Inhibiting DNA methylation as a strategy to enhance adipose-derived stem cells differentiation: Focus on the role of Akt/mTOR and Wnt/ß-catenin pathways on adipogenesis.

Adipose-derived mesenchymal stem cells (ASCs) represent a valid therapeutic option for clinical application in several diseases, due to their ability to repair damaged tissues and to mitigate the inflammatory/immune response. A better understanding of the underlying mechanisms regulating ASC biology might represent the chance to modulate their in vitro characteristics and differentiation potential for regenerative medicine purposes. Herein, we investigated the effects of the demethylating agent 5-azacytidine (5-aza) on proliferation, clonogenicity, migration, adipogenic differentiation and senescence of ASCs, to identify the molecular pathways involved. Through functional assays, we observed a detrimental effect of 5-aza on ASC self-renewal capacity and migration, accompanied by actin cytoskeleton reorganization, with decreased stress fibers. Conversely, 5-aza treatment enhanced ASC adipogenic differentiation, as assessed by lipid accumulation and expression of lineage-specific markers. We analyzed the involvement of the Akt/mTOR, MAPK and Wnt/ß-catenin pathways in these processes. Our results indicated impairment of Akt and ERK phosphorylation, potentially explaining the reduced cell proliferation and migration. We observed a 5-aza-mediated inhibition of the Wnt signaling pathway, this potentially explaining the pro-adipogenic effect of the drug. Finally, 5-aza treatment significantly induced ASC senescence, through upregulation of the p53/p21 axis. Our data may have important translational implications, by helping in clarifying the potential risks and advantages of using epigenetic treatment to improve ASC characteristics for cell-based clinical approaches.

Single-nucleotide polymorphisms in 3'-untranslated region inducible costimulator gene and the important roles of miRNA in alopecia areata.

Background: Alopecia areata (AA) spares the stem cell compartment and attacks only the base of the hair follicle, which is surrounded by infiltrating lymphocytes. AA is associated with polymorphisms in immune-related genes and with decreased function of CD4+CD25+ T regulatory (Treg) cells. Treg function is modulated by the costimulatory molecules, like inducible costimulator (ICOS) that are crucial in orienting T cell differentiation and function so that they strongly impact on the immunologic decision between tolerance or autoimmunity development. Objective: The aim of our study was to investigate the possible association of AA with single-nucleotide polymorphisms (SNP) present in the ICOS 3'-untranslated region (3'UTR) region and to elucidate how SNPs modulate ICOS gene expression by affecting miRNA binding sites. Methods: This is a case-control study performed in 184 patients with AA and 200 controls. ICOS gene and miRNA expression were analyzed by real-time polymerase chain reaction. Results: The genotype carrying the rs4404254(C) [p = 0.012, OR (95% CI): 0.5 (0.3-0.8)] and rs4675379(C) [p = 0.015, OR (95% CI): 0.3 (0.1-0.8)] 3' UTR alleles was more frequently observed in AA patients than in controls and correlated with a reduced ICOS expression. miR-1276 significantly suppressed ICOS expression by binding to the 3'UTR of ICOS mRNA. Also, we observed that, miR-101 and miR-27b are upregulated, while miR-103 and miR-2355-3p are downregulated in peripheral blood mononuclear cells of AA patients compared to controls. Conclusion: Our data show that rs4404254 and rs4675379 SNPs of ICOS gene are associated with AA and also reveal that the presence of rs4404254 polymorphism correlates with ICOS post-transcriptional repression by microRNA binding.

Treatment of the Mayer-Rokitansky-Küster-Hauser syndrome with autologous in vitro cultured vaginal tissue: descriptive study of long-term results and patient outcomes.

OBJECTIVE: Evaluating sexual function and quality of life (QoL) in patients treated with a modified Abbé-McIndoe technique using in vitro cultured autologous vaginal mucosa. DESIGN: Descriptive study. SETTING: Policlinico Umberto I, Sapienza University of Rome. POPULATION: From 2006 to 2016, 39 women affected by Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) underwent vaginoplasty at our centre using a modified Abbé-McIndoe technique with in vitro cultured autologous vaginal tissue. METHODS: For each patient, vaginal tissue was obtained by full-thickness biopsy of the vaginal vestibule. Following enzymatic dissociation, cells were cultured for 2-3 weeks before the transplant. MAIN OUTCOME MEASURES: Each patient completed two validated questionnaires to quantify sexual function and QoL: the Female Sexual Function Index (FSFI), administered at 12, 36, and 60 months, and the Psychological General Well Being Index (PGWBI) administered at 0, 6, and 36 months after surgery. RESULTS: Twelve months after surgery, 29 patients were engaging in regular sexual activity. The FSFI test results showed a satisfactory sexual function compared to the general population, with median values of 25.85 (range 4.6-30.5) at 12 months, 27.2 (range 4.4-33.6) at 36 months, and 29.6 (range 23.9-33.6) at 60 months. The PGWBI questionnaire showed a median score of 420.5 (range 108-540) before surgery, and 459 (range 252-533) at the 60-month follow-up. CONCLUSIONS: Vaginoplasty performed with the use of autologous vaginal tissue, besides ensuring a long-term satisfying sex life, helps in achieving an improvement in QoL that is maintained over time. TWEETABLE ABSTRACT: Vaginoplasty using in vitro vaginal tissue ensures a satisfactory sexual function and improves quality of life.

HCV derived from sera of HCV-infected patients induces pro-fibrotic effects in human primary fibroblasts by activating GLI2.

Hepatitis C virus (HCV) infection is a leading cause of liver fibrosis, especially in developing countries. The process is characterized by the excess accumulation of ECM that may lead, over time, to hepatic cirrhosis, liver failure and also to hepatocarcinoma. The direct role of HCV in promoting fibroblasts trans-differentiation into myofibroblasts, the major fibrogenic cells, has not been fully clarified. In this study, we found that HCV derived from HCV-infected patients infected and directly induced the trans-differentiation of human primary fibroblasts into myofibroblasts, promoting fibrogenesis. This effect correlated with the activation of GLI2, one of the targets of Hedgehog signaling pathway previously reported to be involved in myofibroblast generation. Moreover, GLI2 activation by HCV correlated with a reduction of autophagy in fibroblasts, that may further promoted fibrosis. GLI2 inhibition by Gant 61 counteracted the pro-fibrotic effects and autophagy inhibition mediated by HCV, suggesting that targeting HH/GLI2 pathway might represent a promising strategy to reduce the HCV-induced fibrosis.

Analytical and clinical comparison of different immunoassay systems for the detection of antiphospholipid antibodies.

INTRODUCTION: We evaluated analytical and clinical performances of IgG and IgM anticardiolipin (aCL) antibodies and anti-ß2-glycoprotein I (a-ß2GpI) antibodies and upper limit reference ranges (99th percentiles) in comparison with manufacturer's cutoff values with different commercial methods. METHODS: We assayed aCL and a-ß2GpI in serum samples from 30 healthy individuals, 77 patients with antiphospholipid syndrome (APS) diagnosed according to the Sydney criteria, 51 patients with autoimmune diseases, eight patients with previous thrombotic events, six patients with other diseases, and 18 patients with infectious diseases, using ELISA Inova Diagnostics; EliA Phadia Laboratory Systems; CliA Zenit-RA; and CliA Bio-Flash. RESULTS: Anticardiolipin and a-ß2GpI IgG and IgM immunoassays showed good analytic performances with both 99th percentile and manufacturer's cutoff reference values. Our results showed fair to moderate agreement among assays. In-house cutoff values gave significantly better performances only for a-ß2GpI IgG with all the immunoassays analyzed with the exception of Inova CliA Bio-Flash where we obtained the same performances with in-house and manufacturer's cutoffs. CONCLUSIONS: By guidelines, all laboratories are strongly advised to validate/verify the manufacturer's cutoff values. We recommend establishing low-positive, medium-/high-positive, and high-positive CliA IgG aCL and a-ß2GpI ranges in order to help clinicians in the diagnosis and treatment of APS.

Predictive factors for postoperative wound complications after neck dissection.

The objective of this retrospective study was to evaluate risk factors for wound complications after neck dissection. One hundred and nineteen patients were treated with neck dissection for squamous-cell carcinoma of the upper aerodigestive tract at the National Cancer Institute in Rome between 2006 and 2009. Postoperative wound complications were divided into major or minor and were related to different variables to identify risk factors. Postoperative wound complications were found in 20.2% of patients with an individual patient probability for different risk factors ranging from 2% to 34.1%. Preoperative chemoradiation therapy (CRT) and the type of neck dissection were associated with a higher risk of major complications (p ≤ 0.05). Previous CRT and radical neck dissection/modified radical neck dissection are risk factors for major wound complications in patients with head and neck squamous cell carcinoma undergoing neck dissection. Patients requiring neck dissection after CRT should be informed about the increased risk of the procedure, and selective neck dissection, if oncologically appropriate, should be considered to reduce complications.

Anti-cardiolipin and anti-ß2-glycoprotein I antibodies: normal reference ranges in northwestern Italy.

Laboratory tests for anticardiolipin antibodies (aCL) and anti-ß2glycoprotein I antibodies (a-ß2GPI) face problems common to many autoantibody assays: the lack of a reference standard and the need for each laboratory to assess assay-specific cut-off values. The aims of the study were to evaluate the reference range upper limits (99th percentile) used for aCL and a-ß2GPI in the northwest of Italy and to investigate the analytical performances of these assays with the newly obtained reference ranges. We assayed aCL and a-ß2GPI in 104 serum samples from patients without a history of thrombosis, pregnancy morbidity, tumours, infections and/or autoimmune diseases (30 males and 74 non-pregnant females). We tested all the commercial assays available in our regions (i.e. Orgentec Diagnostika, Aesku Diagnostics and Inova Diagnostics ELISA; CliA Zenit-RA and EliA Phadia Laboratory Systems). A further 30 serum samples, including 10 from healthy subjects, 10 from antiphospholipid syndrome (APS) patients and 10 from septic patients were assessed to investigate the analytical performance of the obtained cut-off limits. Reference range upper limits obtained with the commercial kits differ among assays and from the values reported by the manufacturer. Moreover, normal reference ranges calculated for IgG and IgM aCL differed from the arbitrary selected laboratory classification values suggested in the guidelines of 40 GPL and MPL.

Italian cross-cultural adaptation and validation of three different scales for the evaluation of shoulder pain and dysfunction after neck dissection: University of California - Los Angeles (UCLA) Shoulder Scale, Shoulder Pain and Disability Index (SPADI) and Simple Shoulder Test (SST).

Shoulder syndrome after neck dissection is a well known entity, but its incidence and prognostic factors influencing recovery have not been clearly assessed due to the heterogeneity of possible evaluations. The University of California - Los Angeles (UCLA) Shoulder Scale, the Shoulder Pain and Disability Index (SPADI) and the Simple Shoulder Test (SST) are three English-language questionnaires commonly used to test shoulder impairment. An Italian version of these scales is not available. The aim of the present study was to translate, culturally adapt and validate an Italian version of UCLA Shoulder Scale, SPADI and SST. Translation and cross-cultural adaptation of the SPADI, the UCLA shoulder scale and the SST was performed according to the international guidelines. Sixty-six patients treated with neck dissection for head and neck cancer were called to draw up these scales. Forty patients completed the same questionnaires a second time one week after the first to test the reproducibility of the Italian versions. All the English-speaking Italian patients (n = 11) were asked to complete both the English and the Italian versions of the three questionnaires to validate the scales. No major problems regarding the content or the language were found during the translation of the 3 questionnaires. For all three scales, Cronbach's α was > 0.89. The Pearson correlation coefficient was r > 0.91. With respect to validity, there was a significant correlation between the Italian and the English versions of all three scales. This study shows that the Italian versions of UCLA Shoulder Scale, SPADI and SST are valid instruments for the evaluation of shoulder dysfunction after neck dissection in Italian patients.

Clinical manifestations, co-sensitizations, and immunoblotting profiles of buckwheat-allergic patients.

BACKGROUND: Buckwheat allergy is a rare food allergy in Europe and North America, whereas it is often described and studied in Asia. The aim of this study was to describe a series of patients with proven buckwheat allergy evaluated in an Italian allergy clinic. Co-sensitization to other food and inhalant allergens and immunoblotting profiles of buckwheat-allergic patients were studied. METHODS: Patients with suspected buckwheat allergy who attended the allergy clinic between January 1, 2006, and September 30, 2008, were evaluated. All patients underwent skin prick tests for a standard panel of inhalant and food allergens, prick-by-prick with buckwheat flour, buckwheat-specific IgE determinations, and double-blind placebo-controlled food challenge (DBPCFC) with buckwheat flour. Immunoblotting with buckwheat flour extract was performed on sera from buckwheat-allergic patients. RESULTS: Among 72 patients with suspected buckwheat allergy, 30 (41.7%) were sensitized to buckwheat and 24 had a positive DBPCFC. The mean buckwheat IgE level was 6.23 kUA/l (range, 0.16 to >100 kUA/l). Several IgE-binding proteins were identified and grouped into three patterns: a 16-kDa band in patients with predominantly gastrointestinal symptoms with grass and wheat flour co-sensitization, a 25-kDa band in patients with predominantly cutaneous symptoms and a low frequency of co-sensitization, and a 40-kDa band in patients with anaphylaxis and a low frequency of co-sensitization. CONCLUSIONS: Buckwheat allergy is an emerging food allergy in Italy. We identified three distinct patterns of clinical and laboratory characteristics, suggesting that specific allergens could be more frequently associated with clinical manifestations of different severity.

Complex multipathways alterations and oxidative stress are associated with Hailey-Hailey disease.

BACKGROUND: Hailey-Hailey disease (HHD) is an autosomal dominant disorder characterized by suprabasal cutaneous cell separation (acantholysis) leading to the development of erosive and oozing skin lesions. While a strong relationship exists between mutations in the gene that encodes the Ca(2+)/Mn(2+)-adenosine triphosphatase ATP2C1 and HHD, we still have little understanding of how these mutations affect manifestations of the disease. OBJECTIVES: This study was designed to determine early signalling events that affect epithelial cell growth and differentiation during HHD development. METHODS: Expression of key regulatory signals important for maintaining skin homeostasis were evaluated by Western blot analysis and by reverse transcriptase-polymerase chain reaction in primary keratinocytes obtained from skin biopsies of patients with HHD. Reactive oxygen species accumulation in primary keratinocytes derived from lesional skin of patients with HHD was assessed by dihydrorhodamine 123 (DHR) assay. RESULTS: HHD-derived keratinocytes showed downregulation of both Notch1 and differential regulation of different p63 isoforms. Itch and p63 are co-expressed in the epidermis and in primary keratinocytes where Itch controls the p63 protein steady-state level. We found that the Itch protein was significantly decreased in HHD-derived keratinocytes whereas the expression of its target, c-Jun, remained unaffected. We also found that HHD-derived keratinocytes undergo oxidative stress, which may explain both Notch1 and Itch downregulation. CONCLUSIONS: Our attempt to explore the molecular mechanism underlying HHD indicates a complex puzzle in which multi-hit combinations of altered signal pathways may explain the wide spectrum of defects in HHD.

Association between multiple intestinal atresia and omphalocele: a case report.

Multiple intestinal atresia is a rare disorder with vascular or hereditary etiology. So far, the occurrence of this malformation along with omphalocele has not been reported. We describe a boy born from a nonconsanguineous gypsy couple with intrauterine growth retardation, omphalocele, and multiple intestinal atresia from the pylorus to the rectum. The microscopic examination of the intestine shows multiple small lumina with a sieve-like appearance. This is characteristic of the hereditary atresias and suggests development of a defect in (re)canalization during embryogenesis. The association with omphalocele indicates a common developmental defect may be present.

Fibroblast growth factor 10 induces proliferation and differentiation of human primary cultured keratinocytes.

Fibroblast growth factor 10 is a novel member of the fibroblast growth factor family, which is involved in morphogenesis and epithelial proliferation. It is highly homologous to the keratinocyte growth factor (or fibroblast growth factor 7), a key mediator of keratinocyte growth and differentiation. Both fibroblast growth factor 10 and keratinocyte growth factor bind with high affinity to the tyrosine kinase keratinocyte growth factor receptor. Here we analyzed the effect of fibroblast growth factor 10 on primary cultures of human keratinocytes, grown in chemically defined medium, and we compared the proliferative and differentiative cell responses to fibroblast growth factor 10 with those induced by keratinocyte growth factor and epidermal growth factor. Cell counting, 5-bromo-2'-deoxyuridine incorporation, and western blot analysis showed that fibroblast growth factor 10, similarly to keratinocyte growth factor, not only is a potent mitogen for human keratinocytes, but also promotes the expression of both early differentiation markers K1 and K10 and late differentiation marker filaggrin in response to the Ca2+ signal, and seems to sustain the proliferative activity in suprabasal stratified cells. Immunoprecipitation/western blot analysis revealed that fibroblast growth factor 10, similarly to keratinocyte growth factor, is able to induce tyrosine phosphorylation of keratinocyte growth factor receptor and of cellular substrates such as PLCgamma.

Up-modulation of the expression of functional keratinocyte growth factor receptors induced by high cell density in the human keratinocyte HaCaT cell line.

Keratinocyte growth factor (KGF) is involved in the control of proliferation and differentiation of human keratinocytes. It binds to, and activates, the tyrosine kinase KGF receptor (KGFR), a splicing transcript variant of the fibroblast growth factor receptor 2. We have previously shown (C. Marchese et al., Cell Growth Differ., 8: 989-997, 1997) that differentiation of primary cultured keratinocytes triggered by high Ca2+ concentrations in the growing medium induced up-regulation of KGFR expression, which suggested that KGFR may play a crucial role in the control of the proliferative/differentiative program during transition from basal to suprabasal cells. Here we analyzed the process of modulation of the expression of KGFRs in the human keratinocyte cell line HaCaT, widely used as a model to study keratinocyte differentiation. Western blot and double immunofluorescence for KGFR and the K1 differentiation marker showed that cell differentiation and stratification induced by confluence and high cell density correlated with an increase in KGFR expression. KGFRs, present on suprabasal differentiated cells, appeared to be efficiently tyrosine-phosphorylated by KGF, which indicated that the receptors up-regulated by differentiation can be functionally activated by ligand binding. Bromodeoxyuridine incorporation assay revealed that a significant portion of suprabasal differentiated cells expressing KGFR seemed to be still able to synthesize DNA and to proliferate in response to KGF, which suggested that increased KGFR expression may be required for retention of the proliferative activity.

Changes in autonomic and EEG patterns induced by hypnotic imagination of aversive stimuli in man.

Autonomic and electroencephalographic (EEG) responses to aversive stimuli presented by means of hypnotic suggestion have been studied in man.Healthy volunteers with simple phobia were screened for susceptibility to hypnosis. The experimental paradigm included periods of rest during which the hypnotized subjects were asked to produce an emotionally neutral mental image and periods of emotional activation in which they were asked to image a phobic object. Heart rate (HR), respiratory frequency (RF) and EEG were processed to obtain the HR-related indexes of sympatho-vagal balance and the EEG spectral components. The results showed a significant increase in HR and RF with a shift of the sympatho-vagal indexes towards a sympathetic predominance during the hypnotic emotional activation. EEG activity showed a significant increase in the gamma band with a left fronto-central prevalence. There was also a less pronounced increase in the beta band. In conclusion, by means of hypnosis, autonomic and behavioral responses to fear-like stimuli can be induced in man in a reproducible and controlled manner. Such a paradigm could be applied in human neuroimaging studies to identify central nervous structures that modulate stress and fear-related reactions.

Expression of functional mGlu5 metabotropic glutamate receptors in human melanocytes.

Cultured human melanocytes express mGlu5 metabotropic glutamate (mGlu) receptors, as shown by RT-PCR, immunocytochemistry, Western blot analysis, and measurement of agonist-stimulated polyphosphoinositide hydrolysis. The mGlu5 receptor agonists (S)-3, 5-dihydroxyphenylglycine and quisqualate increased [(3)H-methyl]thymidine incorporation and melanocyte proliferation in subconfluent cultures, but impaired cell viability in confluent cultures. Both effects were prevented by 2-methyl-6-(2-phenyl-1-ethynyl)-pyridine, a potent and highly selective mGlu5 receptor antagonist. Agonists of other mGlu receptor subtypes (such as the mGlu2/3 receptor agonist, 2S,2'R,3'R-2-2', 3'-dicarboxycyclopropylglycine, or the mGlu4/6/7/8 receptor agonist, L-2-amino-4-phosphonobutanoate) or selective agonists of ionotropic glutamate receptors (N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, and kainate) did not affect melanocyte proliferation or viability. The presence of a receptor for glutamate, the major excitatory neurotransmitter, in human melanocytes is intriguing. mGlu5 receptors may be involved in the control of melanocyte proliferation (and perhaps in other functions), but harbor a potential toxicity and may therefore contribute to cell damage under pathological conditions.

Expression of keratinocyte growth factor receptor compared with that of epidermal growth factor receptor and erbB-2 in endometrial adenocarcinoma.

Immunohistochemical analysis of the expression of keratinocyte growth factor receptor (KGFR) was performed in human endometrial carcinomas from 18 patients and in normal proliferative and secretory endometrium. The level of immunostaining was correlated with the clinico-pathological characteristics of the endometrial carcinoma patients and with the parallel expression of the epidermal growth factor receptor (EGFR) and erbB-2. The results showed that KGFR expression increased with the stage of the tumor and that the simultaneous overexpression of the three growth factor receptors appeared to be related to the depth of myometrial invasion. Taken together, these observations suggest that KGFR may represent an additional prognostic indicator in endometrial cancer.

Allelic imbalance at the LKB1 (STK11) locus in tumours from patients with Peutz-Jeghers' syndrome provides evidence for a hamartoma-(adenoma)-carcinoma sequence.

Patients with Peutz-Jeghers' syndrome (PJS) develop hamartomatous gastrointestinal polyps and characteristic pigmentation, as a result of germline mutations in the LKB1 gene. The hamartomas in PJS were long considered to be without malignant potential. There is, however, accumulating epidemiological evidence to suggest that PJS predisposes to cancers at several different sites (colon, pancreas, breast, ovary, testis, and cervix), although large enough patient samples are rarely available to prove this. Allelic imbalance [allele loss, loss of heterozygosity (LOH)] has previously been reported in a small number of PJS polyps, suggesting that LKB1 acts as a tumour suppressor in these tumours. This study confirms allelic loss at LKB1 in PJS polyps and shows that LOH also occurs in cancers of the colon, breast, and cervix in PJS patients. Allele loss was additionally found in a colonic adenoma from a PJS patient, strongly suggesting the existence of a hamartoma-(adenoma)-carcinoma sequence in tumourigenesis. These results provide molecular evidence that PJS patients are predisposed to cancers at several sites, as a direct result of selection for loss of the 'wild-type' LKB1 allele in tumours. Given the rare involvement of LKB1 in sporadic cancers, these data also suggest that the indirect effect on cancer risk (or 'bystander effect') proposed for hamartomas in juvenile polyposis does not apply to carcinomas in PJS.

Receptor-mediated endocytosis of keratinocyte growth factor.

Keratinocyte growth factor (KGF) is a fibroblast growth factor which acts specifically on epithelial cells, regulating their proliferation and differentiation. KGF elicits its activity through binding to and activation of KGF receptor, a splicing transcript variant of fibroblast growth factor receptor 2 (FGFR2). Here we analyzed the pathway of internalization of KGF and its receptor using several approaches, including the utilization in immunofluorescence and in immunoelectron microscopy of a functional KGF-HFc chimeric protein as a specific tool to follow the endocytosis of the growth factor and of its receptor. Western blot analysis with anti-FGFR2 and anti-phosphotyrosine antibodies, as well as parallel double immunofluorescence and confocal analysis of NIH3T3 KGFR transfectants treated with KGF at 4 degrees C, followed by incubations at 37 degrees C for different time points, showed that KGF induced endocytosis of tyrosine activated KGFRs. The use of KGF-HFc in immunofluorescence and in immunogold electron microscopy on KGFR transfectants, A253 epithelial tumor cells and human cultured keratinocytes allowed us to follow the early steps of KGF internalization and revealed that this process occurred through clathrin-coated pits. A quantitative ELISA assay confirmed that KGF-HFc binding on the cell surface rapidly decreased because of internalization. Our results demonstrate that KGF is internalized by receptor-mediated endocytosis and illustrate the involvement of clathrin-coated pits in this process.