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Background: The COVID-19 pandemic has been a health emergency with a significant impact on the world due to its high infectiousness. The disease, primarily identified in the lower respiratory tract, develops with numerous clinical symptoms affecting multiple organs and displays a clinical finding of anosmia. Several authors have investigated the pathogenetic mechanisms of the olfactory disturbances caused by SARS-CoV-2 infection, proposing different hypotheses and showing contradictory results. Since uncertainties remain about possible virus neurotropism and direct damage to the olfactory bulb, we investigated the expression of SARS-CoV-2 as well as ACE2 receptor transcripts in autoptic lung and olfactory bulb tissues, with respect to the histopathological features., Methods: Twenty-five COVID-19 olfactory bulbs and lung tissues were randomly collected from 200 initial autopsies performed during the COVID-19 pandemic. Routine diagnosis was based on clinical and radiological findings and were confirmed with post-mortem swabs. Real-time RT-PCR for SARS-CoV-2 and ACE2 receptor RNA was carried out on autoptic FFPE lung and olfactory bulb tissues. Histological staining was performed on tissue specimens and compared with the molecular data., Results: While real-time RT-PCR for SARS-CoV-2 was positive in 23 out of 25 lung samples, the viral RNA expression was absent in olfactory bulbs. ACE2-receptor RNA was present in all tissues examined, being highly expressed in lung samples than olfactory bulbs., Conclusions: Our finding suggests that COVID-19 anosmia is not only due to neurotropism and the direct action of SARS-CoV-2 entering the olfactory bulb. The mechanism of SARS-CoV-2 neuropathogenesis in the olfactory bulb requires a better elucidation and further research studies to mitigate the olfactory bulb damage associated with virus action.

A growing body of evidence indicates that a neuropathological cross-talk takes place between the coronavirus disease 2019 (COVID-19) -the pandemic severe pneumonia that has had a tremendous impact on the global economy and health since three years after its outbreak in December 2019- and Alzheimer's Disease (AD), the leading cause of dementia among human beings, reaching 139 million by the year 2050. Even though COVID-19 is a primary respiratory disease, its causative agent, the so-called Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), is also endowed with high neuro-invasive potential (Neurocovid). The neurological complications of COVID-19, resulting from the direct viral entry into the Central Nervous System (CNS) and/or indirect systemic inflammation and dysregulated activation of immune response, encompass memory decline and anosmia which are typically associated with AD symptomatology. In addition, patients diagnosed with AD are more vulnerable to SARS-CoV-2 infection and are inclined to more severe clinical outcomes. In the present review, we better elucidate the intimate connection between COVID-19 and AD by summarizing the involved risk factors/targets and the underlying biological mechanisms shared by these two disorders with a particular focus on the Angiotensin-Converting Enzyme 2 (ACE2) receptor, APOlipoprotein E (APOE), aging, neuroinflammation and cellular pathways associated with the Amyloid Precursor Protein (APP)/Amyloid beta (Aβ) and tau neuropathologies. Finally, the involvement of ophthalmological manifestations, including vitreo-retinal abnormalities and visual deficits, in both COVID-19 and AD are also discussed. Understanding the common physiopathological aspects linking COVID-19 and AD will pave the way to novel management and diagnostic/therapeutic approaches to cope with them in the post-pandemic future.

Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. EVs from pathogenic bacteria play functions in host immune modulation, elimination of host defenses, and acquisition of nutrients from the host. Here, we observed EV production of the bacterial speck disease causal agent, Pseudomonas syringae pv. tomato ( Pto ) DC3000, as outer membrane vesicle release. Mass spectrometry identified 369 proteins enriched in Pto DC3000 EVs. The EV samples contained known immunomodulatory proteins and could induce plant immune responses mediated by bacterial flagellin. Having identified two biomarkers for EV detection, we provide evidence for Pto DC3000 releasing EVs during plant infection. Bioinformatic analysis of the EV-enriched proteins suggests a role for EVs in antibiotic defense and iron acquisition. Thus, our data provide insights into the strategies this pathogen may use to develop in a plant environment. IMPORTANCE The release of extracellular vesicles (EVs) into the environment is ubiquitous among bacteria. Vesiculation has been recognized as an important mechanism of bacterial pathogenesis and human disease but is poorly understood in phytopathogenic bacteria. Our research addresses the role of bacterial EVs in plant infection. In this work, we show that the causal agent of bacterial speck disease, Pseudomonas syringae pv. tomato , produces EVs during plant infection. Our data suggest that EVs may help the bacteria to adapt to environments, e.g., when iron could be limiting such as the plant apoplast, laying the foundation for studying the factors that phytopathogenic bacteria use to thrive in the plant environment., Competing Interests: The authors declare no conflict of interest.

Dittrichia viscosa uptake and translocation of the metalloid As is not fully understood and some data are contradictory, but its adaptability to this pollutant is known and is dependent on its genetic variability. D. viscosa is not a hyperaccumulator plant, but it can grow in high-drought conditions while still producing large biomass, even tolerating significant concentrations of As 3+ and As 5+ . In spite of these remarkable characteristics, adaptive modification of performances is not predictable in wild populations. In previous work, we established experimental clonal populations to perform a functional study on the aquaporin NIP1.1. Here, we propose a strategy to select a clonal population of D. viscosa with a defined phenotype related to As tolerance and to reduced NIP1.1 expression levels for phytoremediation applications. From the previous work, we selected four independent clones, two of them belonging to the weak population (W8 and W9) and the other two belonging to the strong population (S1 and S3). The weak and strong populations differ for a different expression ratio root/shoot of DvNip1;1 that brings a different tolerance to As presence. The stress response of the populations, revealed by the CAT enzymatic test, was statistically correlated to the clones, but not to As uptake. Performance of the selected plants on a second unrelated metallic pollutant, Cd, was evaluated, showing that Cd uptake is also independent from the tolerant phenotype. In vitro culture methods using solid media and temporary immersion bioreactors were compared to propose an optimized combined protocol. The procedure yielded propagation of genetically stable tolerant clonal lines with good uptake of As and Cd. The plants, mass-produced with the developed in vitro protocol, were able to maintain their acquired abilities and are potentially able be later applied in phytoremediation or contaminated areas' re-naturalization.

Beyond deficits in hippocampal-dependent episodic memory, Alzheimer's Disease (AD) features sensory impairment in visual cognition consistent with extensive neuropathology in the retina. 12A12 is a monoclonal cleavage specific antibody (mAb) that in vivo selectively neutralizes the AD-relevant, harmful N-terminal 20-22 kDa tau fragment(s) (i.e., NH 2 htau) without affecting the full-length normal protein. When systemically injected into the Tg2576 mouse model overexpressing a mutant form of Amyloid Precursor Protein (APP), APPK670/671L linked to early onset familial AD, this conformation-specific tau mAb successfully reduces the NH 2 htau accumulating both in their brain and retina and, thus, markedly alleviates the phenotype-associated signs. By means of a combined biochemical and metabolic experimental approach, we report that 12A12mAb downregulates the steady state expression levels of APP and Beta-Secretase 1 (BACE-1) and, thus, limits the Amyloid beta (Aβ) production both in the hippocampus and retina from this AD animal model. The local, antibody-mediated anti-amyloidogenic action is paralleled in vivo by coordinated modulation of the endocytic (BIN1, RIN3) and bioenergetic (glycolysis and L-Lactate) pathways. These findings indicate for the first time that similar molecular and metabolic retino-cerebral pathways are modulated in a coordinated fashion in response to 12A12mAb treatment to tackle the neurosensorial Aβ accumulation in AD neurodegeneration.

Background: White Sponge Nevus (WSN) is a rare benign disorder associated with mutations in genes coding for cytokeratin 4 (KRT4) and 13 (KRT13) characterized by dyskeratotic hyperplasia of mucous membranes. This study was aimed at examining different approaches (cytology, pathology and genetic analysis) to WSN diagnosis., Methods: A series of four patients with asymptomatic white diffuse oral lesions were evaluated and, before performing an incisional biopsy for pathology, an oral brush Thin Prep was collected for exfoliative liquid-based cytology (LBC). DNA for genetic analysis was also obtained from patients and both their parents, using buccal swabs., Results: Pathology and cytology showed similar results, leading to the same diagnosis of hyperkeratotic epithelium with acanthosis and spongiosis, without atypia, demonstrating the efficiency of LBC for the differential diagnosis. Sequencing analysis revealed at least 6 rare variants in the KRT4 and KRT13 genes in each patient, contributed in part by both unaffected parents., Conclusions: Thin Prep for oral exfoliative cytology and genetic analysis are sufficient for an accurate diagnosis of WSN. The combination of cytological and genetic analyses could substitute the histologic exam, providing a non-invasive alternative for incisional biopsy.

Tau cleavage plays a crucial role in the onset and progression of Alzheimer's Disease (AD), a widespread neurodegenerative disease whose incidence is expected to increase in the next years. While genetic and familial forms of AD (fAD) occurring early in life represent less than 1%, the sporadic and late-onset ones (sAD) are the most common, with ageing being an important risk factor. Intracerebroventricular (ICV) infusion of streptozotocin (STZ)-a compound used in the systemic induction of diabetes due to its ability to damage the pancreatic β cells and to induce insulin resistance-mimics in rodents several behavioral, molecular and histopathological hallmarks of sAD, including memory/learning disturbance, amyloid-β (Aβ) accumulation, tau hyperphosphorylation, oxidative stress and brain glucose hypometabolism. We have demonstrated that pathological truncation of tau at its N-terminal domain occurs into hippocampi from two well-established transgenic lines of fAD animal models, such as Tg2576 and 3xTg mice, and that it's in vivo neutralization via intravenous (i.v.) administration of the cleavage-specific anti-tau 12A12 monoclonal antibody (mAb) is strongly neuroprotective. Here, we report the therapeutic efficacy of 12A12mAb in STZ-infused mice after 14 days (short-term immunization, STIR) and 21 days (long-term immunization regimen, LTIR) of i.v. delivery. A virtually complete recovery was detected after three weeks of 12A12mAb immunization in both novel object recognition test (NORT) and object place recognition task (OPRT). Consistently, three weeks of this immunization regimen relieved in hippocampi from ICV-STZ mice the AD-like up-regulation of amyloid precursor protein (APP), the tau hyperphosphorylation and neuroinflammation, likely due to modulation of the PI3K/AKT/GSK3-β axis and the AMP-activated protein kinase (AMPK) activities. Cerebral oxidative stress, mitochondrial impairment, synaptic and histological alterations occurring in STZ-infused mice were also strongly attenuated by 12A12mAb delivery. These results further strengthen the causal role of N-terminal tau cleavage in AD pathogenesis and indicate that its specific neutralization by non-invasive administration of 12A12mAb can be a therapeutic option for both fAD and sAD patients, as well as for those showing type 2 diabetes as a comorbidity.

Introduction: Primary autoimmune hypophysitis (PAHs) is a rare inflammatory disease of the pituitary gland. Although largely investigated, the pathogenesis of PAH is not completely clarified. We aimed to investigate the immune response in PAHs., Material and Methods: Serum anti-pituitary and anti-hypothalamus antibodies (respectively APAs and AHAs) were investigated though an indirect immunofluorescence on monkey hypophysis and hypothalamus slides, serum cytokines though an array membrane and cell-mediated immunity though the white blood cells count., Results: Nineteen PAH cases entered the study. APA or AHA were identified in all cases. APA were detected in 13 patients (68.4%) and AHA in 13 patients (68.4%). Ten patients (52.6%) were simultaneously positive for both APA and AHA. The prevalence of APAs and AHAs was higher as compared to those observed in 50 health controls (respectively 14% p < 0.001 and 24% p = 0.004) and in 100 not-secreting pituitary adenoma (NFPAs) (respectively 22% p = 0.002 and 8% p < 0.001). Similarly, the prevalence of simultaneous positivity for APA and AHA (52.9%) was higher as compared to the those detected in patients affected by NFPAs (0%; p < 0.001) and in health controls (16% p = 0.002). No differences were identified between PAHs and controls at qualitative and quantitative analysis of serum cytokines and white blood cells count., Conclusions: This study suggest that APA and AHA may be detected in an high percentage of PAH cases and that their simultaneous identification may be useful for the differential diagnosis between PAH and NFPAs, in an appropriate clinical context.

We explored the potential link between chronic inflammatory arthritis and COVID-19 pathogenic and resolving macrophage pathways and their role in COVID-19 pathogenesis. We found that bronchoalveolar lavage fluid (BALF) macrophage clusters FCN1+ and FCN1+SPP1+ predominant in severe COVID-19 were transcriptionally related to synovial tissue macrophage (STM) clusters CD48hiS100A12+ and CD48+SPP1+ that drive rheumatoid arthritis (RA) synovitis. BALF macrophage cluster FABP4+ predominant in healthy lung was transcriptionally related to STM cluster TREM2+ that governs resolution of synovitis in RA remission. Plasma concentrations of SPP1 and S100A12 (key products of macrophage clusters shared with active RA) were high in severe COVID-19 and predicted the need for Intensive Care Unit transfer, and they remained high in the post-COVID-19 stage. High plasma levels of SPP1 were unique to severe COVID-19 when compared with other causes of severe pneumonia, and IHC localized SPP1+ macrophages in the alveoli of COVID-19 lung. Investigation into SPP1 mechanisms of action revealed that it drives proinflammatory activation of CD14+ monocytes and development of PD-L1+ neutrophils, both hallmarks of severe COVID-19. In summary, COVID-19 pneumonitis appears driven by similar pathogenic myeloid cell pathways as those in RA, and their mediators such as SPP1 might be an upstream activator of the aberrant innate response in severe COVID-19 and predictive of disease trajectory including post-COVID-19 pathology.

We previously described the profibrogenic effect of NGF on conjunctival Fibroblasts (FBs) and its ability to trigger apoptosis in TGFβ1-induced myofibroblasts (myoFBs). Herein, cell apoptosis/signalling, cytokines' signature in conditioned media and inflammatory as well as angiogenic pathway were investigated. Experimental myoFBs were exposed to NGF (0.1-100 ng/mL), at defined time-point for confocal and biomolecular analysis. Cells were analysed for apoptotic and cell signalling activation in cell extracts and for some inflammatory and proinflammatory/angiogenic factors' activations. NGF triggered cJun overexpression and phospho-p65-NFkB nuclear translocation. A decreased Bcl2:Bax ratio and a significant expression of smad7 were confirmed in early AnnexinV-positive myoFBs. A specific protein signature characterised the conditioned media: a dose dependent decrease occurred for IL8, IL6 while a selective increase was observed for VEGF and cyr61 (protein/mRNA). TIMP1 levels were unaffected. Herein, NGF modulation of smad7, the specific IL8 and IL6 as well as VEGF and cyr61 modulation deserve more attention as opening to alternative approaches to counteract fibrosis.

Context.—: Clinical autopsies have historically provided a fundamental contribution in the definition of the clinicopathologic basis of infectious diseases. Even though we are witnessing the decline of the clinical autopsy, its importance remains unchanged as it is the most exhaustive way to investigate diseases. The identification of the virus in postmortem tissues is a fundamental step in the definition of its clinical features., Objective.—: To investigate the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in postmortem examination with swabs., Design.—: We performed postmortem swabs in 12 autopsy cases of patients with a clinical diagnosis of SARS-CoV-2-related pneumonia. Our protocol consisted of a rhinopharyngeal and a tracheal swab in order to search for the virus in the upper airways, and of 2 swabs on the parenchyma of each lung. We also performed a fifth swab on the parenchyma of both lungs in order to search for other viruses that could evolve in a clinical picture of interstitial pneumonia., Results.—: Overall, we found 9 of 12 cases had at least 1 postmortem swab positive for SARS-CoV-2. Moreover, we evaluated the time between the antemortem and postmortem swabs, the time between death and the postmortem swabs, and the time between the postmortem swabs and acceptance to the microbiology laboratory. Of note, we did not find a relationship between the results of the swabs and either the time elapsed from their collection or the time elapsed before their acceptance in the microbiology laboratory., Conclusions.—: A thorough knowledge of the eventual persistence of pathogens in deaths related to infectious diseases is fundamental for the safety of the operators during the autopsy practice, especially when referring to emergent pathogens, such as SARS-CoV-2. Our study highlights the importance in performing multiple swabs in the postmortem examination, because SARS-CoV-2 swab positivity can be limited to only a single swab., (© 2020 College of American Pathologists.)

Cardiac myxoma is a rare benign neoplasm of the heart. Historically myxomas were incidental findings during autopsies, however improved imaging techniques made these diagnosis possible in living patients, making the surgical treatment of these neoplasms achievable. Cardiac myxomas may occur both sporadically and in a familial context, often in the clinico-pathological picture of the Carney complex. While familial myxomas occur in the context of well-known genetic mutations, the molecular etiology of sporadically occurring myxomas is still not completely clear. We must note however that many of the patients affected by myxomas are asymptomatic; when symptoms are present they are often nonspecific and hard to decipher, especially when referring to sporadically occurring heart myxomas. In this paper we describe a case of sudden death from the massive embolization of a left atrial cardiac myxoma. We also reviewed all the cases in the literature of sudden death from heart myxoma embolism. An accurate epidemiology of heart myxomas would be the key to outline the best treatment practices and the etiology of sporadic myxomas, nevertheless this target could only be pursued with a deep revaluation of the clinical autopsy as a fundamental diagnostic tool., (Copyright © 2020. Published by Elsevier Inc.)

Efficient DNA extraction procedures is a critical step involved in the process of successful DNA analysis of such samples. Various protocols have been devised for the genomic DNA extraction from human tissues and forensic stains, such as dental tissue that is the skeletal part that better preserves DNA over time. However DNA recovery is low and protocols require labor-intensive and time-consuming step prior to isolating genetic material. Herein, we describe an extremely fast procedure of DNA extraction from teeth compared to classical method. Sixteen teeth of 100-year-old human remains were divided into two groups of 8 teeth and we compared DNA yield, in term of quantity and quality, starting from two different sample preparation steps. Specifically, teeth of group 1 were treated with a classic technique based on several steps of pulverization and decalcification, while teeth of group 2 were processed following a new procedure to withdraw dental pulp. In the next phase, the samples of both group underwent the same procedure of extraction, quantification and DNA profile analysis. Our findings provide an alternative protocol to obtain a higher amount of good quality DNA in a fast time procedure, helpful for forensic and anthropological studies., (© 2019 The Authors. Electrophoresis published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bacteria belonging to the Pseudomonas genus are highly successful colonizers of the plant rhizosphere. The ability of different Pseudomonas species to live either commensal lifestyles or to act as agents of plant-growth promotion or disease is reflected in a large, highly flexible accessory genome. Nevertheless, adaptation to the plant environment involves a commonality of phenotypic outputs such as changes to motility, coupled with synthesis of nutrient uptake systems, stress-response molecules and adherence factors including exopolysaccharides. Cyclic-di-GMP (cdG) is a highly important second messenger involved in the integration of environmental signals with appropriate adaptive responses and is known to play a central role in mediating effective rhizosphere colonization. In this study, we examined the transcription of multiple, reportedly plant-upregulated cdG metabolism genes during colonization of the wheat rhizosphere by the plant-growth-promoting strain P. fluorescens SBW25. While transcription of the tested genes generally increased in the rhizosphere environment, we additionally observed a tightly orchestrated response to environmental cues, with a distinct transcriptional pattern seen for each gene throughout the colonization process. Extensive phenotypical analysis of deletion and overexpression strains was then conducted and used to propose cellular functions for individual cdG signaling genes. Finally, in-depth genetic analysis of an important rhizosphere colonization regulator revealed a link between cdG control of growth, motility and stress response, and the carbon sources available in the rhizosphere.

Background: Magnesium (Mg) is essential for human health and is absorbed mainly in the intestine. In view of the likely occurrence of an Mg deficit in inflammatory bowel disease (IBD) and the documented role of Mg in modulating inflammation, the present study addresses whether Mg availability can affect the onset and progression of intestinal inflammation., Methods: To study the correlation between Mg status and disease activity, we measured magnesemia by atomic absorption spectroscopy in a cohort of IBD patients. The effects of dietary Mg modulation were assessed in a murine model of dextran sodium sulfate (DSS)-induced colitis by monitoring magnesemia, weight, fecal occult blood, diarrhea, colon length, and histology. Expression of the transient receptor potential melastatin (TRPM) 6 channel was assessed by real-time reverse transcription polymerase chain reaction and immunohistochemistry in murine colon tissues. The effect of Mg on epithelial barrier formation/repair was evaluated in human colon cell lines., Results: Inflammatory bowel disease patients presented with a substantial Mg deficit, and serum Mg levels were inversely correlated with disease activity. In mice, an Mg-deficient diet caused hypomagnesemia and aggravated DSS-induced colitis. Colitis severely compromised intestinal Mg2+ absorption due to mucosal damage and reduction in TRPM6 expression, but Mg supplementation resulted in better restoration of mucosal integrity and channel expression., Conclusions: Our results highlight the importance of evaluating and correcting magnesemia in IBD patients. The murine model suggests that Mg supplementation may represent a safe and cost-effective strategy to reduce inflammation and restore normal mucosal function.

Peripheral arterial disease (PAD), is a major health problem. Many studies have been focused on the possibilities of treatment offered by vascular regeneration. Human adipose-derived stem cells (HASCs), multipotent CD34+ stem cells found in the stromal-vascular fraction of adipose tissues, which are capable to differentiate into multiple mesenchymal cell types. The High mobility group box 1 protein (HMGB1) is a nuclear protein involved in angiogenesis. The aim of the study was to define the role of HMGB1 in cell therapy with HASCs, in an animal model of PAD. We induced unilateral ischemia in mice and we treated them with HASCs, with the specific HMGB1-inihibitor BoxA, with HMGB1 protein, and with the specific VEGF inhibitor sFlt1, alternately or concurrently. We measured the blood flow recovery in all mice. Immunohistochemical and ELISA analyses was performed to evaluate the number of vessels and the VEGF tissue content. None auto-amputation occurred and there have been no rejection reactions to the administration of HASCs. Animals co-treated with HASCs and HMGB1 protein had an improved blood flow recovery, compared to HASCs-treated mice. The post-ischemic angiogenesis was reduced when the HMGB1 pathway was blocked or when the VEGF activity was inhibited, in mice co-treated with HASCs and HMGB1. In conclusion, the HASCs treatment can be used in a mouse model of PAD to induce post-ischemic angiogenesis, modulating angiogenesis by HMGB1. This effect is mediated by VEGF activity. Although further data are needed, these findings shed light on possible new cell treatments for patients with PAD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This study aims at investigating the epigenetic landscape of cardiomyocytes exposed to elevated glucose levels. High glucose (30 mM) for 72 hours determined some epigenetic changes in mouse HL-1 and rat differentiated H9C2 cardiomyocytes including upregulation of class I and III histone deacetylase protein levels and activity, inhibition of histone acetylase p300 activity, increase in histone H3 lysine 27 trimethylation, and reduction in H3 lysine 9 acetylation. Gene expression analysis focused on cardiotoxicity revealed that high glucose induced markers associated with tissue damage, fibrosis, and cardiac remodeling such as Nexilin (NEXN), versican, cyclic adenosine 5'-monophosphate-responsive element modulator (CREM), and adrenoceptor α2A (ADRA2). Notably, the transcription factor CREM was found to be important in the regulation of cardiotoxicity-associated genes as assessed by specific small interfering RNA and chromatin immunoprecipitation experiments. In CD1 mice, made hyperglycemic by streptozotoicin (STZ) injection, cardiac structural alterations were evident at 6 months after STZ treatment and were associated with a significant increase of H3 lysine 27 trimethylation and reduction of H3 lysine 9 acetylation. Consistently, NEXN, CREM, and ADRA2 expression was significantly induced at the RNA and protein levels. Confocal microscopy analysis of NEXN localization showed this protein irregularly distributed along the sarcomeres in the heart of hyperglycemic mice. This evidence suggested a structural alteration of cardiac Z-disk with potential consequences on contractility. In conclusion, high glucose may alter the epigenetic landscape of cardiac cells. Sildenafil, restoring guanosine 3', 5'-cyclic monophosphate levels, counteracted the increase of CREM and NEXN, providing a protective effect in the presence of hyperglycemia., (Copyright © 2017 Endocrine Society.)

Targeting a host factor essential for the replication of different viruses but not for the cells offers a higher genetic barrier to the development of resistance, may simplify therapy regimens for coinfections, and facilitates management of emerging viral diseases. DEAD-box polypeptide 3 (DDX3) is a human host factor required for the replication of several DNA and RNA viruses, including some of the most challenging human pathogens currently circulating, such as HIV-1, Hepatitis C virus, Dengue virus, and West Nile virus. Herein, we showed for the first time, to our knowledge, that the inhibition of DDX3 by a small molecule could be successfully exploited for the development of a broad spectrum antiviral agent. In addition to the multiple antiviral activities, hit compound 16d retained full activity against drug-resistant HIV-1 strains in the absence of cellular toxicity. Pharmacokinetics and toxicity studies in rats confirmed a good safety profile and bioavailability of 16d. Thus, DDX3 is here validated as a valuable therapeutic target.

Background: Infliximab is effective in inflammatory bowel disease through several mechanisms, possibly acting at the mucosal level., Aim: To assess the role of infliximab on intestinal mucosa and whether it contributes to mucosal healing., Methods: Human colonic mucosal biopsies were incubated with or without infliximab. Cultured biopsies were evaluated for histological staining, CD68, CD3, E-cadherin and phospho-extracellular signal-regulated kinases (ERK) expression, and apoptosis. A scratch assay and MTT assay were performed with Caco2 cells in the presence of infliximab and/or tumour necrosis factor (TNF)-α or treated with supernatants obtained from human peripheral blood mononuclear cells or human intestinal fibroblasts treated with TNF-α and infliximab alone or in association., Results: Infliximab-treated biopsies displayed a better histological appearance, reduced inflammation with an increase of E-cadherin, phospho-ERK and apoptosis. Supernatants showed lower TNF-α, IL-17, IL-6 and IL-8 concentration, with an increase in fibroblast-growth-factor. Motility at scratch assay and proliferation at MTT assay of Caco2 cells displayed differential modulation by TNF-α and infliximab, directly or through supernatants of human intestinal fibroblasts and human peripheral blood mononuclear cells exposed to them., Conclusion: Infliximab contributes to the mucosal healing process by acting directly at an intestinal mucosal level; infliximab indirectly affects epithelial cell migration and proliferation by acting on both fibroblasts and leukocytes., (Copyright © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Aims: Male sex is a risk factor for medulloblastoma (MB), and is also a negative predictor for clinical outcome. The aim of this study was to assess sex differences in tumour biological features and hormone receptor profiles in a cohort of MB patients., Methods and Results: Sixty-four MBs and five normal cerebella were included in the study. Cell proliferation (Ki67), apoptosis (cleaved caspase-3) and microvessel density (CD31) were evaluated in tumours by immunohistochemistry. Tissues were analysed for oestrogen receptor (ER)α, ERβ1, ERβ2, ERβ5 and androgen receptor (AR) expression. The results demonstrated sex-specific features in MBs, with tumours from females showing a higher apoptosis/proliferation ratio and less tumour vascularization than tumours from males. MBs were negative for ERα and AR, but expressed ERβ isoforms at similar levels between the sexes. Altogether, these findings indicate that signalling mechanisms that control cell turnover and angiogenesis operate more efficiently in females than in males. The lack of sex differences in the hormone receptor profiles suggests that circulating oestrogens could be the major determinants of the sexual dimorphism observed in MB features., Conclusions: Here, we provide molecular support for epidemiological data showing sex differences in MB incidence and outcome, completely defining the hormone receptor profile of the tumours., (© 2015 John Wiley & Sons Ltd.)

Background: The object of this study was to investigate the potential role of FGF23 on plaque stability in type 2 diabetic patients with internal carotid artery stenosis., Methods: In this retrospective observational study, we analyzed FGF23 serum level in 361 type 2 diabetic patients with internal carotid artery stenosis undergoing carotid endarterectomy and in 598 diabetic controls without carotid atherosclerosis., Results: We found that FGF23 median serum levels was significantly higher in patients than in diabetic controls [67.7 (59.5-77.8) pg/mL and 43.89 (37.5-50.4), P < 0.001] and was significantly and independently associated with unstable plaque in patients with internal carotid artery stenosis [OR, 5,71 (95% CI, 2.09-15.29]., Conclusions: We have found, for the first time, that FGF23 could be associated with unstable plaque in type 2 diabetic patients with internal carotid artery stenosis.

Background Aims: Subcutaneous fat represents a valuable reservoir of adipose-derived stem cells (ASCs) in the stromal vascular fraction (SVF), widely exploited in regenerative medicine applications, being easily harvested through lipoaspiration. The lack of standardized procedures for autologous fat grafting guided research efforts aimed at identifying possible differences related to the harvesting site, which may affect cell isolation yield, cell growth properties and clinical outcomes. Subcutaneous fat features a complex architecture: the superficial fascia separates superficial adipose tissue (SAT) from deep layer tissue (DAT). We aimed to unravel the differences between SAT and DAT, considering morphological structure, SVF composition, and ASC properties., Methods: SAT and DAT were collected from female donors and comparatively analyzed to evaluate cellular yield and viability, morphology, immunophenotype and molecular profile. ASCs were isolated in primary culture and used for in vitro differentiation assays. SAT and DAT from cadaver donors were also analyzed through histology and immunohistochemistry to assess morphology and cell localization within the hypoderm., Results: Liposuctioned SAT contained a higher stromal tissue compound, along with a higher proportion of CD105-positive cells, compared with DAT from the same harvesting site. Also, cells isolated from SAT displayed increased multipotency and stemness features. All differences were mainly evidenced in specimens harvested from the abdominal region. According to our results, SAT features overall increased stem properties., Conclusions: Given that subcutaneous adipose tissue is currently exploited as the gold standard source for high-yield isolation of adult stem cells, these results may provide precious hints toward the definition of standardized protocols for microharvesting., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Plant chitinases have been studied for their importance in the defense of crop plants from pathogen attacks and for their peculiar vacuolar sorting determinants. A peculiarity of the sequence of many family 19 chitinases is the presence of a C-terminal extension that seems to be important for their correct recognition by the vacuole sorting machinery. The 7 amino acids long C-terminal vacuolar sorting determinant (CtVSD) of tobacco chitinase A is necessary and sufficient for the transport to the vacuole. This VSD shares no homology with other CtVSDs such as the phaseolin's tetrapeptide AFVY (AlaPheValTyr) and it is also sorted by different mechanisms. While a receptor for this signal has not yet been convincingly identified, the research using the chitinase CtVSD has been very informative, leading to the observation of phenomena otherwise difficult to observe such as the presence of separate vacuoles in differentiating cells and the existence of a Golgi-independent route to the vacuole. Thanks to these new insights in the endoplasmic reticulum (ER)-to-vacuole transport, GFPChi (Green Fluorescent Protein carrying the chitinase A CtVSD) and other markers based on chitinase signals will continue to help the investigation of vacuolar biogenesis in plants.

Malignant gliomas are primary brain tumors characterized by morphological and genetic complexities, as well as diffuse infiltration into normal brain parenchyma. Within gliomas, microglia/macrophages represent the largest tumor-infiltrating cell population, contributing by at least one-third to the total tumor mass. Bi-directional interactions between glioma cells and microglia may therefore play an important role on tumor growth and biology. In the present study, we have characterized the influence of glioma-soluble factors on microglial function, comparing the effects of media harvested under basal conditions with those of media obtained after inducing a pro-inflammatory activation state in glioma cells. We found that microglial cells undergo a different pattern of activation depending on the stimulus; in the presence of activated glioma-derived factors, i.e. a condition mimicking the late stage of pathology, microglia presents as a mixture of polarization phenotypes (M1 and M2a/b), with up-regulation of iNOS (inducible nitric oxide synthase), ARG (arginase) and IL (interleukine)-10. At variance, microglia exposed to basal glioma-derived factors, i.e. a condition resembling the early stage of pathology, shows a more specific pattern of activation, with increased M2b polarization status and up-regulation of IL-10 only. As far as viability and cell proliferation are concerned, both LI-CM [LPS (lipopolysaccharide)-IFNγ (interferon γ) conditioned media] and C-CM (control-conditioned media) induce similar effects on microglial morphology. Finally, in human glioma tissue obtained from surgical resection of patients with IV grade glioblastoma, we detected a significant amount of CD68 positive cells, which is a marker of macrophage/microglial phagocytic activity, suggesting that in vitro findings presented here might have a relevance in the human pathology as well.

Gliotoxin (GT) belongs to the epipolythiodioxopiperazine class of toxins secreted from certain fungi including Aspergillus fumigatus, which is the most prolific producer of this secondary metabolite. Recently, enhanced amounts of GT were found in in vitro biofilm-grown A. fumigatus mycelium. To further correlate the A. fumigatus biofilm growth phenotype with the enhanced secretion of GT, a polyclonal antibody (pAb) was produced by immunizing mice against GT. By an indirect immunofluorescent assay, pAb was then able to recognize specifically GT onto A. fumigatus Af293 biofilm formed on human pulmonary epithelial cells. Then, treating Af293 biofilms with a compound which reduces the GT disulfide bonds provoked shutdown of the GT-specific immunofluorescence (IF) signals along the hyphae. To explore the potential of GT for diagnostic use, pAb was shown to react with GT on hyphae into Aspergillus culture-positive respiratory tract specimens from patients with probable invasive aspergillosis (IA) and into tissue specimens from the lungs of patients with proven IA. As the presence of fungal hyphae in clinical specimens strongly indicates the in vivo A. fumigatus growth as a biofilm, anti-GT antibodies could be a specific and sensitive diagnostic tool for detecting A. fumigatus biofilm-associated clinical infections., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Background: Gelatin tannate, a gelatin powder containing tannic acids, is commonly employed as an intestinal astringent. Neither information nor animal model exist to confirm its efficacy or unravel mechanisms of action., Objective: To evaluate the action of gelatin tannate in murine dextran sodium sulphate (DSS)-induced acute colitis., Methods: Mice were exposed to DSS and received gelatin tannate by gavage. At sacrifice, colon histological degree of inflammation was assessed. Stool samples were cultured for microbiological analysis. Colon samples were analysed by two-photon confocal microscopy and atomic force microscopy. Elisa was performed on murine serum to assess lipopolysaccharide and peptidoglycan levels., Results: Gelatin tannate treatment reduced disease activity, bodyweight loss, and preserved colonic length. It produced a decrease in the amount of enterobacteria and enterococci. At confocal microscopy, intestinal samples from healthy and treated mice displayed similar structure in mucus layer thickness and composition; samples from placebo group had no mucus layer or a thinner stratus. Atomic force microscopy confirmed these findings. Treated mice showed lower blood LPS levels vs. control., Conclusions: Gelatin tannate decreased the severity of colitis. Acting as a gut barrier enhancer, it re-establishes gut homeostasis by recovering intestinal permeability and mucus layer integrity in gut mucosa and by modulating microbiota composition.

Background: Endoscopy represents both an alternative and useful complement to the standard microsurgical approach to the anterior craniovertebral junction (CVJ). Nevertheless, few studies provide an experimental comparison between transnasal and transoral endoscopic control on CVJ. We compared the surgical exposition angle and the working channel volume of both the transnasal and transoral approaches in the cadaver., Methods: Eleven fresh non-perfused cadavers were studied. Transnasal and transoral linear and angled exposure of the CVJ were evaluated by means of X-ray and CT scan both in sagittal and lateral planes., Results: The transoral endoscopic surgical exposition was wider compared with the transnasal in anterior and lateral projections:(1)in the sagittal plane, both in vertical exposition (transnasal inferior to transoral from 5.89 % to 76.48 %, average 35.89 %) and in vertical surgical angle (from 22 % to 77.42 %, average 56.53 %); (2)in the coronal plane, both in coronal exposition (transnasal inferior to transoral from 50.77 % to 83.88 %, average 70.34 %) and in coronal surgical angle (from 65.58 % to 86.71 %, average 76.70 %). The sagittal surgical domain was found to spanning from the inferior third of the clivus to C3 with the transoral and from the middle third of the clivus to the nasopalatal line (NPL) with the transnasal approach. The overlapping surgical domain area was found to be the inferior third of the clivus., Conclusions: The endoscope assisted transoral approach allows a better surgical control of the CVJ. It provides a better CVJ exposure, in sagittal and transverse planes, providing a larger working channel and an easier manoeuvrability. The transnasal approach is limited in caudal direction down to the NPL, otherwise the transoral approach is limited in the rostral direction with a maximum to the foramen magnum in normal specimen. In every individual case, pros and cons of the appropriate approach have to be taken into account as well as the choice of a combined transnasal and transoral approaches strategy.

Cilostazol is effective for the treatment of peripheral ischemia. This compound has several beneficial effects on platelet aggregation, serum lipids and endothelial cells, and we recently found that it enhances collateral blood flow in the ischemic hind limbs of mice. Peroxisome proliferator-activated receptor (PPAR)γ, a receptor for thiazolidinediones, plays a role in angiogenesis. The aim of this work was to investigate the underlying molecular mechanisms and effects of cilostazol in a model of peripheral ischemia in diabetic mice. We induced diabetes in mice by streptozotocin (STZ) administration and studied ischemia-induced angiogenesis in the ischemic hind limbs of cilostazol-treated and untreated control mice. We found that perfusion recovery was significantly improved in treated compared with control diabetic mice. Interestingly, we found that the expression of PPARγ is reduced in ischemic tissues of diabetic mice. Furthermore, we discovered that local inhibition of the activity of this nuclear receptor decreased the angiogenic response to cilostazol treatment. Finally, we noted that this phenomenon is dependent on VEGF and modulated by PPARγ. Cilostazol administration enhances collateral blood flow in the ischemic hind limbs of STZ-induced diabetic mice through a PPARγ-dependent mechanism., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: Infliximab is effective in human and murine IBD, but its pharmacodynamic is still poorly known. The aim of this study was to assess the affinity of infliximab to murine TNF-α, its role in murine colitis when administered intra-rectally and its levels in the blood, gut mucosa and stool of healthy and sick mice., Methods: An ELISA kit was built in order to assess the affinity of infliximab to human or murine-TNF-α. Human IgG were used as controls. DSS model of colitis on C57BL/6 mice was used to assess clinical efficacy of infliximab administered intravenously or by enema. Stool, serum and colon samples were collected to assess infliximab levels and histology for Rachmilewitz score., Results: Infliximab showed a good affinity both for human-TNF-α and murine-TNF-α. In DSS colitic mice infliximab ameliorated the severity of colitis, regardless of the administration route. In comparison with colitic mice, healthy mice displayed higher serum and mucosal infliximab levels, while detectable levels of infliximab were found in faeces, particularly in colitic mice., Conclusion: Our data support murine models to study infliximab pharmacokinetics and dynamics. Measurable levels of infliximab can be found at different concentrations in blood, intestinal mucosa and stool from healthy and sick mice, thus infliximab pharmacokinetics could have a major impact in human IBD., (Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Objective: Dystrophin, the missing or defective protein in Duchenne muscular dystrophy, is expressed not only in muscle cells but also in vascular endothelial cells (ECs). In this study, we assessed the effects of dystrophin deficiency on the angiogenic capacities of ECs., Approach and Results: We isolated vascular ECs from mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, and wild-type controls, and we found that mdx-derived ECs have impaired angiogenic properties, in terms of migration, proliferation, and tube formation. They also undergo increased apoptosis in vitro compared with wild-type cells and have increased senescence-associated β-galactosidase activity. Mdx-derived ECs also display reduced ability to support myoblast proliferation when cocultured with satellite cell-derived primary myoblasts. These endothelial defects are mirrored by systemic impairment of angiogenesis in vivo, both on induction of ischemia, stimulation with growth factors in the corneal model and matrigel plug assays, and tumor growth. We also found that dystrophin forms a complex with endothelial NO synthase and caveolin-1 in ECs, and that NO production and cGMP formation are compromised in ECs isolated from mdx mice. Interestingly, treatment with aspirin enhances production of both cGMP and NO in dystrophic ECs, whereas low-dose aspirin improves the dystrophic phenotype of mdx mice in vivo, in terms of resistance to physical exercise, muscle fiber permeability, and capillary density., Conclusions: These findings demonstrate that impaired angiogenesis is a novel player and potential therapeutic target in Duchenne muscular dystrophy.

Vacuolar Sorting Determinants (VSDs) have been extensively studied in plants but the mechanisms for the accumulation of storage proteins in somatic tissues are not yet fully understood. In this work we used two mutated versions of well-documented vacuolar fluorescent reporters, a GFP fusion in frame with the C-terminal VSD of tobacco chitinase (GFPChi) and an N-terminal fusion in frame with the sequence-specific VSD of the barley cysteine protease aleurain (AleuGFP). The GFP sequence was mutated to present an N-glycosylation site at the amino-acid position 133. The reporters were transiently expressed in Nicotiana tabacum protoplasts and agroinfiltrated in Nicotiana benthamiana leaves and their distribution was identical to that of the non-glycosylated versions. With the glycosylated GFPs we could highlight a differential ENDO-H sensitivity and therefore differential glycan modifications. This finding suggests two different and independent routes to the vacuole for the two reporters. BFA also had a differential effect on the two markers and further, inhibition of COPII trafficking by a specific dominant-negative mutant (NtSar1h74l) confirmed that GFPChi transport from the ER to the vacuole is not fully dependent on the Golgi apparatus., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Background: Laser ablation (LA) with a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser is a minimally invasive approach able to achieve a high rate of complete tissue necrosis. In a previous study we described the feasibility of EUS-guided Nd:YAG pancreas LA performed in vivo in a porcine model., Objective: To establish the best laser setting of Nd:YAG lasers for pancreatic tissue ablation. A secondary aim was to investigate the prediction capability of a mathematical model on ablation volume., Design: Ex vivo animal study., Setting: Hospital animal laboratory., Subjects: Explanted pancreatic glands from 60 healthy farm pigs., Intervention: Laser output powers (OP) of 1.5, 3, 6, 10, 15, and 20 W were supplied. Ten trials for each OP were performed under US guidance on ex vivo healthy porcine pancreatic tissue., Main Outcome Measurements: Ablation volume (Va) and central carbonization volume (Vc) were measured on histologic specimens as the sum of the lesion areas multiplied by the thickness of each slide. The theoretical model of the laser-tissue interaction was based on the Pennes equation., Results: A circumscribed ablation zone was observed in all histologic specimens. Va values grow with the increase of the OP up to 10 W and reach a plateau between 10 and 20 W. The trend of Vc values rises constantly until 20 W. The theoretical model shows a good agreement with experimental Va and Vc for OP between 1.5 and 10 W., Limitations: Ex vivo study., Conclusion: Volumes recorded suggest that the best laser OP could be the lowest one to obtain similar Va with smaller Vc in order to avoid the risk of thermal injury to the surrounding tissue. The good agreement between the two models demonstrates the prediction capability of the theoretical model on laser-induced ablation volume in an ex vivo animal model and supports its potential use for estimating the ablation size at different laser OPs., (Copyright © 2013 American Society for Gastrointestinal Endoscopy. Published by Mosby, Inc. All rights reserved.)

Background/objectives: Cilostazol has been found to be effective for the treatment of intermittent claudication (IC). This compound has several beneficial effects on platelet aggregation, serum lipids and endothelial cells, but how these might relate to improvements in walking is not entirely understood. The aim of this work was to investigate the effects of cilostazol on angiogenic response in a murine model of peripheral ischemia and to clarify the underlying molecular mechanisms of that response., Methods: We studied ischemia-induced neovascularization in the ischemic hindlimb of cilostazol-treated and untreated control mice., Results: We found that the perfusion recovery was significantly improved in treated compared with control mice. Interestingly, there was a higher level of circulating endothelial progenitor cells (EPCs) in mice treated with cilostazol than in untreated mice. Furthermore, cilostazol administration resulted in upregulation of granulocyte colony-stimulating factor (G-CSF) and vascular endothelial growth factor (VEGF) in the ischemic muscle of treated mice. Finally, inhibiting VEGF activity significantly reduced cilostazol-induced angiogenesis., Conclusions: The results of this study show that cilostazol administration enhances collateral blood flow in the ischemic hindlimbs of mice through a VEGF-dependent mechanism. These data may help to explain the beneficial effects that this drug has on patients with peripheral arterial disease (PAD) and IC., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Plant sensitive factor attachment protein receptors (SNAREs) encoded by genes of the same sub-family are generally considered as redundant in promoting vesicle-associated membrane fusion events. Nonetheless, the application of innovative experimental approaches highlighted that members of the same gene sub-family often have different functional specificities. In this work, two closely related Qc-SNAREs--the AtSYP51 and the AtSYP52--are compared in their ability to influence different secretory pathways. Their role in the vesicle sorting to the central vacuole has been revised and they were found to have a novel inhibitory function. When transiently overexpressed, the SYP51 and the SYP52 distributed between the TGN and the tonoplast. Our data demonstrate that these SYPs (syntaxin of plants) act as t-SNARE when present on the membrane of TGN/PVC, whereas they behave as inhibitory or interfering SNAREs (i-SNAREs) when they accumulate on the tonoplast. Moreover, the performed functional analysis indicated that the AtSYP51 and the AtSYP52 roles differ in the traffic to the vacuole. The findings are a novel contribution to the functional characterization of plant SNAREs that reveals additional non-fusogenic roles.

Aim: Thyroid cancer represents the first endocrine malignant neoplasm, accounting for 1% of human malignancy. The majority of which are well-differentiated cancer representing up to 90% of thyroid cancer and pursuing a favorable clinical course. The groups of poorly differentiated thyroid cancer (PDC) and anaplastic thyroid cancer (ATC) have a poor outcome and need a strict clinical surveillance., Materials and Methods: Thirty-four cases including 23 PDC/insular cancer and 9 ATC were examined for the expression of an immunohistochemical panel made up by HBME-1, galectin-3, and β-catenin and correlated either with histologic prognostic parameters or the overall surveillance., Results: HBME-1 and galectin-3 were expressed in 100% of the PDC/insular cases and in none of the ATC cases. The data for β-catenin pointed out an 80% expression (12/15) in the PDCs and only a focal and nonspecific positivity in the ATCs. A β-catenin-positive expression was found in all patients with a worse outcome/death and in the presence of vascular invasion and metastatic disease. All 3 PDC patients with β-catenin negativity are alive, whereas only 41% (5/12) are alive in the β-catenin-positive group., Conclusions: Our data set up the idea that PDC represents an intermediate step in the biological process of dedifferentiation of thyroid tumors toward ATC. This shift is underlined by the β-catenin expression, which seems to be related to a worse prognostic behavior. HBME-1 and galectin-3 show a similar pattern in PDC compared with well-differentiated carcinoma, whereas they are not expressed, as well as β-catenin, in anaplastic carcinomas.

Fine-needle aspiration biopsy is regarded as an important tool for diagnosing thyroid lesions because of its simplicity, safety, and cost-effectiveness. Its role in correctly characterizing the group of indeterminate lesions or follicular-patterned neoplasms (FN) might be more decisive. Liquid-based cytology (LBC) is a technique based on the use of a semi-automated device that has gained popularity as a method of collecting and processing both gynecologic and non-gynecologic cytologic specimens. It achieves a diagnostic sensitivity as accurate as conventional preparations especially for its excellent cell preservation and for the lack of background which decrease the amount of inadequate diagnoses. Moreover, the cellular material which has been stored in the preservative solution could be effectively used for the application of immunocytochemical and molecular techniques especially for the Follicular proliferations. In many cases the cytologic features are similar in both methods but the colloid film and the lymphocytic component are more easily evaluated on direct smears whereas nuclear details and colloid globules are better evaluated in LBC slides. The LBC-processed biopsies represent a valid alternative to conventional cytology. The possibility of applying special techniques enhance the efficacy of the cytological diagnosis of thyroid lesions.

We have previously demonstrated that sonic hedgehog (Shh) gene transfer improves angiogenesis in the setting of ischemia by upregulating the expression of multiple growth factors and enhancing the incorporation of endogenous bone marrow (BM)-derived endothelial progenitor cells (EPCs). In this study, we hypothesized that combined therapy with Shh gene transfer and BM-derived EPCs is more effective than Shh gene therapy alone in an experimental model of peripheral limb ischemia. We used old mice, which have a significantly reduced angiogenic response to ischemia, and compared the ability of Shh gene transfer, exogenous EPCs, or both to improve regeneration after ischemia. We found a significantly higher capillary density in the Shh + EPC-treated muscles compared to the other experimental groups. We also found that Shh gene transfer increases the incorporation and survival of transplanted EPCs. Finally, we found a significantly higher number of regenerating myofibers in the ischemic muscles of mice receiving combined treatment with Shh and BM-derived EPCs. In summary, the combination of Shh gene transfer and BM-derived EPCs more effectively promotes angiogenesis and muscle regeneration than each treatment individually and merits further investigation for its potential beneficial effects in ischemic diseases., (Copyright © 2012 S. Karger AG, Basel.)

The aim of the present study was to investigate the role of GV (glycaemic variability) in diabetic vascular complications and to explore the molecular pathways modulated by glycaemic 'swings'. We developed a murine model. A total of 30 diabetic mice received once daily basal insulin administration plus two oral boluses of glucose solution (GV group, named 'V') and 30 diabetic mice received once daily basal insulin plus two oral boluses of saline solution (stable hyperglycaemia group, named 'S') for a period of 30 days. Glycaemia was measured eight times daily to detect GV. Finally, postischaemic vascularization, induced by hindlimb ischaemia 30 days after diabetes onset, was evaluated. We found that GV was significantly different between S and V groups, whereas no significant difference in the mean glycaemic values was detected. Laser Doppler perfusion imaging and histological analyses revealed that the ischaemia-induced angiogenesis was significantly impaired in V mice compared with S group, after ischaemic injury. In addition, immunostaining and Western blot analyses revealed that impaired angiogenic response in V mice occurred in association with reduced VEGF (vascular endothelial growth factor) production and decreased eNOS (endothelial nitric oxide synthase) and Akt (also called protein kinase B) phosphorylation. In conclusion, we describe a murine model of GV. GV causes an impairment of ischaemia-induced angiogenesis in diabetes, likely to be independent of changes in average blood glucose levels, and this impaired collateral vessel formation is associated with an alteration of the VEGF pathway.

Background/aim: The cancer stem cell model suggests that only a rare subpopulation, known as cancer stem cells (CSC) are responsible for tumor initiation. CSC from several human carcinomas are characterized by specific cell surface markers, such as CD133. The CD133 role in colon tumorigenesis remains controversial., Materials and Methods: CD133 was evaluated by immunohistochemistry in a mouse model of colitis-related colon tumorigenesis induced by a combined treatment with azoxymethane (AOM) and dextran sodium sulphate (DSS)., Results: In normal tissue rare scattered positive cells were detectable at the bottom of the crypts. The percentage of positive cells significantly increased in dysplastic lesions and appeared to progressively decrease in the passage from dysplasia to adenoma and then to cancer, although always remaining greater in number than in the normal tissue., Conclusion: An increased CD133 expression occurs at early stages of colon tumorigenesis in the mouse. CD133-expressing cells might play an important role from the earlier phase and throughout the entire process of colon cancer development.

Background and Purpose: Osteoprotegerin (OPG) is a secretory glycoprotein which belongs to the tumor necrosis factor receptor family. Various mechanisms have been suggested by which calcification might alter atherosclerotic plaque stability, but the significance of this intimal calcification is controversial. High concentrations of OPG have been associated with the presence of vascular and cardiovascular diseases. This study was designed to assess the association between gene polymorphisms of the OPG gene (TNFRSF11B), the serum OPG level, and plaque stability in patients with carotid atherosclerosis., Methods: We studied 177 patients with internal carotid artery stenosis who underwent carotid endarterectomy and also 303 controls. Carotid endarterectomy samples removed from patients were assessed by immunohistochemistry. Concentrations of OPG were measured and gene polymorphisms were examined by polymerase chain reaction and restriction enzyme analysis and were compared, initially between patients with carotid atherosclerosis and controls, and subsequently between stable and unstable carotid plaques., Results: We found that the GG genotype of the T245G polymorphism, the CC genotype of the T950C polymorphism, and the CC genotype of the G1181C polymorphism were significantly higher in patients with carotid plaque than in controls (21.5% versus 10.9% , P<0.01; 15.8% versus 7.6%, P<0.01; and 20.3% versus 10.9%, P<0.01, respectively) and that these polymorphisms were associated with high serum OPG levels (4.02 [3.07] versus 2.94 [1.81] pmol/L; P<0.01), which were significantly higher in patients with unstable atherosclerotic plaques (5.86 [4.02] versus 3.53 [1.87] pmol/L; P<0.01)., Conclusions: The TNFRSF11B gene polymorphisms studied are associated with high serum OPG levels and might be potential markers for plaque instability.

Congenital coronary anomalies represent a condition often unrecognized in the living and in the dead. Investigating this condition is relevant for both clinicians and pathologists in order to identify potentially unrecognized coronary causes of sudden death. Hypoplastic coronary artery disease (HCAD) is a rare congenital abnormality reported to be associated with sudden death. We report two additional cases of previously apparently healthy people who died suddenly and were found to have HCAD at postmortem evaluation. The clinicopathologic findings are discussed along with a review of the literature.

Objective: High-mobility group box-1 (HMGB1) protein is a nuclear DNA-binding protein released from necrotic cells, inducing inflammatory responses and promoting tissue repair and angiogenesis. Diabetic human and mouse tissues contain lower levels of HMGB1 than their normoglycemic counterparts. Deficient angiogenesis after ischemia contributes to worse outcomes of peripheral arterial disease in patients with diabetes. To test the hypothesis that HMGB1 enhances ischemia-induced angiogenesis in diabetes, we administered HMGB1 protein in a mouse hind limb ischemia model using diabetic mice., Research Design and Methods: After the induction of diabetes by streptozotocin, we studied ischemia-induced neovascularization in the ischemic hind limb of normoglycemic, diabetic, and HMGB1-treated diabetic mice., Results: We found that the perfusion recovery was significantly attenuated in diabetic mice compared with normoglycemic control mice. Interestingly, HMGB1 protein expression was lower in the ischemic tissue of diabetic mice than in normoglycemic mice. Furthermore, we observed that HMGB1 administration restored the blood flow recovery and capillary density in the ischemic muscle of diabetic mice, that this process was associated with the increased expression of vascular endothelial growth factor (VEGF), and that HMGB1-induced angiogenesis was significantly reduced by inhibiting VEGF activity., Conclusions: The results of this study show that endogenous HMGB1 is crucial for ischemia-induced angiogenesis in diabetic mice and that HMGB1 protein administration enhances collateral blood flow in the ischemic hind limbs of diabetic mice through a VEGF-dependent mechanism.