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BACKGROUND The persisting organ shortage in the field of transplantation recommends the use of marginal kidneys which poorly tolerate ischemic damage. Adenosine triphosphate (ATP) depletion during cold ischemia time (CIT) is considered crucial for graft function. We tested different strategies of kidney perfusion before transplantation in the attempt to improve the technique. MATERIAL AND METHODS Twenty human discarded kidneys from donors after brain death and with at least 20 hours of CIT were randomized to the following experimental groups (treatment time three-hours at 4°C): a) static cold storage (CS); b) static cold hyperbaric oxygenation (Hyp); c) hypothermic perfusion (PE); d) hypothermic perfusion in hyperbaric oxygenation (PE-Hyp); and e) hypothermic oxygenated perfusion (PE-O2). RESULTS Histological results showed that perfusion with or without oxygen did not produce any endothelial damage. A depletion of ATP content following the preservation procedure was observed in CS, PE, and Hyp, while PE-Hyp and PE-O2 were associated with a net increase of ATP content with respect to baseline level. In addition, PE-Hyp was associated with a significant downregulation of endothelial isoform of nitric oxide synthase (eNOS) gene expression and of hypoxia inducible factor-1α (HIF-1α). CONCLUSIONS Hyperbaric or normobaric oxygenation with perfusion improves organ metabolic preservation compared to other methods. This approach may prevent the onset of delayed graft function, but clinical trials are needed to confirm this.

Introduction. High-sensitivity C-Reactive Protein (hsCRP) levels are correlated with vulnerable carotid plaques, although their impact on the outcome of carotid revascularization is unknown. The aim of our study was to investigate the correlation between hsCRP and embolization during carotid artery stenting (CAS). Methods. Patients with symptomatic carotid stenosis were submitted to CAS with distal protection filters. Serum hsCRP was analysed prior to CAS and patients were divided into two groups: Class I, patients presenting hsCRP

The maintenance of the mechanical properties of the vessels results from the correct arrangement of smooth muscle cells and extracellular fibrous proteins (elastin and collagen) in their wall. The morphology of extracellular matrix modifications, particularly of elastin, was investigated in inflammatory (IA) and non specific (NSA) abdominal aortic aneurysms by scanning (SEM), transmission (TEM) and immunoelectron microscopy. Both NSAs and IAs were significantly characterized by extensive extracellular matrix remodelling, including different patterns of elastin degradation. Elastic and collagen fibres distribution appeared to be extensively altered in IAs, while it conformed more to a normal pattern in NSAs. With respect to NSAs, the morphology of elastic fibres in IAs was modified to such an extent that their identification by TEM had a rely on immunocytochemical methods and by SEM on back-scattered electron analysis. The observed ultrastructural changes are indicative of the central role of extracellular matrix modifications in the pathogenesis of IAs and NSAs.

An important inflammatory reaction was histologically observed in the wall of an abdominal aortic aneurysm in a 42 year-old patient suffering from multifocal atherosclerotic lesions. Histochemical and ultrastructural studies showed an active-stage wall lymphomonoplasmacellular component. The histological appearance and biochemical and microbiological studies excluded specific aortitis, a rheumatic disease or concomitant infectious processes. The morphological relationships between the atherosclerotic lesion and the infiltrate together with the immunophenotyping features of the infiltrate suggest the possibility of a close correlation between the inflammatory process and the atherosclerotic lesion of the wall.

Experimental evidence indicates that chronic mechanical sub-occlusion of the intestine may damage the enteric nervous system (ENS), although data in humans are lacking. We here describe the first case of enteric degenerative neuropathy related to a congenital obstruction of the gut. A 3-year and 9-mo old girl began to complain of vomiting, abdominal distension, constipation with air-fluid levels at plane abdominal radiology. Her subsequent medical history was characterized by 3 operations: the first showed dilated duodeno-jejunal loops in the absence of occlusive lesions; the second (2 years later) was performed to obtain full-thickness biopsies of the dilated intestinal loops and revealed hyperganglionosis at histopathology; the third (9 years after the hyperganglionosis was identified) disclosed a Ladd's band which was removed and the associated gut malrotation was corrected. Repeated intraoperative full-thickness biopsies showed enteric degenerative neuropathy along with reduced interstitial cells of Cajal network in dilated loops above the obstruction and a normal neuromuscular layer below the Ladd's band. One year after the latest surgery the patient tolerated oral feeding and did well, suggesting that congenital (partial) mechanical obstruction of the small bowel in humans can evoke progressive adaptive changes of the ENS which are similar to those found in animal models of intestinal mechanical occlusion. Such ENS changes mimic neuronal abnormalities observed in intestinal pseudo-obstruction.

The effect of Ranitidine on the gastric and duodenal lesions induced by Aspirin or Piroxicam was studied by scanning electron microscopy (SEM). Twelve patients were entered into the study. The first group consisted of 6 patients with normal basic morphology, of which 3 were treated with 1.5 g of Aspirin and Ranitidine 300 mg/day, and 3 with 20 mg of Piroxicam and Ranitidine 300 mg/day, for one month. The second group was composed of 6 rheumatic patients, 3 of which took 1.5 g of Aspirin and the other 3 received 20 mg of Piroxicam per day for at least four months. Ranitidine (300 mg/day) was then administered concomitantly for one month to all these 6 patients. Overall, Ranitidine appeared to have a positive effect in preventing the development of gastroduodenal lesions induced by these anti-inflammatory drugs in the first group of patients. No differences were observed in the ability of Ranitidine to promote the healing of lesions induced by chronic treatment. Ranitidine, however, appeared to be more effective in preventing lesions induced by Aspirin rather than those induced by Piroxicam.

Our personal experience on the application of scanning electron microscopy in cardiology, gastroenterology and ophthalmology is reviewed. SEM has not yet significantly contributed to myocardium pathology. However, in the near future, SEM could be a reliable technique to complete the information available from other sources. As to atherosclerosis, SEM allowed us to improve our knowledge of the early stages of the disease; some pathological features, not always detected by conventional morphological examinations, can be documented. An important contribution to gastrointestinal pathology was made by SEM investigations both in the staging of some important diseases (i.e., coeliac disease, peptic ulcer, Crohn's disease, ulcerative colitis) and in the follow-up of mucosal changes during therapy. In the ophthalmological field, SEM provided three-dimensional new information to clinicians, who are familiar with the biomicroscopic images. Our experience in hematology is still limited. However, in the last few years SEM joined to immunocytochemistry allowed us to characterize cell populations in several blood diseases. Some procedures of particular interest in the management of human bioptic specimens are stressed in order to get to a complete correlative microscopy. We conclude that continuous and simultaneous correlations have to be carried out between SEM and other methods and instruments available for morphological investigation.

The filipin probe associated with tannic acid stain was used to study intra- and extracellular lipids in surgically removed human atherosclerotic lesions (n = 20). In particular, intimal thickenings, fatty streaks and fibrolipidic plaques have been investigated by using mainly transmission and scanning electron microscopy. In the intimal thickenings, the lipid deposits were mainly localized in the subendothelial space as homogeneously sized particles (40-140 nm) and more heterogeneous uni-multilamellar vesicles (35-700 nm). Intermediate lipid forms were also observed. In the fatty streaks, the lipid deposits were intracellular and mainly observed in cells with a monocyte/macrophagic phenotype. Lipid inclusions, lipid lysosomal bodies and intracellular cholesterol crystals very similar to those observed in experimentally induced atherosclerosis were documented. In the fibrolipidic plaque the lipid deposits were found both in the intracellular and in the extracellular compartments. Lipids accumulated within arterial macrophages and smooth muscle cells, usually as lipid droplets. Clusters of lipoprotein-like particles (50 nm in diameter) as well as larger uni-multilamellar lipids (700 nm) with an occasional compound appearance were particularly observed bound to elastic tissue and collagen fibers. These morphological observations outline the complexity of lipid metabolism in the various histological aspects of human atherosclerosis.

We considered the role of scanning electron microscopy (SEM) in clinical investigation of different gastrointestinal diseases. The following clinical applications of SEM may be suggested on the basis of our original data and those reported in literature: in peptic ulcer: assessment of the completeness of healing, by observing the mucosal surface architecture of the scars; identification of mucosal changes, namely enterocytic surface membrane alterations, predictive of recurrence; in coeliac disease: early assessment of the response to gluten-free diet and follow-up of the patients by staging the process of mucosal repair in cerebriform, intermediate and villous patterns; in ulcerative and Crohn's colitis: enhancement of the diagnostic sensitivity of perendoscopic biopsy, by detecting differences in surface structure of mucosa surrounding ulcers in both diseases. This is subverted in ulcerative colitis and preserved in Crohn's colitis. Finally the complementary role of SEM in relation to endoscopy and light microscopy is emphasized.

Inflammatory aneurysms are characterised by a peculiar clinical (i.e. abdominal-lumbar pain, weight loss and increased ESR) and morphological picture (whitish wall, adhesion to the surrounding organs and thickness greater than 0.5 cm). The lymphomonoplasmacellular infiltrate and the interstitial deposits of collagen define the histological picture of these lesions. The authors describe three abdominal aortic aneurysms macroscopically characterised by parietal edema, hyperemia and hypertrophy of the preaortic lymphnodes. Histological study revealed a conspicuous and widespread lymphomonoplasmacellular infiltrate and interstitial edema. The abdominal-lumbar pain, the increase in ESR and the reactive C protein defined the clinical and laboratory aspects. Serological tests for syphilis, rheumatoid arthritis and lupus erythematosis were negative. The question which arises from these observations is whether these forms represent separate entities or an early stage in the evolution of inflammatory abdominal aortic aneurysms with fibrosis.

Nanobacteria are controversial infectious agents with nanometric size, the capacity to nucleate hydroxyapatite and grow in culture, and present in human diseases associated with calcification and psammoma bodies. The authors report a case of pathological placental calcifications associated with nanobacteria. Electron microscopy and electron energy loss spectroscopy imaging were used to recognize 160-nm-sized calcium-free bodies mainly presenting as extracellular fibrillary tangles and 500-nm-sized calcified bodies; they encrusted the syncito-trophoblast basal membrane and aggregated into miniaturized psammoma bodies. Nanobacteria may be composed of a prionoid protein with self-assembling and self-propagating abilities whose growth is associated with the formation of psammoma bodies.

The ability to form spheroids under non-adherent conditions is a well-known property of human mesenchymal stem cells (hMSCs), in addition to stemness and multilineage differentiation features. In the present study, we tested the ability of hMSCs isolated from the vascular wall (hVW-MSCs) to grow as spheres, and provide a characterization of this 3D model. hVW-MSCs were isolated from femoral arteries through enzymatic digestion. Spheres were obtained using ultra-low attachment and hanging drop methods. Immunophenotype and pluripotent genes (SOX-2, OCT-4, NANOG) were analyzed by immunocytochemistry and real-time PCR, respectively. Spheres histological and ultrastructural architecture were examined. Cell viability and proliferative capacity were measured using LIVE/DEATH assay and ki-67 proliferation marker. Metabolomic profile was obtained with liquid chromatography-mass spectrometry. In 2D, hVW-MSCs were spindle-shaped, expressed mesenchymal antigens, and displayed mesengenic potential. 3D cultures of hVW-MSCs were CD44+ , CD105low , CD90low , exhibited a low propensity to enter the cell cycle as indicated by low percentage of ki-67 expression and accumulated intermediate metabolites pointing to slowed metabolism. The 3D model of hVW-MSCs exhibits stemness, dormancy and slow metabolism, typically observed in stem cell niches. This culture strategy can represent an accurate model to investigate hMSCs features for future clinical applications in the vascular field.

HIV infection is an independent risk factor for atherosclerosis development and cardiovascular damage. As vessel wall mesenchymal stem cells (MSCs) are involved in the regulation of vessel structure homeostasis, we investigated the role of Tat, a key factor in HIV replication and pathogenesis, in MSC survival and differentiation. The survival of subconfluent MSCs was impaired when Tat was added at high concentrations (200–1,000 ng/ml), whereas lower Tat concentrations (1–100 ng/ml) did not promote apoptosis. Tat enhanced the differentiation of MSC toward adipogenesis by the transcription and activity upregulation of PPARγ. This Tat-related modulation of adipogenesis was tackled by treatment with antagonists of Tat-specific receptors such as SU5416 and RGD Fc. In contrast, Tat inhibited the differentiation of MSCs to endothelial cells by downregulating the expression of VEGF-induced endothelial markers such as Flt-1, KDR, and vWF. The treatment of MSCs with Tat-derived peptides corresponding to the cysteine-rich, basic, and RGD domains indicated that these Tat regions are involved in the inhibition of endothelial marker expression. The Tat-related impairment of MSC survival and differentiation might play an important role in vessel damage and formation of the atherosclerotic lesions observed in HIV-infected patients. J. Cell. Biochem. 113: 1132–1141, 2012. © 2011 Wiley Periodicals, Inc.

Background Neuroblastoma is a deadly childhood cancer, and MYCN-amplified neuroblastoma (MNA-NB) patients have the worst prognoses and are therapy-resistant. While retinoic acid (RA) is beneficial for some neuroblastoma patients, the cause of RA resistance is unknown. Thus, there remains a need for new therapies to treat neuroblastoma. Here we explored the possibility of combining a MYCN-specific antigene oligonucleotide BGA002 and RA as therapeutic approach to restore sensitivity to RA in NB. Methods By molecular and cellular biology techniques, we assessed the combined effect of the two compounds in NB cell lines and in a xenograft mouse model MNA-NB. Results We found that MYCN-specific inhibition by BGA002 in combination with RA (BGA002-RA) act synergistically and overcame resistance in NB cell lines. BGA002-RA also reactivated neuron differentiation (or led to apoptosis) and inhibited invasiveness capacity in MNA-NB. Moreover, we found that neuroblastoma had the highest level of mRNA expression of mTOR pathway genes, and that BGA002 led to mTOR pathway inhibition followed by autophagy reactivation in MNA-NB cells, which was strengthened by BGA002-RA. BGA002-RA in vivo treatment also eliminated tumor vascularization in a MNA-NB mouse model and significantly increased survival. Conclusion Taken together, MYCN modulation mediates the therapeutic efficacy of RA and the development of RA resistance in MNA-NB. Furthermore, by targeting MYCN, a cancer-specific mTOR pathway inhibition occurs only in MNA-NB, thus avoiding the side effects of targeting mTOR in normal cells. These findings warrant clinical testing of BGA002-RA as a strategy for overcoming RA resistance in MNA-NB.

We present an unusual case of liver involvement in monoclonal gammopathy with generalized crystal-storing histiocytosis (G-CSH). A bone marrow storage disease was diagnosed in a 79-year-old man with monoclonal gammopathy of uncertain significance (MGUS). The patient presented with pleural effusion, an osteolytic lesion of the humerus, and an increase of aspartate transaminase and cholestatic markers that raised the clinical suspect of liver disease. A second bone marrow biopsy confirmed the diagnosis of MGUS with a histiocytic component suggestive for G-CSH. Liver biopsy showed an unremarkable histology, no significant inflammatory infiltrates, and intrasinusoidal foamy histiocytes. PAS and Masson’s trichrome stains, showed, in the cytoplasm of both histiocytes and hepatocytes, rod-shaped eosinophilic crystals, which were immunoreactive for kappa light chains. Transmission electron microscopy performed on reprocessed histological sections confirmed the presence of crystals in the hepatocyte cytoplasms. Immunogold labeling intensely stained crystals for kappa light chains. To the best of our knowledge, this is the second case in which an intrahepatocellular crystal storage is described during liver involvement in G-CSH. The present case also suggests that an increase in liver serum enzymes may support the clinical diagnosis of liver CSH in a patient with MGUS.

Introduction: Different models have been proposed for the prediction of the risk/benefit ratio of surgery in patients with carotid atheromasic disease, mainly based on clinical patients’ characteristics and risk factors, but no definite biological markers predictive of plaque instability and disease evolution have emerged so far, able to help the surgeon in the choice and timing of treatment. The main purpose of the present study was to assess the role of the polymorphism for genes commonly implicated in cell proliferation and neoangiogenesis in the clinical and histopathological carotid plaque vulnerability. Materials and methods: We retrospectively studied 29 consecutive patients who underwent carotid endarterectomy in 6 months. All histological variables were collected, as well as patients’ cardiovascular risk factors, clinical presentation, and brain computed tomography (CT) for the presence of ischemic lesions. Next-Generation Sequencing (NGS) was performed on 10-µm FFPE sections by means of a multi-gene panel used for sequencing 343 amplicons in 28 genes. Results: Among the gene variants observed, the polymorphism p.(Gln787=) in the EGFR gene was inversely correlated with intraplaque hemorrhage (p = 0.014), but also with the presence of ischemic brain lesions at CT (p = 0.001). Also p.(Gly105=) polymorphism in the IDH1 gene was inversely correlated with the presence of ischemic brain lesions (p = 0.038). Conclusions: The variant p.(Gln787=) in the EGFR gene seems to play a role in plaque stability in patients with carotid atheromasic disease, on both histopathological and clinical grounds, probably acting on plaque matrix remodeling. This can open new scenarios on the pre-surgical management of these patients.

Background: In the absence of a histological diagnosis, persistent albuminuria is globally accepted as the main diagnostic criteria for diabetic kidney disease (DKD). Methods: In the present retrospective study, we evaluated data from an Italian cohort of 42 deceased diabetic donors (mainly with type 2 diabetes). Using the kidney biopsies obtained at the time of donation to evaluate single or double allocation based on Karpinski score, we determined the prevalence of histological lesions attributable to diabetes. Results: All 42 donors presented with proteinuria in the normal range and an estimated glomerular filtration rate (eGFR) (chronic kidney disease [CKD]-EPI) >60 mL/min/1.73 m2. A kidney biopsy was available for 36 patients; of these, one was not interpretable and 32 showed histopathological lesions consistent with DKD and encompassing all histological classes. Thus, we found a relatively high proportion of histologically proven DKD that had been clinically undiagnosed, as none of the patient had significant proteinuria and eGFR 2. Conclusions: The data we present here support the need to implement routine kidney biopsies in normoalbuminuric diabetic subjects in the early stages of CKD. Such strategy may help to improve risk stratification in diabetic patients and guide therapeutic decisions during the early stages of the disease.

Atherosclerosis may starts early in life and each artery has peculiar characteristics likely affecting atherogenesis. The primary objective of the work was to underpin the microRNA (miR)-profiling differences in human normal femoral, abdominal aortic, and carotid arteries. The secondary aim was to investigate if those identified miRs, differently expressed in normal conditions, may also have a role in atherosclerotic arteries at adult ages. MiR-profiles were performed on normal tissues, revealing that aorta and carotid arteries are more similar than femoral arteries. MiRs emerging from profiling comparisons, i.e., miR-155-5p, -27a-5p, and -139-5p, were subjected to validation by RT-qPCR in normal arteries and also in pathological/atheroma counterparts, considering all the available 20 artery specimens. The three miRs were confirmed to be differentially expressed in normal femoral vs aorta/carotid arteries. Differential expression of those miRs was also observed in atherosclerotic arteries, together with some miR-target proteins, such as vimentin, CD44, E-cadherin and an additional marker SLUG. The different expression of miRs and targets/markers suggests that aorta/carotid and femoral arteries differently activate molecular drivers of pathological condition, thus conditioning the morphology of atheroma in adult life and likely suggesting the future use of artery-specific treatment to counteract atherosclerosis.

Introduction: Electron microscopy (EM) represents an indispensable technique for the diagnosis of kidney glomerular diseases. When dedicated tissue is not available, histological and cryostat sections can be reprocessed for EM using the pop-off technique. Here the practical value of this technique is analysed with emphasis on its accuracy in measuring basement membrane thickness and detecting immune deposits. Methods: Ninety-four histological sections of kidney tissues fixed in Serra’s solution, stained with H&E, PAS, and Masson's Trichrome; for EM analysis, the sections were recovered from either treated or untreated microscope slides through the pop-off technique. Some sections were recovered from cryosections allocated for immunofluorescence. Results: The ultrastructural details were sufficiently maintained on tissues fixed with Serra's solution despite being considered disadvantageous for EM. The type of microscope slides and the time of biopsy storage did not affect the quality of section recovery. The histological stains had only moderate effects on the electron-density of the glomerular basement membrane (GBM). The pop-off technique reduced the GBM thickness when compared to the conventional EM processing but preserved the electron density of immune deposits. Conclusions: The application of the pop-off method to renal biopsy is a useful recovery method that produces limited but satisfactory results when there is no suitable material for EM. The ultrastructural morphology was retained even from tissues fixed with Serra's solution, and deposits maintained the expected electron density, however, we observed an overall thickness reduction of the GBM that could have a potential impact on thin membrane disease diagnosis. Graphic abstract: [Figure not available: see fulltext.]

Background and aim of the work: Every year around 275 thousand people in Europe and 420 thousand in the United States are affected by sudden cardiac arrest. Early electrical defibrillation before the arrival of emergency services can improve survival. Training the population to use the AED is essential. The training method currently in use is the BLSD course, which limits training to a population cohort and may not be enough to meet the requirements of the proposed Law no. 1839/2019. This study aims to verify the effectiveness of an online course that illustrates the practical use of the AED to a population of laypeople. Methods: An observational study was conducted to compare a lay population undergoing the view of a video spot and a cohort of people who had participated in BLSD Category A courses. The performances of the two groups were measured immediately after the course and 6 months later. Results: Overall, the video lesson reported positive results. Six months later the skills were partially retained. The cohort that followed the video lesson showed significant deterioration in the ability to correctly position the pads and in safety. Conclusions: Although improved through significant reinforcements, the video spot represents a valid alternative training method for spreading defibrillation with public access and could facilitate the culture of defibrillation as required by the new Italian law proposal. (www.actabiomedica.it).

Arterial calcification is an actively regulated process, with different morphological manifestations. Micro-RNAs emerged as potential regulators of vascular calcification; they may become novel diagnostic tools and be used for a finest staging of the carotid plaque progression. The present study aimed at characterizing the different miRNA-mRNA axes in carotid plaques according to their histological patterns of calcification. Histopathological analysis was performed on 124 retrospective carotid plaques, with clinical data and preoperatory angio-CT. miRNA analysis was carried out with microfluidic cards. Real-time PCR was performed for selected miRNAs validation and for RUNX-2 and SOX-9 mRNA levels. CD31, CD68, SMA, and SOX-9 were analyzed by immunohistochemistry. miRNA levels on HUVEC cells were analyzed for confirming results under in vitro osteogenic conditions. Histopathological analysis revealed two main calcification subtypes of plaques: calcific cores (CC) and protruding nodules (PN). miRNA array and PCR validation of miR-1275, miR-30a-5p, and miR-30d indicated a significant upregulation of miR-30a-5p and miR-30d in the PN plaques. Likewise, the miRNA targets RUNX-2 and SOX-9 resulted poorly expressed in PN plaques. The inverse correlation between miRNA and RUNX-2 levels was confirmed on osteogenic- differentiated HUVEC. miR-30a-5p and miR-30d directly correlated with calcification extension and thickness at angio-CT imaging. Our study demonstrated the presence of two distinct morphological subtypes of calcification in carotid atheromatous plaques, supported by different miRNA signatures, and by different angio-CT features. These results shed the light on the use of miRNA as novel diagnostic markers, suggestive of plaque evolution.

Background and aim of the study: Nursing has not yet received full social recognition and in general the public opinion does not have a completely positive perception of it, which regards it as an auxiliary profession to the medical profession. This study aims to investigate the image of the nurse among year 4 and 5 high school students. Methods: Two groups of students were interviewed, one at their institution without any previous orientation (n = 102) and one at the University of Bologna during orientation day (n = 388). A validated questionnaire (Nursing Attitude Questionnaire) of 30 items was delivered. Two additional questions were added to explore the possibility of advising relatives and acquaintances to become a nurse and therefore to choose the nursing degree course. Results: All areas of the NAQ scored significantly higher in the group where students attended the orientation day, especially the ones related to the professional role, stereotypes and professional value items. Female students ≥ 21 had a more positive image of the nurse. Both groups recognized that nurses are important figures for patients and that they are intelligent people, who must have a degree in order to carry out their work duties. Differences between the groups were recorded. Students in group 1 did not accept nursing autonomy while students in group 2 understood differences between nursing and medical careers, attributing to them the same value. A positive correlation between the positive image of the nursing profession and the interest in becoming nurses or advising others to undertake the nursing course was found. In conclusion, orientation is effective in helping high school students in the choice of their university courses. Regarding nursing, strategies to improve the image of the nurse and enrollment should be combined during orientation day with current up to date communication and learning tools, such as social media and simulation laboratories. Furthermore, it appears increasingly important to extend orientation and tutoring activities to junior high school students, families and teachers. (www.actabiomedica.it).

Mesenchymal stem cells (MSCs) share many properties with other tissue stromal cells, including cell morphology, immunophenotype, differentiation and immunologic properties. In this study, we compared the immunophenotype and the differentiation potential of human vascular wall mesenchymal stem cells (hVW-MSCs) with those of human dermal fibroblasts and myofibroblasts. Cell morphology and surface markers were evaluated by immunofluorescence and flow cytome- try; functional assays for immunomodulation, angiogenesis, adipogenesis and osteogenesis were performed, together with the mRNA analysis of the critical differentiation genes. hVW-MSCs, dermal fibroblasts and myofibroblasts were all negative to CD34, whereas the expression of CD44 stemness marker was more intense in hVW-MSCs. As expected, hVW-MSC plasticity was wide and the angiogenic, adipogenic, osteogenic features were confirmed. Fibroblasts were the less effective in terms of immunomodulation, angiogenesis and adipogenic differentiation; differ- ently from fibroblasts, the myofibroblasts showed a poor angiogenic commitment. The miner- alization assay was positive in all the three cell types, but ultrastructure interestingly evidenced differential osteogenic patterns among them. Our study supports the higher anti-inflammatory and wound healing repair features of hVW-MSCs, in comparison to the other stromal cells investigated. Moreover, we underline the importance of ultrastructure for investigating the specific osteogenic pattern for each cell type.

Parathyroidectomy is a standard practice to treat recurrent or persistent hyperparathyroidism. However, this can lead to the onset of hypoparathyroidism, treatable with the autotransplantation of parathyroid tissue (PT). Tissue can be transplanted immediately after parathyroidectomy or cryopreserved and transplanted only in case of necessity. Since 2011, the Cord Blood Bank and Cardiovascular Tissue Bank of Emilia-Romagna has been storing PT for potential autologous transplantation. To date, there are highly variable data about the viability and function of PT after thawing. However, it is not clear if the PT quality is affected by different cryopreservation protocols and/or by the storage time. The aim of this study was to assess the ex vivo function and viability of the PTs of ten patients stored in the Bank. Tissue morphology was evaluated before and after cryopreservation through histological investigations. PT function was analyzed by assessing the ability of cryopreserved PT to synthesize and secrete parathyroid hormone (PTH) in response to different calcium concentrations. Moreover, viability and function were also investigated on tissue-isolated cells in culture. These data show that tested tissues appear to be viable and able to produce PTH even after 5 years of storage, and the histological architecture is well preserved.

Little is known about the real incidence and the clinical relevance of the enigmatic Monckeberg's medial calcification in the patency of the femoral artery allograft. Here we present a retrospective study on 143 multiorgan donors (mean age 38 years, range 14-59 years), to describe the incidence and the morphological features of vascular calcifications in banked femoral arteries suitable for clinical use. In the present series, focal vascular calcifications were present in 36 (25 %) cases, 23 cases localized in the intima, 7 in the media, and 6 were mixed. No correlation was found between the incidence of calcifications and the classical cardiovascular clinical risk factors (n = 9); only hypertension correlated with the medial localization, but not with the incidence, of the calcification (P = 0.017). While the macroscopic exclusion criteria of vascular grafts include atheromatous and not-atheromatous lesions, we ignore the actual impact of Monckeberg's medial calcification on vessel transplantation and allograft life. In our opinion this is a very important topic, since when the histological criteria for Monckeberg's calcification diagnosis are used, 25 % of our young donors population was affected. Whether Monckeberg's medial calcification is a stable arterial condition, apparently underestimated in the general population, or a dynamic process evolving with age and atherosclerosis, or a banking-related vascular alteration, still remain an open issue deserving further studies with subjects of different ages.

Objective Malignant aortic tumors (MATs) are exceedingly rare, and a comprehensive review of clinical and therapeutic aspects is lacking in the literature. The aim of this study was to analyze all known cases of MATs and to identify predictors of patients' survival. Methods All patients diagnosed with an aortic tumor treated in a single center along with all case reports and reviews available in the literature through a specific PubMed search using keywords such as “malignant” and “aorta” or “aortic,” “tumor,” or “sarcoma” or “angiosarcoma” were analyzed. The tumor's primary location, clinical presentation, histologic subtype, and treatment choice were examined. Survival at 1 year, 3 years, and 5 years and the possible preoperative and operative outcome predictors were evaluated using Kaplan-Meier analysis with a log-rank test and by Cox regression for multivariate analysis. Results In addition to the 5 cases treated in our center, 218 other cases of MAT were reported in the literature from 1873 to 2017. The mean age of the patients was 60.1 ± 11.9 years, and the male to female ratio was 1.59:1. The median overall survival from diagnosis was 8 (7-9) months; 1-, 3-, and 5-year survival rates were 26%, 7.6%, and 3.5%, respectively. Chronic hypertension (P = .03), fever (P = .03), back pain (P = .01), asthenia (P = .04), and signs of peripheral embolization (P = .007) were significant predictors of a poor result. Histologic subtypes had a different impact on survival, with no statistical significance. Compared with other treatment strategies, combined surgical-medical therapy had the best impact on the median survival rate (surgical-medical, 12 [8-24] months; medical, 8 [5-10] months; surgical 7 [2-16] months; no treatment, 2 [0.5-15] months; P = .001). Analyzing exclusively medical approaches, chemotherapy and radiotherapy had the best impact on median survival rate compared with untreated patients (chemotherapy-radiotherapy, 18 [10-26] months; radiotherapy, 16 [8-20] months; chemotherapy, 10 [7-24] months; no medical treatment, 6 [2-16] months; P = .005); these data were not sustained by multivariate analysis. Conclusions Aortic tumors are a malignant pathologic condition with a short survival rate after initial diagnosis. Survival is further diminished in the presence of clinical factors such as hypertension, fever, back pain, asthenia, and signs of peripheral embolization. Combined surgical and medical treatment, particularly with chemotherapy and radiotherapy, has shown the highest survival rate.

Extraneuraxial hemangioblastoma occurs in nervous paraneuraxial structures, somatic tissues, and visceral organs, as part of von Hippel-Lindau disease (VHLD) or in sporadic cases. The VHL gene plausibly plays a key role in the initiation and tumorigenesis of both central nervous system and extraneuraxial hemangioblastoma, therefore, the underlying molecular and genetic mechanisms of the tumor growth are initially reviewed. The clinical criteria for the diagnosis of VHLD are summarized, with emphasis on the distinction of sporadic hemangioblastoma from the form fruste of VHLD (eg, hemangioblastoma-only VHLD). The world literature on the topic of extraneuraxial hemangioblastomas has been comprehensively reviewed with ∼200 cases reported to date: up to 140 paraneuraxial, mostly of proximal spinal nerve roots, and 65 peripheral, 15 of soft tissue, 6 peripheral nerve, 5 bone, and 39 of internal viscera, including 26 renal and 13 nonrenal. A handful of possible yet uncertain cases from older literature are not included in this review. The clinicopathologic features of extraneuraxial hemangioblastoma are selectively presented by anatomic site of origin, and the differential diagnosis is emphasized in these subsets. Reference is made also to 10 of the authors' personal cases of extraneuraxial hemangioblastomas, which include 4 paraneuraxial and 6 peripheral (2 soft tissue hemangioblastoma and 4 renal).

Vascular calcification is related to vascular diseases, for example, atherosclerosis, and its comorbidities, such as diabetes and chronic kidney disease. In each condition, a distinctive histological pattern can be recognised that may influence technical choices, possible intra-operative complications, and procedure outcomes, no matter if the intervention is performed by open or endovascular means. This review considers the classification and initiating mechanisms of vascular calcification. Dystrophic and metastatic calcifications, Monckeberg's calcification, and genetic forms are firstly outlined, followed by their alleged initiation mechanisms; these include (a) ineffective macrophage efferocytosis; (b) ectopic osteogenesis driven by modified resident or circulating osteoprogenitors. As in physiological bio-mineralisation, active calcification starts with the deposition of cell derived matrix vesicles into the extracellular matrix. To substantiate this belief, an in depth ultra-structural documentation of hydroxyapatite crystal deposition on such vesicles is provided in an ex-vivo human vascular cell model. Revealing the vesicle composition and phenotype in normal and pathological vascular conditions will be essential for the development of new therapeutic strategies, in order to prevent and treat vascular calcification.

Background/Aims: The increase in the survival rate of patients with chronic renal failure due to substitution treatment prompts an investigation of their quality of life (QoL), a key measure to evaluate the outcomes of chronic disease treatment. To determine whether hemodialysis or peritoneal dialysis provide a better QoL, a systematic meta-analysis was performed. Methods: We searched through the database Cinahl, Medline, PubMed, Scopus and Proquest, including articles published from 2011 until June 2016. We selected articles that compared, through KDQOL-SF 1.3 or 36 questionnaires, QoL among patients undergoing hemodialysis and peritoneal dialysis. The data was collected using Excel Office, and t-test has been performed on independent samples to identify significant differences. Results: Only some of the seven articles found significant differences between the two treatments. One of the studies showed a better QoL for peritoneal dialysis patients, while, on the contrary, two other studies support that the best QoL is in patients receiving hemodialysis. Another article displayed significant difference only for satisfaction in relation to care, better in patients on peritoneal dialysis, and for physical health, better in hemodialysis. Conclusions: The analysis has not led to a unanimous conclusion. Quantitative analysis showed that the only statistically significant difference between the QoL of patients on hemodialysis and peritoneal dialysis regards the effect of kidney disease, which happens to be better in patients undergoing peritoneal dialysis.

Chronic kidney disease (CKD) exacerbating vascular disease poses a major challenge to nephrology. Surgically placed vascular fistulas, as an aid to hemodialysis prior to kidney transplant, have extended many lives, while post-surgical restenosis closure of the fistula by smooth muscle cells affects many lives. When post-surgical restenosis is developed, palliative measures are almost always surgical: there are no effective drug treatments. In this study, we offer a testable hypothesis that effects of CKD on widely distributed vascular diseases and the phenomenon of fistula restenosis are both driven by the pathologic creation of non-dividing smooth muscle cells via asymmetric division of exponentially increasing metakaryotic stem cells. In slow growing atherosclerotic plaques, the Benditts demonstrated clonality of smooth muscle cells that we posit originate in a single mutated metakaryotic stem cell of fetal/juvenile vasculogenesis. In the fast process of fistula restenosis, we posit quiescent metakaryotic stem cells "on call" for wound healing among which are rare stem cells that have lost the ability to cease division. These hypotheses and suggestions for specific research paths toward development of effective drug therapies are built on (a) our shared discoveries of the role of metakaryotic stem cells in organogenesis, carcinogenesis, and atherosclerotic plaque formation and (b) the recent finding that metakaryotic cancer stem cells are constitutively resistant to radio- and chemotherapies yet sensitive to killing by a wide range of existing drugs. We propose to test these hypotheses in discarded fistulas and stem cells derived therefrom, and, if supported, to test drug-eluting devices to block fistula restenosis.

ABSTRACT: Aim: To evaluate if anti-Müllerian hormone (AMH) is a reliable marker of ovarian reserve in young women undergoing ovarian tissue cryopreservation. Patients & methods: Relationships of serum AMH levels with primordial follicle density, age and reproductive hormones were investigated using the Pearson or Spearman correlation coefficient in 86 women with cancer (12–38 years) undergoing ovarian tissue cryopreservation. AMH variations through the menstrual cycle were assessed by the Kruskal–Wallis test. p

ABSTRACT: Aim: To present preliminary results of autotransplantation of cryopreserved ovarian tissue performed at Sant’Orsola-Malpighi Hospital, Bologna, Italy. Materials & methods: Orthotopic transplantation was performed in two women with colorectal and breast cancer, and heterotopic transplantation was performed in one Hodgkin’s lymphoma woman. The presence of micrometastasis in the ovarian tissue was checked, and morphological features of ovarian tissue were evaluated before transplantation. Ovarian function was monitored by hormonal and ultrasound-color Doppler examination after transplantation. Results: In all three women, no micrometastasis was found; light and transmission electron microscopy showed well-preserved thawed ovarian tissue. Ovarian function recovery was observed 2–4 months after transplantation. Spontaneous menstrual cycles occurred in two women with normal follicular densities. No periods occurred in the woman with low follicular density at the time of tissue collection. Conclusion: Ovarian tissue cryopreservation and transplantation is a promising approach for preserving ovarian function in women with cancer.

Mesenchymal stromal (stem) cells (MSCs) are defined by plastic adherent growth, multiple phenotype expressions, and tripotential mesodermal capability. The authors report examples where electron microscopy (EM) plays a role in stem cell research. MSCs isolated from human arteries are ultrastructurally heterogeneous and become more homogenous after plastic adhesion. EM shows a moderate complement of organelles, mainly mitochondria, rough endoplasmic reticulum, and glycogen aggregates. Clear vacuoles and vesicles are prominent when cells are recovered from plates using an enzymatic method. Since the mesengenic plasticity is the single most important criterion to define a cell as mesenchymal stromal, the authors induced experimentally adipogenic, leiomyogenic, cardiomyogenic, osteo-chondrogenic differentiations. In no case did EM reveal the achievement of complete differentiation. The authors obtained multivacuolated pre-adipocytes and never univacuolated adipocytes typical of mature white fat; myofibroblast and rhabdomyoblast morphotypes, where contractile filaments were not organized to form functional complexes, i.e., dense bodies and sarcomeres. Chondrogenesis and osteogenesis assays resulted in extracellular matrix changes. Collagen and proteoglycan filament/particle deposition was seen when chondrogenesis was promoted. Hydroxyapatite crystals, psammoma bodies, and plaque-like calcified matrix deposits were found in the osteogenic matrix. EM provides detailed structural information on the degree of differentiation induced in stem cells and demonstrates that the methods so far developed are not able to promote complete cell differentiation. These observations contribute to explain why clinical applications with hMSCs have produced results far lower than initial expectations.

Fibromuscular dysplasia (FMD) of the splenic artery is a rare underdiagnosed condition. Here, we report two cases of FMD affecting the splenic artery: one alone and one concomitantly with the renal artery. Histology revealed fibromuscular thickening of the media layer alternating with a circumferential calcification of the whole artery thickness. Ultrastructurally, FMD showed matrix vesicles and dense bodies in the extracellular matrix. A diagnosis of FMD with calcification was made. This is the first report to document circumferential lamellar calcifications alternating with the more typical fibrotic medial areas in the rare FMD localized to splenic artery.

Aim: To investigate mechanisms by which doxorubicin (DOX) and cisplatin (CIS) cause human ovarian stroma injury. Patients & methods: Stromal cells from human cryopreserved ovarian tissue were cultured in the presence of 1 µM DOX and 10 µM CIS. Ovarian damage induced by treatments was evaluated by ‘Live/Dead’ and sulforhodamine-B assays, the expression of different apoptosis markers. Results: Stromal cell growth was inhibited by DOX and CIS, and this effect was accompanied by apoptosis through mitochondrial pathway activation: Bax, cleaved-caspase 9, cleaved-PARP1 induction and Akt1, Bcl2, phospho-44/42-MAPK/ERK1/2 reduction were observed. Conclusion: DOX and CIS induced apoptosis in human ovarian stromal cells. Knowledge of mechanisms by which the drugs act is important to identify possible ways to counteract side effects of chemotherapy on ovaries.

Study question Which is the best method for human ovarian tissue cryopreservation: slow freezing/rapid thawing (SF/RT) or vitrification/warming (V/W)? Summary answer The conventional SF/RT protocol used in this study seems to better preserve the morpho-functional status of human cryopreserved ovarian tissue than the used open carrier V/W protocol. What is known already Cryopreservation of human ovarian tissue is generally performed using the SF/RT method. However, reduction in the follicular pool and stroma damage are often observed. An emerging alternative procedure is represented by V/W which seems to allow the maintenance of the morphological integrity of the stroma. Study design, size, duration This is a retrospective cohort study including six patients affected by oncological diseases and enrolled from January to December 2014. Participants/materials, setting, methods Ovarian tissue was laparoscopically harvested from the right and left ovaries and was cryopreserved using a routinary SF/RT protocol or a V/W method, involving tissue incubation in two solutions (containing propylene glycol, ethylene glycol and sucrose at different concentrations) and vitrification in an open system. For each patient, three pieces from each ovary were collected at the time of laparoscopy (fresh tissue) and after storage (SF/RT or V/W) and processed for light microscopy (LM) and transmission electron microscopy (TEM), to assess the morphological and ultrastructural features of follicles and stroma, and for laser scanning confocal microscopy (LSCM), to determine the functional energetic/redox stroma status. The preservation status of SF/RT and V/W ovarian tissues was compared with that of fresh ones, as well as between them. Main results and the role of chance By LM and TEM, SF/RT and V/W samples showed cryodamage of small entity. Interstitial oedema and increased stromal cell vacuolization and chromatin clumping were observed in SF/RT samples; in contrast, V/W samples showed oocyte nuclei with slightly thickened chromatin and irregular shapes. The functional imaging analysis by LSCM revealed that the mitochondrial activity and intracellular reactive oxygen species levels were reduced both in SF/RT and in V/W samples compared with fresh samples. The study also showed progressive dysfunction of the mitochondrial activity going from the outer to the inner serial section of the ovarian cortex. The reduction of mitochondrial activity of V/W samples compared with fresh samples was significantly higher in the inner section than in the outer section. Limitations, reasons for caution The results report the bioenergetic and oxidative status assessment of fresh and cryopreserved human ovarian tissue by LSCM, a technique recently applied to tissue samples. The use of LSCM on human ovarian tissues after SF/RT or V/W is a new application that requires validation. The procedures for mitochondrial staining with functional probes and fixing are not yet standardized. Xenografting of the cryopreserved ovarian tissue in severe combined immunodeficient mice and in vitro culture have not yet been performed. Wider implications of the findings The identification of a cryopreservation method able to maintain the morpho-functional integrity of the ovarian tissue and a number of follicles comparable with those observed in fresh tissue might optimize results in clinical practice, in terms of recovery, duration of ovarian function and increased delivery outcomes after replanting. The SF/RT protocol allowed better morpho-functional tissue integrity than the V/W procedure. Study funding/competing interests Funding was provided by Fondazione del Monte di Bologna e Ravenna, Italy. Dr N.A.M. was granted by the project ONEV MIUR PONa3 00134-n.254/R&C 18 5 2011 and the project GR-2011-02351396 (Ministry of Health, Young Researchers Grant 2011/2012). There are no competing interests. Trial registration number Clinical trial 74/2001/0 (approved:13 2 2002): 'Pilot study on cryopreservation of human ovarian tissue: morphological and immunohistochemical analysis before and after cryopreservation'.

Primary malignant melanoma originating in the digestive tract is particularly rare, mainly affecting the anorectum and oral cavity. Primary malignant melanoma of the esophagus has been the subject mostly of case reports. This tumor has a dismal prognosis with a frequency estimated to be approximately 0.1% to 0.2% of all esophageal malignancies. According to the review by Volpin et al of November 2002, 238 cases of primary malignant melanoma of the esophagus have been published up to early 2001. We present an additional case of primary malignant melanoma of the esophagus of the amelanotic variant in a 69-year-old man. The patient was preoperatively investigated by esophageal endoscopy and endoscopic ultrasound. The surgically resected tumor specimen was examined histologically and supplemented by immunohistochemical and ultrastructural analysis. Intra-abdominal relapse occurred after 8 months at the site of surgery, necessitating repeat resection. The patient died of advanced intra-abdominal disease 14 months after the primary diagnosis. A comprehensive computerized (PubMed/Medline) review of the world literature was also carried out and 99 additional cases (after the review by Volpin et al) were found, 9 of them from the 1990s which escaped previous tabulations, and 90 from the years 2000 to 2010, amounting to a grand total of 337 ever published.

Lipofuscin-like granules, first described by Biava and West in 1965, are a subcellular, quasi-physiologic finding mainly seen in the smooth muscle cells of renal arterioles, but also in juxtaglomerular cells and the lacis cells of human kidneys. They increase in number in subjects affected by arterial hypertension and diabetes. They do not correlate with a specific primary renal disease. Lipofuscin-like granules are not related to renin granules. The world literature on this subject is almost non-existent, and the awareness of this finding or its clinical significance among either pathologists or nephrologists is very poor. We incidentally observed these lipofuscin-like granules in 8 cases during the routine electron microscope examination of 440 renal biopsies, and report herein on their ultrastructural features. Six of these 8 patients were affected by arterial hypertension, one of whom was also concomitantly affected by diabetes mellitus. These lipofuscin-like granules appear as dense bodies with a lipid component, a coarsely granular matrix, and a crystalloid component which may appear in a band or dot pattern, according to the plane of sectioning. The pathologist has to be aware of these lipofuscin-like granules in order not to confuse them with the semicircularly organized (fingerprint) linear immune deposits associated with some specific glomerulopathies.

Background aims. The presence of ectopic tissues in the pathologic artery wall raises the issue of whether multipotent stem cells may reside in the vasculature itself. Recently mesenchymal stromal cells (MSC) have been isolated from different human vascular segments (VW MSC), belying the previous view that the vessel wall is a relatively quiescent tissue. Methods. Resident multipotent cells were recovered from fresh arterial segments (aortic arches, thoracic and femoral arteries) collected in a tissue-banking facility and used to establish an in situ and in vitr o study of the stemness features and multipotency of these multidistrict MSC populations. Results. Notch-1 � , Stro-1 � , Sca-1 � and Oct-4 � cells were distributed along an arterial wall vasculogenic niche. Multidistrict VW MSC homogeneously expressed markers of stemness (Stro-1, Notch-1 and Oct-4) and MSC lineages (CD44, CD90, CD105, CD73, CD29 and CD166) whilst they were negative for hematopoietic and endothe- lial markers (CD34, CD45, CD31 and vWF). Each VW MSC population had characteristics of stem cells, i.e. a high effl ux capability for Hoechst 33342 dye and the ability to form spheroids when grown in suspension and generate colonies when seeded at low density. Again, VW MSC cultured in induction media exhibited adipogenic, chondrogenic and leiomyogenic potential but less propensity to osteogenic differentiation, as documented by histochemical, immunohistochemical, molecular and electron microscopy analysis. Conclusions. Overall, these fi ndings may enlighten the physiopathologic mechanisms of vascular wall diseases as well as having potential implications for cellular, genetic and tissue engineering approaches to treating vascular pathologies when these are unresponsive to medical and surgical therapies.

Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSC) are currently under evaluation in phase III clinical trials for inflammatory bowel disease and other intestinal disease manifestations. The therapeutic efficacy of these treatments may derive from a combination of the differentiation, trophic and immunomodulatory abilities of the transplanted cells. We investigated intestinal tissues as sources of MSC: such cells may support tissue-specific functions and hold advantages for engraftment and contribution in the gastrointestinal environment.Intestinal specimens were collected, and the mucosa and submucosa mechanically separated and enzymatically digested. Mesenchymal stromal populations were isolated, expanded and characterized under conditions commonly used for MSC. The differentiation potential, trophic effect and immunomodulatory ability were investigated. Results We successfully isolated and extensively expanded populations showing the typical MSC profile: CD29+, CD44+, CD73+, CD105+ and CD166+, and CD14(-), CD34(-) and CD45(-). Intestinal mucosal (IM) MSC were also CD117+, while submucosal cultures (ISM MSC) showed CD34+ subsets. The cells differentiated toward osteogenic, adipogenic and angiogenic commitments. Intestinal-derived MSC were able to induce differentiation and organization of intestinal epithelial cells (Caco-2) in three-dimensional collagen cultures. Immunomodulatory activity was evidenced in co-cultures with normal heterologous phytohemagglutinin-stimulated peripheral blood mononuclear cells. Conclusions Multipotent MSC can be isolated from intestinal mucosal and submucosal tissues. IM MSC and ISM MSC are able to perform trophic and immunomodulatory functions. These findings could open a pathway for novel approaches to intestinal disease treatment.

A case of spinal thoracic chordoma involving the T9 vertebra in a 70-year-old male patient, destroying the vertebral body and invading the vertebral canal with compression of the spinal cord, is presented. The patient was referred to our neurosurgical unit with a history of an irradiated metastatic adenocarcinoma to the thoracic vertebra, a diagnosis that was rendered 3 years earlier at another hospital on presentation. This misdiagnosis was likely due to the absolute rarity of thoracic vertebral chordomas (2%-3% of all chordomas), the higher frequency of metastatic deposits to the vertebrae from visceral cancers in the elderly, the limited amount of biopsy material available for histologic examination, and the epithelial phenotype of the tumor (keratin/EMA positive). The patient underwent second palliative surgery with subtotal piecemeal removal of the tumor bringing relief of the neurologic symptoms. The bulk of the tumor was represented by a high-grade pleomorphic sarcoma with adjacent areas of atypical chordoma. Small foci of conventional chordoma were also found. The previous histologic slides were also reviewed, which were consistent with the areas of atypical chordoma. Small targeted tissue fragments from areas of (atypical) chordoma and from sarcomatous areas were recovered for electron microscopy. The fine features of chordoma and focal rhabdomyoblastic differentiation were found with the latter retrospectively supported by immunohistochemical detection of striated muscle markers. A final diagnosis of dedifferentiated chordoma with rhabdomyoblastic differentiation was finally established. Rhabdomyoblastic metaplasia is a novelty in dedifferentiated chordoma. The patient died after 5 months. Autopsy was not requested.

Cooperation between research communities and software-development teams has led to the creation of novel software. The purpose of this paper is to show an alternative work method based on the usage of ImageJ (http://rsb.info.nih.gov/ij/), which can be effectively employed in solving common microscopic and ultrastructural image analysis tasks. As an open-source software, ImageJ provides the possibility to work in a free-development/sharing world. Its very "friendly" graphical user interface helps users to manage and edit biomedical images. The on-line material such as handbooks, wikis, and plugins leads users through various functions, giving clues about potential new applications. ImageJ is not only a morphometric analysis software, it is sufficiently flexible to be adapted to the numerous requirements tasked in the laboratories as routine as well as research demands. Examples include area measurements on selectively stained tissue components, cell count and area measurements at single cell level, immunohistochemical antigen quantification, and immunoelectron microscopy gold particle count.

Background The surgical treatment of radio-induced carotid stenosis (RICS) is challenging and burdened with high risk of complications. Carotid stenting (CAS) may be a valid alternative, but better approach is still not defined. Two approaches have been assessed in this an observational monocentric study, focusing on perioperative and follow-up complications. Methods From 2005 to 2013, data on patients treated for extracranial carotid stenosis with previous radiotherapy (RT) for head or neck tumor were prospectively collected according to the procedure performed (open repair [OR], endovascular stenting [CAS]). Patient demographics, clinical risk factors, organ of cancer origin, elapsed time from RT, duplex ultrasound (DUS) and clinical presentation of carotid stenosis were preoperatively gathered. Primary outcomes were technical success, 30-day mortality, transient ischemic attack (TIA)/stroke and myocardial infarction (MI). In OR group, laterocervical hematoma and cranial nerve injury were evaluated and, in CAS group, arterial access site complications were considered as well. Secondary outcomes were the mortality, TIA/stroke, restenosis and reintervention during follow-up. Results Fifteen RICS were treated in 12 patients (M/F=9/3; median age: 71 years, range: 51-80). OR was performed to treat 8 RICS in 5 patients (M/F=3/2; median age: 65, range: 50-76) and CAS to treat 7 RICS in 7 patients (M/F=6/1; median age: 75, range 59-80). In OR group, all patients were asymptomatic and all plaques were hypoechogenic at DUS with histological features of vulnerable plaque. Technical success was 100%. Thirty-day mortality was 12.5% (1 death for upper airways complication in patient with previous neck surgery). TIA/stroke and MI rate were 0% and cranial nerve injury 12.5%. In CAS group, carotid stenosis was symptomatic in 2(28.6%) cases, all plaques resulted hypoechogenic at DUS. Technical success was 100%. Thirty-day mortality was 0%. TIA occurred in 4 (57.1%) patients. No stroke, MI or access site hematoma occurred in this group. Mean follow-up was 35.5±18.2 months. At 12 and 36 month, freedom from neurological events, restenosis and reintervention was 100%. At 1 and 3 years, survival was 91.7% and 81.5%, respectively. No patient died for carotid-related or cardiovascular causes. Conclusions According with the plaque morphology and the high rate of cerebrovascular complications of CAS, OR should be suggested as the first choice for treatment of RICS in the majority of cases. In patients with previous neck surgery, assessment of airways is necessary before OR to avoid respiratory complications.

Aims In this paper we aimed to analyse the typology and the phenotype of the different vascular modifications in human hepatocellular carcinomas (HCCs) with a new immunomorphological and gene expression approach. We also attempted to correlate these modifications with the histological parameters of tumour aggressiveness and the surrounding liver parenchyma. Methods Ninety-six HCCs (from 80 patients) were retrospectively enrolled, 46 occurring in non-cirrhotic livers, and 50 in livers transplanted for cirrhosis. Histopathological analysis, immunohistochemistry for CD34, Nestin and WT1 and RT-PCR for Nestin, transforming growth factor-β1 (TGFβ1) and insulin-like growth factor 1 (IGF1R) mRNA were performed in all nodules. Results By correlating the CD34 and Nestin immunoreactivity in HCC vasculature with the tumorous architecture, we identified four vascular patterns (named from ‘a’ to ‘d’). Each of them was characterised by different expressions of TGFβ1 and IGF1R mRNA. Pattern a showed CD34-positive/Nestin-negative sinusoids, and was prevalent in microtrabecular lesions. Pattern b showed similar morphology and architecture as pattern a, but with Nestin-positive sinusoids and a significant ‘boost’ in IGF1R and TGFβ1 mRNAs. In patterns c and d a progressive sinusoid loss and a gain of newly formed arterioles were seen. Notably, HCCs with pattern a arose more frequently in cirrhosis (p=0.024), and showed lower incidence of microvascular invasion (p=0.002) and infiltration (p=0.005) compared with HCCs with other patterns. Conclusions Although future studies are surely required, the identification of different vascular profiles in HCCs from cirrhotic and non-cirrhotic livers may help clarify the relationship between HCC progression and aggressiveness.

Cells use several mechanisms to transfer information to other cells. In this study, we describe micro/nanotubular connections and exosome-like tubule fragments in multipotent mesenchymal stem cells (MSCs) from human arteries. Scanning and transmission electron microscopy allowed characterization of sinusoidal microtubular projections (700 nm average size, 200 µm average length, with bulging mitochondria and actin microfilaments); short, uniform, variously shaped nanotubular projections (100 nm, bidirectional communication); and tubule fragments (50 nm). This is the first study demonstrating that MSCs from human arteries constitutively interact through an articulate and dynamic tubule network allowing long-range cell to cell communication.

The cryopreservation protocol we use for arterial reconstructive surgery has been studied to evaluate smooth muscle cell (SMC) structural integrity and viability before implantation. Samples of human thoracic aortas (HTA) were harvested from five multi-organ donors. Sampling included unfrozen and cryopreserved specimens. Cryopreservation was performed using RPMI with human albumin and 10% Me 2 SO in a controlled-rate freezing apparatus. Thawing was accomplished by submerging bags in a water bath (39 °C) followed by washings in cooled saline. In situ cell preservation as investigated by light and transmission electron microscopy showed that SMCs from cryopreserved HTA had nuclear and cytoplasmic changes. A TUNEL assay, performed to detect DNA fragmentation in situ, showed increased SMC nuclear positivity in cryopreserved HTA when compared to unfrozen samples. 7-AAD flow cytometry assay of cells derived from cryopreserved HTA showed that an average of 49 ± 16% cells were unlabeled after cryopreservation. Organ cultures aimed to study cell ability to recover cryopreservation damage showed a decreasing number of SMCs from day 4 to day 15 in cryopreserved HTA. In conclusion, the cryopreservation protocol applied in this study induces irreversible damage of a significant fraction of arterial SMCs.

INTRODUCTION: This prospective study defined the immunological and clinical results after fresh and cryopreserved arterial homograft replacement due to graft infection. MATERIALS AND METHODS: Thirty patients who underwent ABO-compatible homograft transplantation were studied for anti-human leukocyte antigen (HLA): antibody production and CD3- and CD4- versus CD8-positive lymphocyte subsets. Nine patients (30%) received immunosuppressive treatment with cyclosporine (1 to 3 mg/kg/d). Immunological studies were performed preoperatively, and early (1, 3, 7 days) and late (1, 3, 6, 12, 24, 36, 48 months) during follow-up. Abdominal computed tomography scans were performed postoperatively at 1, 6, 12, 24, 36, and 48 months of follow-up. RESULTS: Preoperatively, antibodies were not detected. Postoperatively, a progressive increase in percent panel reactive antibodies was observed in all patients 1 month after the transplant. There were no difference between fresh and cryopreserved homografts. The antibody response among patients treated with cyclosporine was less pronounced and delayed. Recipient antibodies were directed against donor-specific antigens. During the immediate postoperative period (1, 3, 7 days) there was a slight increase in CD3- and CD4-positive T lymphocytes and a concomitant decrease in the CD8 subset. Later, CD3 and CD4 progressively decreased and the CD8 set increased. Clinically, no patients had signs of recurrent infection upon late follow-up. Four patients died (13%), but only one death was homograft-related (rupture of the graft). At 2-year follow-up, two patients showed stenotic lesions due to chronic rejection. Clinically, no differences were noted between fresh and cryopreserved homografts, or between patients treated with or without cyclosporine. CONCLUSIONS: Fresh and cryopreserved arterial homografts are immunogenic; they induce a strong anti-HLA antibody response, similar to chronic rejection.