Molecular Mechanisms Underlying Loss of Vascular and Epithelial Integrity in Irritable Bowel Syndrome.

BACKGROUND & AIMS: The pathophysiology of irritable bowel syndrome (IBS) is multifactorial and includes epithelial barrier dysfunction, a key element at the interface between the gut lumen and the deeper intestinal layers. Beneath the epithelial barrier there is the vascular one representing the last barrier to avoid luminal antigen dissemination The aims of this study were to correlate morpho-functional aspects of epithelial and vascular barriers with symptom perception in IBS. METHODS: Seventy-eight healthy subjects (controls) and 223 patients with IBS were enrolled in the study and phenotyped according to validated questionnaires. Sugar test was used to evaluate in vivo permeability. Immunohistochemistry, western blot, and electron microscopy were used to characterize the vascular barrier. Vascular permeability was evaluated by assessing the mucosal expression of plasmalemma vesicle-associated protein-1 and vascular endothelial cadherin. Caco-2 or human umbilical vein endothelial cell monolayers were incubated with soluble mediators released by mucosal biopsies to highlight the mechanisms involved in permeability alteration. Correlation analyses have been performed among experimental and clinical data. RESULTS: The intestinal epithelial barrier was compromised in patients with IBS throughout the gastrointestinal tract. IBS-soluble mediators increased Caco-2 permeability via a downregulation of tight junction gene expression. Blood vessel density and vascular permeability were increased in the IBS colonic mucosa. IBS mucosal mediators increased permeability in human umbilical vein endothelial cell monolayers through the activation of protease-activated receptor-2 and histone deacetylase 11, resulting in vascular endothelial cadherin downregulation. Permeability changes correlated with intestinal and behavioral symptoms and health-related quality of life of patients with IBS. CONCLUSIONS: Epithelial and vascular barriers are compromised in patients with IBS and contribute to clinical manifestations.

Fabry Disease Nephropathy: Histological Changes With Nonclassical Mutations and Genetic Variants of Unknown Significance.

RATIONALE & OBJECTIVE: Fabry disease (FD) is an X-linked genetic disorder that causes lysosomal storage of glycosphingolipids, primarily globotriaosylceramide (Gb3) and its derivative globotriaosylsphingosine (lyso-Gb3), with multiorgan dysfunction including chronic kidney disease. Affected individuals may be carriers of gene variants that are of uncertain significance (GVUS). We describe kidney pathology at the early stages of FD-related kidney disease to gain insights into its association with GVUS and sex. STUDY DESIGN: Single-center, case series. SETTING & PARTICIPANTS: Thirty-five consecutively biopsied patients (aged 48.1±15.4 years, 22 females) from among 64 patients with genetically diagnosed FD. Biopsies were retrospectively screened using the International Study Group of Fabry Nephropathy Scoring System. OBSERVATIONS: Genetic mutation type, p.N215S and D313Y, sex, age, estimated glomerular filtration rate (eGFR), plasma lyso-Gb3 (pLyso-Gb3) levels, and histological parameters, including Gb3 deposits were recorded. Genetic analyses showed mostly missense mutations, p.N215S variant in 15, and the "benign polymorphism" D313Y in 4 of the biopsied patients. Morphological lesions were similar for men and women except for interstitial fibrosis and arteriolar hyalinosis being more common in men. Early in their clinical course, patients with normal/mild albuminuria had podocyte, tubular, and peritubular capillary vacuoles/inclusions, and evidence of chronicity, i.e., glomerulosclerosis, interstitial fibrosis, tubular atrophy. These findings appeared to be associated with pLyso-Gb3, eGFR, and age. LIMITATIONS: Retrospective design and inclusion of outpatients partially based on family pedigree. CONCLUSIONS: In early stages of kidney disease in the setting of FD, numerous histological abnormalities are present. These observations suggest that kidney biopsies early in FD may reveal activity of kidney involvement that may inform clinical management.

The Characteristics and Survival Potential Under Sub-lethal Stress of Mesenchymal Stromal/Stem Cells Isolated from the Human Vascular Wall.

Mesenchymal stromal/stem cells (MSCs) have been identified in multiple human tissues, including the vascular wall. High proliferative potential, multilineage, and immunomodulatory properties make vascular MSCs promising candidates for regenerative medicine. Indeed, their location is strategic for controlling vascular and extra-vascular tissue homeostasis. However, the clinical application of MSCs, and in particular vascular MSCs, is still challenging. Current studies are focused on developing strategies to improve MSC therapeutic applications, like priming MSCs with stress conditions (hypoxia, nutrient deprivation) to achieve a higher therapeutic potential. The goal of the present study is to review the main findings regarding the MSCs isolated from the human vascular wall. Further, the main priming strategies tested on MSCs from different sources are reported, together with the experience on vascular MSCs isolated from healthy cryopreserved and pathological arteries. Stress induction can be a priming approach able to improve MSC effectiveness through several mechanisms that are discussed in this review. Nevertheless, these issues have not been completely explored in vascular MSCs and potential side effects need to be investigated.

The PPAR-γ Agonist Pioglitazone Modulates Proliferation and Migration in HUVEC, HAOSMC and Human Arteriovenous Fistula-Derived Cells.

The failure of arteriovenous fistulas (AVFs) following intimal hyperplasia (IH) increases morbidity and mortality rates in patients undergoing hemodialysis for chronic kidney disease. The peroxisome-proliferator associated receptor (PPAR-γ) may be a therapeutic target in IH regulation. In the present study, we investigated PPAR-γ expression and tested the effect of pioglitazone, a PPAR-γ agonist, in different cell types involved in IH. As cell models, we used Human Endothelial Umbilical Vein Cells (HUVEC), Human Aortic Smooth Muscle Cells (HAOSMC), and AVF cells (AVFCs) isolated from (i) normal veins collected at the first AVF establishment (T0), and (ii) failed AVF with IH (T1). PPAR-γ was downregulated in AVF T1 tissues and cells, in comparison to T0 group. HUVEC, HAOSMC, and AVFC (T0 and T1) proliferation and migration were analyzed after pioglitazone administration, alone or in combination with the PPAR-γ inhibitor, GW9662. Pioglitazone negatively regulated HUVEC and HAOSMC proliferation and migration. The effect was antagonized by GW9662. These data were confirmed in AVFCs T1, where pioglitazone induced PPAR-γ expression and downregulated the invasive genes SLUG, MMP-9, and VIMENTIN. In summary, PPAR-γ modulation may represent a promising strategy to reduce the AVF failure risk by modulating cell proliferation and migration.

Molecular Signature of Biological Aggressiveness in Clear Cell Sarcoma of the Kidney (CCSK).

Clear cell sarcoma of the kidney (CCSK) is a rare pediatric renal tumor with a worse prognosis than Wilms' tumor. Although recently, BCOR internal tandem duplication (ITD) has been found as a driver mutation in more than 80% of cases, a deep molecular characterization of this tumor is still lacking, as well as its correlation with the clinical course. The aim of this study was to investigate the differential molecular signature between metastatic and localized BCOR-ITD-positive CCSK at diagnosis. Whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) were performed on six localized and three metastatic BCOR-ITD-positive CCSKs, confirming that this tumor carries a low mutational burden. No significant recurrences of somatic or germline mutations other than BCOR-ITD were identified among the evaluated samples. Supervised analysis of gene expression data showed enrichment of hundreds of genes, with a significant overrepresentation of the MAPK signaling pathway in metastatic cases (p < 0.0001). Within the molecular signature of metastatic CCSK, five genes were highly and significantly over-expressed: FGF3, VEGFA, SPP1, ADM, and JUND. The role of FGF3 in the acquisition of a more aggressive phenotype was investigated in a cell model system obtained by introducing the ITD into the last exon of BCOR by Crispr/Cas9 gene editing of the HEK-293 cell line. Treatment with FGF3 of BCOR-ITD HEK-293 cell line induced a significant increase in cell migration versus both untreated and scramble cell clone. The identification of over-expressed genes in metastatic CCSKs, with a particular focus on FGF3, could offer new prognostic and therapeutic targets in more aggressive cases.

Effect of different fatty acids on Neisseria gonorrhoeae viability.

Neisseria gonorrhoeae (GC) is the agent of one of the most common bacterial sexually transmitted infections worldwide. The possible development of 'untreatable' infections points out the need for antibiotic-sparing methods to reduce the number of gonococcal infections. In this context, fatty acids are interesting candidates as next-generation antibacterial agents. The aim of this study was to investigate the bactericidal effects of selected fatty acids on GC viability, as well as to observe their biological effects by means of transmission electron microscopy. The cytotoxicity of these compounds on human cervical cells (HeLa), chosen as a model of genital mucosa, was assessed as well. Lauric, myristic, and palmitic acid displayed high killing activity against GC in concentrations ranging between 100 µM and 25 µM, whereas the antimicrobial effect of oleic and butyric acids was present in concentrations between 1 mM and 0.25 mM. Modifications induced by fatty acids on the GC cell included the disorganization of the cytoplasmic structure, the distortion of pili/fimbriae, and the separation of the inner and outer membrane layers. For concentrations active against GC, fatty acids were not toxic for cervical cells. Our data can help in promoting innovative antibiotic-free compounds for the treatment of GC infections.

Predictors of survival in malignant aortic tumors.

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.

Different Drugs Effect on Mesenchymal Stem Cells Isolated From Abdominal Aortic Aneurysm.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a progressive dilation of the aortic wall, determined by the unbalanced activity of matrix metalloproteinase (MMPs). In vitro and in vivo studies support the pivotal role of MMP-9 to AAA pathogenesis. In our experience, we elucidated the expression of MMP-9 in an ex vivo model of human mesenchymal stem cells isolated from AAA specimen (AAA-MSCs). Thus, MMP-9 inhibition could be an attractive therapeutic strategy for inhibiting AAA degeneration and rupture. Our study was aimed at testing the effect of 3 different drugs (pioglitazone, doxycycline, simvastatin) on MMP-9 and peroxisome proliferator-activated receptor (PPAR)-γ expression in AAA-MSCs. METHODS: Aneurysmal aortic wall segments were taken from AAA patients after the open surgical treatment. MSCs were isolated from AAA (n = 20) tissues through enzymatic digestion. AAA-MSCs were exposed to different doses of pioglitazone (5-10-25 µM), doxycycline (10-25 µM), and simvastatin (10 µM) for 24 h. The effect of each drug was evaluated in terms of cell survival, by crystal violet stain. MMP-9 and PPAR-γ mRNA were analyzed using real-time PCR. RESULTS: AAA-MSCs were not affected by the exposure to the selected drugs, as shown by the analysis of cell viability. Interestingly, MMP-9 mRNA resulted significantly decreased after each treatment, recording a downregulation of 50% in presence of pioglitazone, 90% with doxycycline, and 40% with exposed to simvastatin, in comparison to untreated cells. We further analyzed the expression of PPAR-γ, target of pioglitazone, observing an upregulation in exposed AAA-MSCs to controls. CONCLUSIONS: Our data support the potential therapeutic effect of pioglitazone, doxycycline, and simvastatin on AAA by reducing the MMP-9 expression in a patient-specific model (AAA-MSCs). In addition, pioglitazone drives the increase of PPAR-G, another promising target for AAA therapy. Further studies are necessary to elucidate the mechanism driving this inhibitory pathway, which can reduces the mortality risk associated with AAA rupture.

Intrahepatocellular crystal storing mimicking a clinical liver disease during monoclonal gammopathy: report of a case and review of the literature.

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.

Pharmacological (or Synthetic) and Nutritional Agonists of PPAR-γ as Candidates for Cytokine Storm Modulation in COVID-19 Disease.

The cytokine storm is an abnormal production of inflammatory cytokines, due to the over-activation of the innate immune response. This mechanism has been recognized as a critical mediator of influenza-induced lung disease, and it could be pivotal for COVID-19 infections. Thus, an immunomodulatory approach targeting the over-production of cytokines could be proposed for viral aggressive pulmonary disease treatment. In this regard, the peroxisome proliferator-activated receptor (PPAR)-γ, a member of the PPAR transcription factor family, could represent a potential target. Beside the well-known regulatory role on lipid and glucose metabolism, PPAR-γ also represses the inflammatory process. Similarly, the PPAR-γ agonist thiazolidinediones (TZDs), like pioglitazone, are anti-inflammatory drugs with ameliorating effects on severe viral pneumonia. In addition to the pharmacological agonists, also nutritional ligands of PPAR-γ, like curcuma, lemongrass, and pomegranate, possess anti-inflammatory properties through PPAR-γ activation. Here, we review the main synthetic and nutritional PPAR-γ ligands, proposing a dual approach based on the strengthening of the immune system using pharmacological and dietary strategies as an attempt to prevent/treat cytokine storm in the case of coronavirus infection.

Histological Evidence of Diabetic Kidney Disease Precede Clinical Diagnosis.

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 <60 mL/min/1.73 m2. 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.

Differentiation and plasticity of human vascular wall mesenchymal stem cells, dermal fibroblasts and myofibroblasts: a critical comparison including ultrastructural evaluation of osteogenic potential.

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 cytometry; 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; differently from fibroblasts, the myofibroblasts showed a poor angiogenic commitment. The mineralization 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.

Extraneuraxial Hemangioblastoma: Clinicopathologic Features and Review of the Literature.

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).

Mechanisms of Arterial Calcification: The Role of Matrix Vesicles.

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.

Activity of synthetic peptides against Chlamydia.

The in vitro activity of six synthetic peptides against 36 strains of Chlamydia from different origins was investigated. Clavanin MO (CMO) proved to be the most active peptide, reducing the inclusion number of all Chlamydia strains from eight different species tested by ≥50% at 10 µg mL-1 . Mastoparan L showed an equal activity against C. trachomatis, C. pneumoniae, C. suis, and C. muridarum, but did not exert any inhibitory effect against C. psittaci, C. pecorum, C. abortus, and C. avium even at 80 µg mL-1 . These data suggest that CMO could be a promising compound in the prevention and treatment of chlamydial infections.

Relationship between Calcification and Vulnerability of the Carotid Plaques.

BACKGROUND: Carotid plaques with a high degree of calcification are usually considered at low embolic risk. However, since a precise evaluation of the extent of calcification is not possible preoperatively through duplex ultrasound and postoperatively by conventional histological examination due to the decalcification process, the relationship between the amount of calcium involvement and plaque vulnerability has not been evaluated yet. This study aims to correlate the extent of carotid plaque calcification with clinical, radiological, and histological complications. METHODS: Symptomatic and asymptomatic consecutive patients submitted to carotid endarterectomy between January to December 2014 were included in the study. The amount of carotid calcification was assessed at preoperative computed tomography (CT) through measurement of thickness and circumferential calcium extension and graded from 1 to 8 accordingly (Babiarz classification). Patients were then categorized into 2 groups (low-level group: grade 1-5; high-level group: grade 6-8) and correlated with clinical characteristics and ipsilateral cerebral ischemic lesions at CT. Vulnerability of the plaque was assessed histologically according with American Heart Association (AHA) Classification. Results were overall blindly correlated. RESULTS: One hundred five patients (81% male; age: 73 ± 8 years) were enrolled in the study. Forty (38%) were symptomatic and 43 (40%) had an ipsilateral focal lesion at preoperative cerebral CT. Thirty-six (38%) patients had high-level carotid calcification degree at CT scan. At histological analysis, 56 (56%) plaques were considered complicated (AHA type VI). Patients with high-level and low-level carotid calcification had similar epidemiological risk factors, preoperative neurological symptoms, and histological complications (17% vs. 15%, P = 0.76 and 50% vs. 55%, P = 0.62, respectively). The high-level calcification group showed a significantly higher incidence of ipsilateral cerebral lesions at preoperative CT (56% vs. 32%, P = 0.01). CONCLUSIONS: A high level of calcification of the carotid plaque is not necessarily associated with lower vulnerability: the incidence of preoperative neurological symptoms and histological complications is similar in patients with and without extensive carotid plaque calcification. Cerebral ischemic lesions may be even more frequent in the presence of highly calcified plaques.

Doxorubicin and cisplatin induce apoptosis in ovarian stromal cells obtained from cryopreserved human ovarian tissue.

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.

The study of calcified atherosclerotic arteries: an alternative to evaluate the composition of a problematic tissue reveals new insight including metakaryotic cells.

BACKGROUND: Calcifications of atherosclerotic plaques represent a controversial issue as they either lead to the stabilization or rupture of the lesion. However, the cellular key players involved in the progression of the calcified plaques have not yet been described. The primary reason for this lacuna is that decalcification procedures impair protein and nucleic acids contained in the calcified tissue. The aim of our study was to preserve the cellular content of heavily calcified plaques with a new rapid fixation in order to simplify the study of calcifications. METHODS: Here we applied a fixation method for fresh calcified tissue using the Carnoy's solution followed by an enzymatic tissue digestion with type II collagenase. Immunohistochemistry was performed to verify the preservation of nuclear and cytoplasmic antigens. DNA content and RNA preservation was evaluated respectively with Feulgen staining and RT-PCR. A checklist of steps for successful image analysis was provided. To present the basic features of the F-DNA analysis we used descriptive statistics, skewness and kurtosis. Differences in DNA content were analysed with Kruskal-Wallis and Dunn's post tests. The value of P < 0.05 was considered significant. RESULTS: Twenty-four vascular adult tissues, sorted as calcified (14) or uncalcified (10), were processed and 17 fetal tissues were used as controls (9 soft and 8 hard). Cells composing the calcified carotid plaques were positive to Desmin, Vimentin, Osteocalcin or Ki-67; the cellular population included smooth muscle cells, osteoblasts and osteoclasts-like cells and metakaryotic cells. The DNA content of each cell type found in the calcified carotid artery was successfully quantified in 7 selected samples. Notably the protocol revealed that DNA content in osteoblasts in fetal control tissues exhibits about half (3.0 ng) of the normal nuclear DNA content (6.0 ng). CONCLUSION: Together with standard histology, this technique could give additional information on the cellular content of calcified plaques and help clarify the calcification process during atherosclerosis.

Unusual lamellar calcifications in two rare cases of splenic aneurysms associated with fibromuscular dysplasia.

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.

The incidence and morphology of Monckeberg's medial calcification in banked vascular segments from a monocentric donor population.

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.