic vesicle trafficking in erythroid maturation.

Aim: To unravel key aspects of the use of lanthanide-doped nanoparticles (NPs) in biomedicine, the interaction with immune and brain cells. Materials & methods: Effects of citrate-stabilized CaF2 and SrF2: Yb, Er NPs (13–15 nm) on human dendritic cells and neurons were assessed in vitro. In vivo distribution was analyzed in mice at tissue and ultrastructural levels, and with glia immunophenotyping. Results: The NPs did not elicit dendritic cell activation and were internalized by cultured neurons, without viability changes. After intravenous injection, NPs were found in the brain parenchyma, without features of glial neuroinflammatory response. Conclusion: Lanthanide-doped NPs do not activate cells protagonists of systemic and brain immune responses, are endocytosed by neurons and can cross an intact blood–brain barrier.

Alice Busato,1,* Roberta Bonafede,1,* Pietro Bontempi,2 Ilaria Scambi,1 Lorenzo Schiaffino,1 Donatella Benati,1 Manuela Malatesta,1 Andrea Sbarbati,1 Pasquina Marzola,3 Raffaella Mariotti1 1Department of Neurosciences, Biomedicine and Movement Sciences, School of Medicine, 2Department of Biotechnology, 3Department of Computer Science, University of Verona, Verona, Italy *These authors contributed equally to this work Purpose: Recent findings indicate that the beneficial effects of adipose stem cells (ASCs), reported in several neurodegenerative experimental models, could be due to their paracrine activity mediated by the release of exosomes. The aim of this study was the development and validation of an innovative exosome-labeling protocol that allows to visualize them with magnetic resonance imaging (MRI).Materials and methods: At first, ASCs were labeled using ultrasmall superparamagnetic iron oxide nanoparticles (USPIO, 4–6nm), and optimal parameters to label ASCs in terms of cell viability, labeling efficiency, iron content, and magnetic resonance (MR) image contrast were investigated. Exosomes were then isolated from labeled ASCs using a standard isolation protocol. The efficiency of exosome labeling was assessed by acquiring MR images in vitro and in vivo as well as by determining their iron content. Transmission electron microscopy images and histological analysis were performed to validate the results obtained.Results: By using optimized experimental parameters for ASC labeling (200µg Fe/mL of USPIO and 72hours of incubation), it was possible to label 100% of the cells, while their viability remained comparable to unlabeled cells; the detection limit of MR images was of 102 and 2.5×103 ASCs in vitro and in vivo, respectively. Exosomes isolated from previously labeled ASCs retain nanoparticles, as demonstrated by transmission electron microscopy images. The detection limit by MRI was 3µg and 5µg of exosomes in vitro and in vivo, respectively.Conclusion: We report a new approach for labeling of exosomes by USPIO that allows detection by MRI while preserving their morphology and physiological characteristics. Keywords: MRI, superparamagnetic iron oxide nanoparticles, cellular imaging, stem cells labeling, exosome labeling

Glucose transport plays an important role in maintaining low sugar concentration in airway surface liquid (ASL), which is critical for mucociliary clearance and bacterial colonization. Experimental evidence indicates that glucose/hexose uptake in lung/airway cells occurs by means of two structurally distinct glucose transporter pathways: the Na+-dependent glucose transporters (SGLT family) and the facilitative glucose transporters (GLUT family). In this study, we examined the expression of the major glucose transporters of the intestine, GLUT2, GLUT5, SGLT1 and T1R3 taste receptor subunit, in the trachea of rats using immunohistochemistry and immunoelectron microscopy, and compared them using double-labeled confocal microscopy. We found that GLUT2, GLUT5, SGLT1 and T1R3 are selectively expressed in different cell types. T1R3 and GLUT2 are predominantly expressed in subsets of solitary chemoreceptor cells (SCCs) and ciliated cells, GLUT5 is present in subsets of SCCs and in secretory cells, and SGLT1 is exclusively expressed in a unique cell type, SCCs. Furthermore, we demonstrated that T1R3 is colocalized with SGLT1 in SCCs and with GLUT2 transporter in ciliated cells. In conclusion, these findings reveal that different cell types are associated with the uptake of glucose in ASL and that, due to their T1R3 expression, SCCs and ciliated cells are most likely to participate in the chemosensory process in ASL.

In the intestine, changes of sugar concentration generated in the lumen during digestion induce adaptive responses of glucose transporters in the epithelium. A close matching between the intestinal expression of glucose transporters and the composition and amount of the diet has been provided by several experiments. Functional evidence has demonstrated that the regulation of glucose transporters into enterocytes is induced by the sensing of sugar of the enteroendocrine cells through activation of sweet taste receptors (T1R2 and T1R3) and their associated elements of G-protein-linked signaling pathways (e.g. α-gustducin, phospholipase C β type 2 and transient receptor potential channel M5), which are signaling molecules also involved in the perception of sweet substances in the taste receptor cells (TRCs) of the tongue. Considering this phenotypical similarity between the intestinal cells and TRCs, we evaluated whether the TRCs themselves possess proteins of the glucose transport mechanism. Therefore, we investigated the expression of the typical intestinal glucose transporters (i.e. GLUT2, GLUT5 and SGLT1) in rat circumvallate papillae, using immunohistochemistry, double-labeling immunofluorescence, immunoelectron microscopy and reverse transcriptase-polymerase chain reaction analysis. The results showed that GLUT2, GLUT5 and SGLT1 are expressed in TRCs; their immunoreactivity was also observed in cells that displayed staining for α-gustducin and T1R3 receptor. The immunoelectron microscopic results confirmed that GLUT2, GLUT5 and SGLT1 were predominantly expressed in cells with ultrastructural characteristics of chemoreceptor cells. The presence of glucose transporters in TRCs adds a further link between chemosensory information and cellular responses to sweet stimuli that may have important roles in glucose homeostasis, contributing to a better understanding of the pathways implicated in glucose metabolism.

The diffuse chemosensory system (DCS) is an anatomical structure composed of solitary chemosensory cells (SCCs, also called solitary chemoreceptor cells), which have analogies with taste cells but are not aggregated in buds. The concept of DCS has been advanced, after the discovery that cells similar to gustatory elements are present in several organs. The elements forming the DCS share common morphological and biochemical characteristics with the taste cells located in taste buds of the oro-pharyngeal cavity but they are localized in internal organs. In particular, they may express molecules of the chemoreceptorial cascade (e.g. trans-membrane taste receptors, the G-protein alpha-gustducin, PLCbeta2, TRPM5). This article will focus on the mammalian DCS in apparatuses of endodermic origin (i.e. digestive and respiratory systems), which is composed of an enormous number of sensory elements and presents a multiplicity of morphological aspects. Recent research has provided an adequate description of these elements, but the functional role for the DCS in these apparatuses is unknown. The initial findings led to the definition of a DCS structured like an iceberg, with a mysterious "submerged" portion localized in the distal part of endodermic apparatuses. Recent work has focussed on the discovery of this submerged portion, which now appears less puzzling. However, the functional roles of the different cytotypes belonging to the DCS are not well known. Recent studies linked chemosensation of the intraluminal content to local control of absorptive and secretory (exocrine and endocrine) processes. Control of the microbial population and detection of irritants seem to be other possible functions of the DCS. In the light of these new findings, the DCS might be thought to be involved in a wide range of diseases of both the respiratory (e.g. asthma, chronic obstructive pulmonary disease, cystic fibrosis) and digestive apparatuses (absorptive or secretive diseases, dysmicrobism), as well as in systemic diseases (e.g. obesity, diabetes). A description of the functional roles of the DCS might be a first step toward the discovery of therapeutic approaches which target chemosensory mechanisms.

Background: There is evidence that stern cells contribute to the restoration of tissue vascularisation and organ function. The objective of this study was to assess the presence of adipose derived adult stern cells left in their natural scaffold in the purified lipoaspirate and to access the clinical effectiveness of lipoaspirate transplantation in the treatment of radiation side effects. Methods: This study was designed beginning with surgical procedures in 2002 and envisaging a continious patient follow up to 31 months. Twenty consecutive patients undergoing therapy for side effects of radiation treatment wiht severe symptoms or irreversible functiomn damage (LENT-SOMAscale grade 3 and 4) were enrolled. Purified autologous lipoaspirates (60 to 120 cc) taken from a healthy donor site were administered by reperted low-invasive computer-assisted injection. Therapy outcomes were assessed by symptoms classification according to the LENT-SOMA scale, cytofluorrimetric characterization, and ultrastructural evaluation of targeetd tissue. Results: In the isolated stromal vascular fraction of 2 cc of human lipoaspirate, cells with mesenchymal stren cell physical proprieties and immunophenotype were in average 1.07 ± 0.5 percent (n=4), with a clonogenic fraction of 0.139 percent. At least 1.02 × 10 3 colony-forming units-fibroblast were present in cach lipoaspirate. Ultrastructure of target tissue systematically exhibited progressive regeneration, including neovessel formation and improved hydratation. Clinical outcomes led to a systematic improvement or remission of symptoms in all evaluated patients, including otherwise untreatable patients exhibiting initial irreversible functional damage. Conclusions: This surgical procedure is a low invasive therapeutic approach for resolving the late side effects of radiotherapy. According to the proposed hypothesis of the ischernic nature of radiolesions treatment with lipoaspirate transplantation is potentially extended to other forms of microangiopathies.

Recent studies indicate that the processes mediated by the (T1R2/T1R3) glucose/sugar receptor of gustatory cells in the tongue, and hormones like leptin and ghrelin contribute to the regulation of glucose homeostasis. Altered plasma levels of leptin and ghrelin are associated with obesity both in humans and rodents. In the present study, we evaluated the ultrastructure of the mucosa, and the expression of molecules implicated in the regulation of glucose homeostasis (GLUT2, SGLT1, T1R3, ghrelin and its receptor) in the trachea of an animal model of obesity (Zucker rats). We found that the tracheal epithelium of obese animals was characterized by the presence of poorly differentiated cells. Ciliated and secretory cells were the cell lineages with greatest loss of differentiation. Severe epithelial alterations were associated with marked deposit of extracellular matrix in the lamina propria. The expression pattern of GLUT2 and SGLT1 glucose transporters was similar in the trachea of both the Zucker rat genotypes, whereas that of T1R3 was reduced in ciliated cells of obese rats. A different immunolocalization for ghrelin was also found in the trachea of obese rats. In conclusion, the tracheal morphological alterations in obese animals seem to compromise the expression of molecules involved in the homeostasis of glucose.

The regenerative property of fat grafting has been described. However, it is not clear whether the clinical results are attributable to the stem cells or are linked to other components of the adipose tissue. This work is aimed at analysis of the histologic and ultrastructural changes of aged facial skin after injection of fat graft in addition to its stromal vascular fraction, obtained by centrifugation, and to compare the results with those obtained by the injection of expanded adipose-derived mesenchymal stem cells.This study was performed in six consecutive patients who were candidates for face lift and whose ages ranged between 45 and 65 years. The patients underwent sampling of fat by liposuction from the abdominal region. The injection of fat and its stromal vascular fraction or expanded mesenchymal stem cells was performed in the preauricular areas. Fragments of skin were removed before and 3 months after each treatment and analyzed by optical and electron microscopy.After treatment with the autologous lipidic component and stromal vascular fraction, the skin showed a decrease in elastic fiber network (elastosis) and the appearance of new oxytalan elastic fibers in papillary dermis. The ultrastructural examination showed a modified tridimensional architecture of the reticular dermis and the presence of a richer microvascular bed. Similar results following treatment with expanded mesenchymal stem cells were observed.This study demonstrates that treatment with either fat and stromal vascular fraction or expanded mesenchymal stem cells modifies the pattern of the dermis, representing a skin rejuvenation effect.

Cryopreservation of subcutaneous white adipose tissue (sWAT) avoids multiple surgeries in patients subjected to reconstructive procedure. Fat grafts were performed subcutaneously on 26 mice treated with fresh (13 mice) or cryopreserved (13 mice) human sWAT. Cytofluorometry for CD marker expression of stem cells, differentiation capability, and in vivo survival of fat grafts were evaluated. In vitro analysis evidenced that cryopreservation did not affect the stem potential of samples. In vivo MRI showed that grafts were well preserved in 13 mice treated with fresh sWAT, whereas in 13 animals treated with thawed fat, graft volumes were strongly reduced after 1 week. Ultrastructural studies performed both on fresh and thawed specimens demonstrated that grafts performed with thawed sWAT are able to store lipids more slowly with respect to grafts performed with fresh sWAT and adipocytes maintained a multilocular appearance. Collected data demonstrated that the protocol of cryopreservation could maintain the regenerative capability of the sWAT, but the rate of reabsorption after fat grafting is higher using cryopreserved sWAT. Maintaining the stem potential of sWAT after cryopreservation is a very important aspect for reconstructive and regenerative medicine. The employment of cryopreserved sWAT represents an interesting goal for surgeons. Surely there is the necessity to improve the protocol of cryopreservation. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

The progression of a lesion to a carcinoma is dependent on the engagement of 'reactive stroma' that provides structural and vascular support for tumour growth and also leads to tissue reorganization and invasiveness. The composition of reactive stroma closely resembles that of granulation tissue, and myofibroblasts are thought to play a critical role in driving the stromal reaction of invasive tumours as well as of physiological wound repair. In the present work, we established a myofibroblast-like cell line, named A17, from a mouse mammary carcinoma model in which tumourigenesis is triggered in a single step by the overexpression of HER-2/neu transgene in the epithelial compartment of mammary glands. We showed that although they derived from a tumour of epithelial origin and did not express HER-2/neu transgene, their subcutaneous injection into the backs of syngeneic mice gave rise to sarcomatoid tumours which expressed alpha-smooth muscle actin at the invasive edge. The expression of cytokeratin 14 suggested a myoepithelial origin but immunophenotypical profile, invasive and neoangiogenic potential of A17 cells and tumours showed many similarities with the reactive stroma that occurs in wound repair and in cancerogenesis. Our results suggest that epithelial tumours have the potential to develop highly tumourigenic and invasive reactive stromal cells and our cell line represents a novel, effective model for studying epithelial-stromal interaction and the role of myofibroblasts in tumour development.

The G-protein subunit α-gustducin is a marker of chemoreceptive cells. In the present study, we examined the immunohistochemical localization of α-gustducin in rat airway epithelium both by light and electron microscopy. α-Gustducin immunoreactivity was found in solitary cells that presented ultrastructural features of chemoreceptor cells, i.e. flask-shaped or pear-shaped, with an apical process with thin microvilli protruding into the lumen. The immunostaining was mainly concentrated in the apical process and along the basolateral cell surface. To investigate whether α-gustducin-immunoreactive cells represented a distinct cell subset in rat airways, we performed double-label immunocytochemistry with antibodies to protein gene groduct (PGP) 9.5, a marker of neuroendocrine cells, and to phospholipase C beta2 (PLCβ2), a component of the bitter signalling pathway. α-Gustducin-immunoreactive cells were present in a subset of PGP-9.5-immunoreactive elements, although not all α-gustducin-positive cells expressed PGP 9.5 labelling. In addition, a subset of α-gustducin-expressing cells colocalized PLCβ2. This work thus demonstrates that solitary α-gustducin-immunoreactive cells exist throughout the airways and represent a specialized cell type with morphological and immunohistochemical characteristics of chemoreceptor cells.

The expression of molecules involved in the transductory cascade of the sense of taste (TRs, alpha-gustducin, PLCbeta2, IP3R3) has been described in lingual taste buds or in solitary chemoreceptor cells located in different organs. At the laryngeal inlet, immunocytochemical staining at the light and electron microscope levels revealed that alpha-gustducin and PLCbeta2 are mainly localized in chemosensory clusters (CCs), which are multicellular organizations differing from taste buds, being generally composed of two or three chemoreceptor cells. Compared with lingual taste buds, CCs are lower in height and smaller in diameter. In laryngeal CCs, immunocytochemistry using the two antibodies identified a similar cell type which appears rather unlike the alpha-gustducin-immunoreactive (IR) and PLCbeta2-IR cells visible in lingual taste buds. The laryngeal IR cells are shorter than the lingual ones, with poorly developed basal processes and their apical process is shorter and thicker. Some cells show a flask-like shape due to the presence of a large body and the absence of basal processes. CCs lack pores and their delimitation from the surrounding epithelium is poorly evident. The demonstration of the existence of CCs strengthens the hypothesis of a phylogenetic link between gustatory and solitary chemosensory cells.

A specific laryngeal sensory epithelium (SLSE), which includes arrays of solitary chemoreceptor cells, is described in the supraglottic region of the rat. Two plates of SLSE were found, one on each side of the larynx. The first plate was located in the ventrolateral wall of the larynx, and the second was located in the interarytenoidal region. In SLSE, immunoblotting showed the presence of α-gustducin and phospholipase C β2 (PLCβ2), which are two markers of chemoreceptor cells. At immunocytochemistry, laryngeal immunoreactivity for α-gustducin was localized mainly in solitary chemosensory cells. Double-label immunocytochemistry using confocal microscopy demonstrated that α-gustducin–expressing cells in large part colocalize type III IP3 receptor (IP3R3), another key molecule in bitter taste perception. However, some IP3R3–expressing cells do not colocalize α-gustducin. At ultrastructural immunocytochemistry, these cells showed packed apical microvilli, clear cytoplasmic vesicles, and cytoneural junctions. SLSE was characterized by high permeability to a tracer due to poorly developed junctional contacts between superficial cells. Junctions were short in length and showed little contact with the terminal web. Ultrastructural analysis showed deep pits among the superficial cells. In SLSE, high density of intraepithelial nerve fibers was found. The lamina propria of the SLSE appeared thicker than that in other supraglottic regions. It was characterized by the presence of a well-developed subepithelial nerve plexus. The immunocytochemical and ultrastructural data suggested that SLSE is a chemoreceptor located in an optimal position for detecting substances entering the larynx from the pharynx or the trachea. J. Comp. Neurol. 475:188–201, 2004. © 2004 Wiley-Liss, Inc.

Contrast-enhanced MRI, immunostaining and electron microscopy were used to detect areas of intense angiogenesis in experimental tumors. This work was also aimed at evaluating the possible effect of the surrounding tissues on tumor microvasculature and at studying the penetration of macromolecules in avascular areas. Human colon carcinoma cells were implanted in subcutaneous tissue of nude mice. Dynamic T1-weigthed 3D pulse sequences were acquired before and after administration of Gd-DTPA-albumin to obtain parametric maps of fractional plasma volume (fpv) and transendothelial permeability (Kps). The maps suggested that tumor can be subdivided into 4 zones located in the peripheral rim (zones I–II) or in the core (zones III–IV) of the tumor itself. Significant differences (p

Region-specific decreases of neurofilament proteins (NF) were described in the ventral tegmental area (VTA) of rats treated chronically with morphine, cocaine or alcohol. In a previous study, we demonstrated that NF levels were also changed in the VTA after chronic treatment with nicotine. The aim of this study was to clarify the submicroscopic basis of decreased immunoreactivity for NF-68, NF-160 and NF-200, as determined by using NR4, BF10 and RT97 antibodies, respectively. Microdensitometric analysis of brain sections showed that immunoreactivity for all NF was reduced in the VTA of animals exposed chronically to nicotine (0.4 mg/kg per day, 6 days of treatment), when compared to rats exposed to saline. Reduction in immunoreactivity was significant for NF-68 (P < 0.05), NF-160 (P < 0.01) and NF-200 (P < 0.05), showing a relative reduction of 34%, 42% and 38%, respectively, when compared to saline-treated rats. No difference was observed for any of the NF under study when immunoreactivity measurements in the substantia nigra were compared. Ultrastructural analysis was applied to evaluate changes in NF-68, NF-160 and NF-200 immunoreactivity in regions of the VTA that contain dopaminergic neurons following chronic nicotine treatment. At the electron microscopic level, no degenerative changes were found in neurons or glial cells of the VTA. With ultrastructural immunohistochemistry, evaluation of the homogeneity parameter of NF distribution showed a loss of homogeneity for NF-68 linked to the nicotine treatment. In areas in which NF organization appeared well preserved, analysis of the numerical density of NF revealed no significant difference for NF-68 (897/ micro m2 vs. 990/ micro m2), NF-160 (970/ micro m2 vs. 820/ micro m2) and NF-200 (1107/ micro m2 vs. 905/ micro m2) in nicotine-treated rats when compared to saline-treated rats. These results confirm that nicotine shares the same properties with cocaine and morphine in reducing NF in the VTA, a key brain structure of the rewards system, and that chronic nicotine treatment changes the axonal distribution of 68 kDa neurofilaments in the rat VTA.

We investigated similarities in the signaling pathways elicited by the F2 isoprostane 8-iso-PGF2alpha and by low doses of U46619 to induce platelet activation. Both 0.01-0.1 micromol/L U46619 and 0.01-1 micromol/L 8-iso-PGF2alpha triggered shape change and filopodia extension, as well as adhesion to immobilized fibrinogen of washed platelets. At these doses the two platelet agonists failed to trigger secretion and aggregation, which were however induced by higher doses of U46619 (0.1-1 micromol/L). SB203580 (1-10 micromol/L), a specific inhibitor of the p38 mitogen activated protein (MAP) kinase blunted platelet shape change and adhesion induced by 0.05-1 micromol/L 8-iso-PGF2alpha and by 0.01 micromol/L U46619. These platelet responses were also inhibited by 20 micromol/L cytochalasin D, an inhibitor of actin polymerization, and 50 micromol/L piceatannol, an inhibitor of the Syk tyrosine kinases. Both 8-iso-PGF2alpha and U46619-induced p38 MAP kinase phosphorylation in suspended platelets and this was inhibited by piceatannol, indicating that Syk activation occurs upstream of p38 MAP kinase phosphorylation. These findings suggest that the signaling pathway triggered by both 8-iso-PGFalpha and low concentrations of U46619 to induce platelet adhesion and shape change implicates Syk, the p38 MAP kinase, and actin polymerization.

Subcutaneous white adipose tissue harvested by liposuction has been studied with the aim to understand how the adipocytes modify their morphology when subjected to the passage in a needle for liposuction and to cryopreservation. The work try to clarify the ultrastructural aspects of adipose tissue, in the conditions described before, examining samples of body fat employed in fat graft procedures, and samples after cryopreservation. Scanning and transmission electron microscopy show that the first event that occur in the adipocytes is a lesion of mild degree detectable early in the samples fixed immediately after liposuction. The sequence of events following the adipocyte stress appeared composed by different phases: plasmatic membrane interruption, loss of lipid charge, formation of cup-like adipocytes and formation of post-adipocytes (i.e. cells that survive to traumatic events and restart to internalize lipid droplets). In conclusion, the study suggests that the loss of lipid charge in adipose cell is an active process that can be due to a small hole in the cytoplasmic membrane with the preservation of a large part of the cytoplasmatic content and that at the end of the process of lipid extrusion the cell can maintain viability.

Octreotide has been found to be effective in the prevention of postoperative complications of pancreatic surgery, but the benefit of preoperative octreotide administration has not been assessed.To evaluate the ability of octreotide in reducing the amount of digestive enzymes in the pancreas before surgery, a morphometric ultrastructural study of the gland was undertaken in patients undergoing demolitive pancreatic surgery.Twenty-three inpatients received saline (n = 8) or octreotide (100 micrograms s.c.) before surgery either once (n = 5), or three (n = 5) or six (n = 5) times at 8-hour intervals. At surgery, biopsies of the pancreas were taken and processed for electron microscopy. Several parameters were assessed in exocrine cells by means of ultrastructural morphometry.A single administration of octreotide significantly reduced the exocrine granule number and the mean and total granular surface sectional area, and the ratio between granule area and cytoplasmic area. Repeated octreotide administrations were associated with partial (3 administrations) and complete (6 administrations) recovery of all parameters to control values.Preoperative administration of octreotide, the synthetic analogue of somatostatin, acutely reduces exocrine granule number and size in the pancreatic cell. This finding can partially explain the prophylactic effect of the drug on early complications of pancreatic surgery. Such an effect is not maintained over multiple administrations of the somatostatin analogue. Possible explanations for this latter finding are discussed.

We have studied the postnatal development of the intrinsic nervous system in the circumvallate papilla-vonEbner gland complex using NADPH-diaphorase cytochemistry, immunocytochemistry (for nitric oxide synthase-1 and alpha-internexin) and electron microscopy. In rats sacrificed in their first day post partum (1 p.p.), only isolated NADPH-diaphorase positive neurons were visible in the organ. At 2 p.p., a small group of neurons was visible at the base of the papillae and positive neurons formed short chains close to the developing glandular tubules. In the following days, the NADPH-diapharase positive cells increased in number and nerve fibres were associated to small ganglia located at the base of the papilla or in the gland. After the first week of extrauterine life, the intrinsic nervous system was similar to the intrinsic system of adult animals. An immunocytochemical positivity for nitric oxide synthase-1 appeared at 4 p.p. in neurons located in the gland and at 7 p.p. in cells located at the base of the papilla. Immunocytochemical staining for alpha-internexin showed that at 1 p.p. developing nerve fibres were present in the connective tissue of the tongue's muscle layer. At 2-3 p.p., developing nerve fibres were also present at the bases and in the core of the papilla. In the following days, the positivity for alpha-internexin was reduced and one week after birth was virtually absent. Ultrastructural examination revealed that since 1 p.p. isolated neurons can be found at the base of the papilla. In conclusion, the intrinsic nervous system originates from neurons present in the organ at the birth which, in the first days, undergo a biochemical and morphological maturation while the nerve fibres rapidly grow. These findings support the hypothesis that the intrinsic nervous system of the circumvallate papilla has a role in the maturation of the vonEbner gland.

The ultrastructural features of membrane-bounded bodies contained in the tubulo-vesicular system in the outer segment of taste bud cells are described. Each body showed a round, fusiform or oval shape, was surrounded by a trilaminar membrane and enclosed an electron dense matrix sometimes containing inclusions. These bodies were found at all ages studied. Similar structures were also found embedded in the material plugging the taste pore. Our findings suggest that these bodies could be secreted at the free surface of the cells and be involved in the concentration of divalent cations.

A facile method for the synthesis of water dispersible Er(3+)/Yb(3+) and Tm(3+)/Yb(3+) doped upconverting GdF(3) nanoparticles is reported. Strong upconversion emissions are observed in the red (for Er/Yb doped) and near-infrared (for Tm/Yb doped) regions upon laser excitation at 980 nm. The PEG coating ensures a good dispersion of the system in water and reduces the radiationless de-excitation of the excited states of the Er(3+) and Tm(3+) ions by water molecules. The r(2) relaxivity values are quite high with respect to the common T(2)-relaxing agents (22.6 ± 3.4 mM(-1) s(-1) and 15.8 ± 3.4 mM(-1) s(-1) for the Tm/Yb and Er/Yb doped samples, respectively), suggesting that the present NPs can be interesting as T(2) weighted contrast agents for proton MRI purpose. Preliminary experiments conducted in vitro, in stem cell cultures, and in vivo, after subcutaneous injection of the lanthanide-doped GdF(3) NPs, indicate scarce toxic effects. After an intravenous injection in mice, the GdF(3) NPs localize mainly in the liver. The present results indicate that the present Er(3+)/Yb(3+) and Tm(3+)/Yb(3+) doped GdF(3) NPs are suitable candidates to be efficiently used as bimodal probes for both in vitro and in vivo optical and magnetic resonance imaging.

Technological developments based on the use of autologous white adipose tissue (WAT) attracted attention to minor fat depots as possible sources of adipose tissue. In plastic surgery, the trochanteric fatty pad is one of the most used WAT depots for its location and organoleptic characteristics that make it particularly suitable for reconstructive procedures. Despite its wide use in clinic, the structure of this depot has never been studied in detail and it is not known if structural differences exist among trochanteric fat and other subcutaneous WAT depots. The present study was performed on trochanteric fat pad with the aim to clarify the morphology of its adipocytes, stroma and microcirculation, with particular reference to the stem niches. Histological and ultrastructural studies showed that the main peculiar feature of the trochanteric fat concerns its stromal component, which appears less dense than in the other subcutaneous WATs studied. The intra-parenchymal collagen stroma is poor and the extracellular compartment shows large spaces, filled with electron-light material, in which isolated collagen bundles are present. The adipocytes are wrapped in weak and easily detachable collagen baskets. These connective sheaths are very thin compared to the sheaths in other subcutaneous WAT depots. The capillaries are covered by large, long and thin elements surrounded by an external lamina; these perivascular cells are poor in organelles and mainly contain poly-ribosomes. In conclusion, when compared to other WAT deposits, the trochanteric fatty pad shows structural peculiarities in its stroma and microcirculation suggesting a high regenerative potential. Resistance, dissociability, microvascular weft and high regenerative potential make the trochanteric fatty pad a privileged source for harvesting in autologous WAT-based regenerative procedures.

In the intestine, changes of sugar concentration generated in the lumen during digestion induce adaptive responses of glucose transporters in the epithelium. A close matching between the intestinal expression of glucose transporters and the composition and amount of the diet has been provided by several experiments. Functional evidence has demonstrated that the regulation of glucose transporters into enterocytes is induced by the sensing of sugar of the enteroendocrine cells through activation of sweet taste receptors (T1R2 and T1R3) and their associated elements of G-protein-linked signaling pathways (e.g. α-gustducin, phospholipase C β type 2 and transient receptor potential channel M5), which are signaling molecules also involved in the perception of sweet substances in the taste receptor cells (TRCs) of the tongue. Considering this phenotypical similarity between the intestinal cells and TRCs, we evaluated whether the TRCs themselves possess proteins of the glucose transport mechanism. Therefore, we investigated the expression of the typical intestinal glucose transporters (i.e. GLUT2, GLUT5 and SGLT1) in rat circumvallate papillae, using immunohistochemistry, double-labeling immunofluorescence, immunoelectron microscopy and reverse transcriptase-polymerase chain reaction analysis. The results showed that GLUT2, GLUT5 and SGLT1 are expressed in TRCs; their immunoreactivity was also observed in cells that displayed staining for α-gustducin and T1R3 receptor. The immunoelectron microscopic results confirmed that GLUT2, GLUT5 and SGLT1 were predominantly expressed in cells with ultrastructural characteristics of chemoreceptor cells. The presence of glucose transporters in TRCs adds a further link between chemosensory information and cellular responses to sweet stimuli that may have important roles in glucose homeostasis, contributing to a better understanding of the pathways implicated in glucose metabolism.

The aim of the work was to compare in vivo MRI visualization of pancreatic islets labeled with clinical-grade superparamagnetic iron oxide (SPIOs) contrast agents with ex vivo examination of liver tissue in an experimental model of marginal mass transplantation in rats. Seven hundred IEq (Islet Equivalent) from Wistar rats, labeled by incubation with Endorem® or Resovist®, were transplanted into Sprague–Dawley rats through the portal vein. Liver MR images of recipient rats were acquired at different time points (3–42 days) after transplantation. Animals were sacrificed during this period and their livers were excised and prepared for histology and electron microscopy. Hypointense spots originating from iron particles were observed in MR images. The number of separate spots was counted. Three days after transplantation one spot for every three or four transplanted islets was observed. Seven days after transplantation, histological sections showed the presence of iron within pancreatic islets. The time course of MR images showed a decrease in the number of spots, at 42 days, amounting to 65 and 22% of the initial value, for Resovist® and Endorem® respectively, while no immunopositive endocrine cells were detected in histological slices. The present work shows that pancreatic islets can be labeled using clinically approved SPIO contrast agents and visualized using in vivo MRI with high sensitivity, consistently with findings in the literature. Differently from reports in the literature, our findings indicate that iron particles could last in the liver for long periods, independently of the presence of intact pancreatic islets. Copyright © 2009 John Wiley & Sons, Ltd.

Acyl homoserine lactones (AHLs) are intercellular signaling molecules used in quorum sensing by Gram-negative bacteria. We studied the early effects on the rat airway of in vivo intratracheal administration of AHLs (i.e., P. aeruginosa and B. cepacia) to test the hypothesis that AHLs also act on the airway cells, modifying secretory mechanisms which are important in mucosal defense. One hour after treatment, N-butyryl-homoserine lactone (C4-HL) had caused dilated extracellular spaces, loss of cilia, reduction of secretory material, and the presence of prenecrotic elements in the epithelium, while N-octanoyl-homoserine lactone (C8-HL) caused a mild lesion in the epithelium. After treatment with either C4- or C8-HL, reduced immunoreactivity was found using CC10 antibody. At ultrastructural examination, dilatation of the mitochondria was evident in ciliate and secretory cells, while solitary chemosensory cells appeared better preserved, showing aspects of nucleocytoplasmic activation. Using microarray analysis, we found down-regulation of early gene Fos and Egr1 in all AHL-treated specimens. In vivo pharmacological magnetic resonance imaging after C4- or C8-HL treatment showed a slight increase in tracheal secretion at a first evaluation 5 min after administration, with no increase in the following minutes. In conclusion, AHLs induce an early mucosal response, and the chondriomas of ciliate and secretory cells are the main cytological target of AHL action. Our results show that AHL action is not limited to activation of conspecific bacteria, but also modifies innate airway defense mechanisms. Anat Rec, 292:439–448, 2009. © 2009 Wiley-Liss, Inc.

The mechanisms involved in the pathogenesis of epilepsy, a chronic neurological disorder that affects approximately one percent of the world population, are not well understood. Using a mouse model of epilepsy, we show that seizures induce elevated expression of vascular cell adhesion molecules and enhanced leukocyte rolling and arrest in brain vessels mediated by the leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-1, encoded by Selplg) and leukocyte integrins alpha(4)beta(1) and alpha(L)beta(2). Inhibition of leukocyte-vascular interactions, either with blocking antibodies or by genetically interfering with PSGL-1 function in mice, markedly reduced seizures. Treatment with blocking antibodies after acute seizures prevented the development of epilepsy. Neutrophil depletion also inhibited acute seizure induction and chronic spontaneous recurrent seizures. Blood-brain barrier (BBB) leakage, which is known to enhance neuronal excitability, was induced by acute seizure activity but was prevented by blockade of leukocyte-vascular adhesion, suggesting a pathogenetic link between leukocyte-vascular interactions, BBB damage and seizure generation. Consistent with the potential leukocyte involvement in epilepsy in humans, leukocytes were more abundant in brains of individuals with epilepsy than in controls. Our results suggest leukocyte-endothelial interaction as a potential target for the prevention and treatment of epilepsy.