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Purpose:Tebentafusp is a first-in-class bispecific fusion protein designed to target gp100 (a melanoma-associated antigen) through a high affinity T-cell receptor (TCR) binding domain and an anti-CD3 T-cell engaging domain, which redirects T cells to kill gp100-expressing tumor cells. Here, we report a multicenter phase I/II trial of tebentafusp in metastatic melanoma (NCT01211262) focusing on the mechanism of action of tebentafusp.Patients and Methods:Eighty-four patients with advanced melanoma received tebentafusp. Treatment efficacy, treatment-related adverse events, and biomarker assessments were performed for blood-derived and tumor biopsy samples obtained at baseline and on-treatment.Results:Tebentafusp was generally well-tolerated and active in both patients with metastatic uveal melanoma and patients with metastatic cutaneous melanoma. A 1-year overall survival rate of 65% was achieved for both patient cohorts. On-treatment cytokine measurements were consistent with the induction of IFNγ pathway–related markers in the periphery and tumor. Notably, tebentafusp induced an increase in serum CXCL10 (a T-cell attractant) and a reduction in circulating CXCR3+ CD8+ T cells together with an increase in cytotoxic T cells in the tumor microenvironment. Furthermore, increased serum CXCL10 or the appearance of rash (likely due to cytotoxic T cells targeting gp100-expressing skin melanocytes) showed a positive association with patient survival.Conclusions:These data suggest that redirecting T cells using a gp100-targeting TCR/anti-CD3 bispecific fusion protein may provide benefit to patients with metastatic melanoma. Furthermore, the activity observed in these two molecularly disparate melanoma classes hints at the broad therapeutic potential of tebentafusp.

Purpose: Tebentafusp is a first-in-class bispecific fusion protein designed to target gp100 (a melanoma-associated antigen) through a high affinity T-cell receptor (TCR) binding domain and an anti-CD3 T-cell engaging domain, which redirects T cells to kill gp100-expressing tumor cells. Here, we report a multicenter phase I/II trial of tebentafusp in metastatic melanoma (NCT01211262) focusing on the mechanism of action of tebentafusp. Patients and Methods: Eighty-four patients with advanced melanoma received tebentafusp. Treatment efficacy, treatment-related adverse events, and biomarker assessments were performed for blood-derived and tumor biopsy samples obtained at baseline and on-treatment. Results: Tebentafusp was generally well-tolerated and active in both patients with metastatic uveal melanoma and patients with metastatic cutaneous melanoma. A 1-year overall survival rate of 65% was achieved for both patient cohorts. On-treatment cytokine measurements were consistent with the induction of IFNγ pathway–related markers in the periphery and tumor. Notably, tebentafusp induced an increase in serum CXCL10 (a T-cell attractant) and a reduction in circulating CXCR3+ CD8+ T cells together with an increase in cytotoxic T cells in the tumor microenvironment. Furthermore, increased serum CXCL10 or the appearance of rash (likely due to cytotoxic T cells targeting gp100-expressing skin melanocytes) showed a positive association with patient survival. Conclusions: These data suggest that redirecting T cells using a gp100-targeting TCR/anti-CD3 bispecific fusion protein may provide benefit to patients with metastatic melanoma. Furthermore, the activity observed in these two molecularly disparate melanoma classes hints at the broad therapeutic potential of tebentafusp.

Nuova Rivista di Storia della Medicina, Vol 3 No 1 (2022)

BackgroundThe use of immunotherapeutic vaccination in prostate cancer is a promising approach that likely requires the induction of functional, cytotoxic T cells . The experimental approach described here uses a well-studied adenovirus-poxvirus heterologous prime-boost regimen, in which the vectors encode a combination of prostate cancer antigens, with the booster dose delivered by either the intravenous or intramuscular (IM) route. This prime-boost regimen was investigated for antigen-specific CD8+ T cell induction.MethodsThe coding sequences for four antigens expressed in prostate cancer, 5T4, PSA, PAP, and STEAP1, were inserted into replication-incompetent chimpanzee adenovirus Oxford 1 (ChAdOx1) and into replication-deficient modified vaccinia Ankara (MVA). In four strains of mice, ChAdOx1 prime was delivered intramuscularly, with an MVA boost delivered by either IM or intravenous routes. Immune responses were measured in splenocytes using ELISpot, multiparameter flow cytometry, and a targeted in vivo killing assay.ResultsThe prime-boost regimen was highly immunogenic, with intravenous administration of the boost resulting in a sixfold increase in the magnitude of antigen-specific T cells induced and increased in vivo killing relative to the intramuscular boosting route. Prostate-specific antigen (PSA)-specific responses were dominant in all mouse strains studied (C57BL/6, BALBc, CD-1 and HLA-A2 transgenic).ConclusionThis quadrivalent immunotherapeutic approach using four antigens expressed in prostate cancer induced high magnitude, functional CD8+ T cells in murine models. The data suggest that comparing the intravenous versus intramuscular boosting routes is worthy of investigation in humans.

La beta thalassemia rappresenta una delle più comuni patologie autosomiche recessive nel mondo. I Paesi mediterranei, del Medio Oriente e del Sud Est Asiatico sono regioni ad alta prevalenza di beta thalassemia. Le più alte incidenze sono riportate a Cipro, nel Sud Est Asiatico e in Sardegna, correlate molto probabilmente alla pressione selettiva esercitata dal Pl. falciparum, agente eziologico della malaria. In Sardegna, per la rilevanza sanitaria della patologia e in seguito alla pubblicazione dei primi studi scientifici sul morbo di Cooley, sono fiorite importanti scuole di pediatria e genetica medica, che contribuiranno alla definizione dei criteri diagnostici, della terapia e della prevenzione, anche neonatale, dell’anemia mediterranea e delle emoglobinopatie.

Nuova Rivista di Storia della Medicina, V. 2 N. 1 (2021)

According to the UNESCO Atlas, Sardinian is an endangered language, and the debate about its promotion in Sardinia is lively and passionate (Moseley 2010). In fact, over the past two generations, the language vitality and vigour of the limba (in Sardinian: language) have been drastically reduced. Nowadays, youngsters tend to abandon the limba when the literacy process towards Italian starts in school. The OCSE-PISA 2012 reports that students in Sardinia are among the lowest in literacy within the Italian state: Bolognesi and Heeringa (2005) argue that youngsters are losing Sardinian without mastering the Italian language. In order to reverse this language shift, a common written standard, the Limba Sarda Comuna (LSC) was made official by the local government in 2006. However, the LSC is still contested: “plastic language”, “Frankenstein monster idiom” are some of the epithets cast against it by the local press. In this contribution, we show a successful pilot experiment of a concrete application of the limba at school, that in our opinion could be easily applied on a wider scale in other parts of Sardinia. In fact, in the academic year 2014–15, three classes in a middle school of Orosei (Nuoro) took part in a laboratory where the limba was used both orally (local variety) and in written form (LSC, for the didactic material) following the so-called CLIL approach. Students learned Sardinian history in a Mediterranean and European perspective, using the LSC in reading and writing. No participant – L2 speakers included – rejected the LSC for being “artificial”, even though the local variety is approximately 85% similar to the LSC, according to Bolognesi (2007). This pilot experiment shows that a concrete application of the LSC in schools is possible and desirable. A discussion on how to expand this pilot experiment in different settings will be provided.

Background Tebentafusp (IMCgp100) is a bispecific T cell redirector comprised of an affinity-enhanced TCR recognising melanocyte lineage antigen gp100 and a T cell engaging anti-CD3 scFv domain. Tebentafusp has shown activity as monotherapy in advanced cutaneous and uveal melanoma (Middleton et al., ASCO 2019), and we have previously reported that over half of uveal melanoma patients treated with tebentafusp display melanocyte-related adverse events (MRAE). These include vitiligo/skin hypopigmentation, leukotrichia, and hyperpigmentation and, collectively, are associated with better overall survival in uveal patients receiving tebentafusp (Orloff et al, AACR 2020). In this study, we dissected the mechanisms by which tebentafusp may induce MRAE and highlight the potential clinical significance. Methods In vitro studies were conducted to assess the direct and indirect effects of tebentafusp on epidermal melanocytes from healthy donors. Expression of gp100 and the gp100:HLA*02:01 target complex by melanocytes were quantified at the mRNA level and on the cell surface by confocal microscopy, respectively. Melanocytes co-cultured with PBMC and increasing concentrations of tebentafusp were assessed for their susceptibility to lysis and/or ability to stimulate cytokine production. These readouts were compared to gp100-positive and negative melanoma cancer cell lines. Melanin production by melanocytes was quantified and the melanin synthesis pathway interrogated at the mRNA and protein level following exposure to secretomes from tebentafusp-redirected PBMC against melanoma cancer cells. Results Healthy melanocytes expressed 2 to 3-fold lower levels of gp100 peptide-HLA complexes on their surface compared to gp100-positive melanoma cell lines. In the presence of tebentafusp, this lower target expression translated into 3–6 fold lower levels of IFNγ and more than 100 fold lower granzyme B production by redirected T cells and these melanocytes were resistant to direct tebentafusp-induced killing (EC50 for melanocytes greater than 1nM vs EC50 melanoma cell lines of 23–50 pM). Supernatants from T cells activated in response to melanoma cancer cells by tebentafusp downregulated the melanin content of healthy melanocytes (20–30% reduction). Western blotting revealed 30–40% inhibition of two key components of the melanin synthesis pathway; the tyrosinase-related protein (TRP)-1 and TRP-2. This inhibition was reversed by blocking IFNγ in supernatants from activated T cells. Conclusions MRAEs, especially vitiligo, associated with response to tebentafusp, may be explained, at least in part, by the downregulation of melanin biosynthesis pathway genes by IFNγ secreted by tebentafusp-activated T cells. Ethics Approval The study was approved by the South Central - Oxford A Research Ethics Committee (UK), REC reference 13/SC/0226 References Middleton, et al., Relationship between clinical efficacy and AEs of IMCgp100, a novel bispecific TCR–anti-CD3, in patients with advanced melanoma. Journal of Clinical Oncology. 2019. Orloff, et al., Vitiligo and other clinical melanocyte-related adverse events following tebentafusp (IMCgp100) exposure in patients with uveal melanoma. AACR (American Association for Cancer Research), 2020.

Tra le principali malattie parassitarie che interessano il cane e colpiscono l’uomo ci sono quelle trasmesse da elminti, in particolare cestodi. La contaminazione ambientale e la mancata applicazione di semplici norme igieniche ne amplificano la trasmissione. L’echinococcosi (endemica in America Latina, Africa sub-Sahariana, Mongolia, Repubblica Popolare Cinese, Asia del sud e sud Est e Australia, e in tutte le regioni, dove, come in Sardegna, è fiorente la pastorizia) è considerata oggi come la zoonosi più importante della Regione Mediterranea. L’anchilostomiasi, patologia causata da un nematode, un tempo considerata patologia esclusiva di minatori, fornaciari, lavoratori della terra, è una parassitosi presente per lo più in aree del mondo dove la scarsità di adeguati presidi igienici e il clima caldo umido ne facilitano la diffusione; l’uomo malato trasmette con le feci le uova del parassita nell’ambiente, diventando contagioso per i propri simili. Mentre l’anchilostomiasi è ormai limitata ad aree del mondo depresse e a un’area circoscritta del territorio nazionale, l’echinococcosi pur in calo, è ancora presente in modo significativo soprattutto in Sardegna, nonostante le campagne di prevenzione e le innumerevoli risorse stanziate, economiche e umane. In questo lavoro si esamina l’utilizzo dei filmati prodotti e diffusi per informazione e prevenzione da ENPI, INAIL e Università degli Studi di Cagliari nella seconda metà del Novecento, alla luce di due inchieste territoriali condotte da Università di Cagliari e Istituto Zooprofilattico Sperimentale della Sardegna., Nuova Rivista di Storia della Medicina, V. 1 N. 1 (2020)

PURPOSE: Tebentafusp is a first-in-class bispecific fusion protein designed to target gp100 (a melanoma-associated antigen) through a high affinity T cell receptor (TCR) binding domain, and an anti-CD3 T cell engaging domain which redirects T cells to kill gp100-expressing tumor cells regardless of their intrinsic specificity. Here we report data from a multicentre Phase 1/2 trial of tebentafusp in metastatic melanoma (NCT01211262) focusing on the mechanism of action of tebentafusp. PATIENTS AND METHODS: 84 patients with advanced melanoma (n=61 cutaneous, n=19 uveal, n=4 other origin) received tebentafusp. Treatment efficacy and treatment-related AEs were assessed and coded, and biomarker assessments were performed for blood-derived and tumor biopsy samples obtained at baseline and on-treatment. RESULTS: Tebentafusp was generally well tolerated and active in both metastatic Uveal melanoma (mUM) and metastatic Uveal melanoma (mCM) patients. A 65% 1-year overall survival rate was achieved for both patient cohorts. On-treatment cytokine measurements were consistent with the induction of IFNγ pathway-related markers in the periphery and tumor. Notably, tebentafusp induced an increase in serum CXCL10 (a T cell attractant), and a reduction in circulating CXCR3+ CD8+ T cells together with an increase in cytotoxic T cells in the tumor microenvironment. Furthermore, increased serum CXCL10 or the appearance of rash, (likely due to cytotoxic T cells targeting of gp100-expressing skin melanocytes) showed a positive association with patient survival. CONCLUSIONS: These data suggest that re-directing T cells using a gp100-targeting T cell receptor/anti-CD3 bispecific fusion protein may provide benefit to patients with metastatic melanoma. Furthermore, the activity observed in these two molecularly disparate melanoma classes hints at the broad therapeutic potential of tebentafusp.

In our paper we report a brief history of the X-rays discovery and discuss the implications of their use and abuse in the Italian pedriatic schools of the early 20th century. Indeed, history of the X-ray treatment in the Italian Pediatric School has not yet been well studied. Even if the scientific experience of many physicians is well known in literature, a summary was missing. In Italy, in 1900, exposure to Röntgenand ultraviolet radiation or to large amounts of solar rays was a widespread medical practice, especially in several pediatric schools. During those years, diagnosis and treatment of childhood pathologies underwent considerable changes, especially after the twenties, when scientists developed an unquestionable trust in the therapeutic properties of radiation, considered harmless at that time. We report the main steps of the scientific research of the early 20th century in Italy. (www.actabiomedica.it)

Beta thalassaemia represents one of the most common autosomal recessive disorders worldwide. High prevalence is present in the Mediterranean, Middle East and Far East. The highest incidences are reported in Cyprus, South East Asia and Sardinia and are most likely related to the selective pressure from Pl. falciparum, the causative agent of malaria. InSardinia, because of the health relevance of beta thalassaemia and haemoglobinopathies and after the publication of the first scientific research on Cooley's anaemia, important Schools of Paediatrics and Clinical Genetics have been set up, which have contributed to defining diagnostic criteria, therapeutic and preventive measures (especially, newborn screening). The aim of the present study is to examine the results of the first scientific research made by the Sardinian Schools of Paediatrics and Clinical Genetics, from 1929 to 1957.La beta thalassemia rappresenta una delle piů comuni patologie autosomiche recessive nel mondo. I Paesi mediterranei, del Medio Oriente e del Sud Est Asiatico sono regioni ad alta prevalenza di beta thalassemia. Le piů alte incidenze sono riportate a Cipro, nel Sud Est Asiatico e in Sardegna, correlate molto probabilmente alla pressione selettiva esercitata dal Pl. falciparum, agente eziologico della malaria. In Sardegna, per la rilevanza sanitaria della patologia e in seguito alla pubblicazione dei primi studi scientifici sul morbo di Cooley, sono fiorite importanti scuole di pediatria e genetica medica, che contribuiranno alla definizione dei criteri diagnostici, della terapia e della prevenzione, anche neonatale, dell’anemia mediterranea e delle emoglobinopatie.

La beta thalassemia rappresenta una delle più comuni patologie autosomiche recessive nel mondo. I Paesi mediterranei, del Medio Oriente e del Sud Est Asiatico sono regioni ad alta prevalenza di beta thalassemia. Le più alte incidenze sono riportate a Cipro, nel Sud Est Asiatico e in Sardegna, correlate molto probabilmente alla pressione selettiva esercitata dal Pl. falciparum, agente eziologico della malaria. In Sardegna, per la rilevanza sanitaria della patologia e in seguito alla pubblicazione dei primi studi scientifici sul morbo di Cooley, sono fiorite importanti scuole di pediatria e genetica medica, che contribuiranno alla definizione dei criteri diagnostici, della terapia e della prevenzione, anche neonatale, dell’anemia mediterranea e delle emoglobinopatie., Beta thalassaemia represents one of the most common autosomal recessive disorders worldwide. High prevalence is present in the Mediterranean, Middle East and Far East. The highest incidences are reported in Cyprus, South East Asia and Sardinia and are most likely related to the selective pressure from Pl. falciparum, the causative agent of malaria. In Sardinia, because of the health relevance of beta thalassaemia and haemoglobinopathies and after the publication of the first scientific research on Cooley’s anaemia, important Schools of Paediatrics and Clinical Genetics have been set up, which have contributed to defining diagnostic criteria, therapeutic and preventive measures (especially, newborn screening). The aim of the present study is to examine the results of the first scientific research made by the Sardinian Schools of Paediatrics and Clinical Genetics, from 1929 to 1957.

Introduction: Bispecific T cell engagers hold promise for effective cancer treatment. The anti-tumor activity of these therapeutics molecules predominantly relies on their capacity to induce direct killing of tumor cells by engaging the cytotoxic machinery of polyclonal CD8+ T cells. However, the impact of additional functionally different T cell populations that can be engaged alongside CD8+ T cells remains unclear. ImmTAC molecules are a novel class of bispecific fusion proteins that use an affinity-enhanced monoclonal TCR to target tumor antigen-derived peptides presented by HLA, and an anti-CD3 monomeric antibody to engage T cells. Tebentafusp, our lead clinical candidate, targets a gp100-derived peptide and is under clinical investigation in metastatic melanoma. Preliminary data revealed T cell activation, trafficking and infiltration into the tumor bed in patients responding to treatment. Importantly not only CD8+ T cells, but also different subsets of CD4+ T cells appeared to be activated, mobilized and redirected to the tumor site following tebentafusp treatment, suggesting a potential contribution of these T cell subsets to the anti-tumor response. The aim of this study was to investigate in vitro the functional capacity of different T cell populations, besides CD8+ T cells, following ImmTAC-mediated redirection toward cancer cells. Methods: Th1, Th2, Th17 CD4+ T cells, γδ T cells and Mucosal-Associated Invariant T cells (MAIT) were isolated from healthy volunteers and their functional response following exposure to ImmTAC in the presence of target cells was analysed. Results: In addition to CD8+ T cells, other T cell populations, were activated in vitro by ImmTAC and elicited a range of effector functions that may differentially contribute to anti-tumor responses. For example, we found that γδ T cells were able to mount rapid and potent cytokine and cytotoxic responses against cancer cells upon redirection with ImmTAC. The mechanisms that underpin our in vitro observations and the potential correlations in patients treated with tebentafusp are under investigation. Conclusions: Multiple T cells populations display functionally distinct responses following ImmTAC-mediated redirection. These results will add valuable insight to our understanding of bispecific T cell engager mechanism of action. In addition, they will help guide rationale for improving the design of these therapeutic molecules and select appropriate combination strategies. Citation Format: Adel Benlahrech, David K. Cole, Christopher J. Holland, Rupert Kenefeck, Rahul C. Khanolkar, Cheryl McAlpine, Sion Lewis, Angela Valerio-Fernandes, Mariantonella Vardeu, David Berman, Marco Lepore. Functionally distinct T cell subsets contribute to ImmTAC-mediated anti-tumor response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4554.

Objective: Pediatric congenital heart surgery (CHS)involves intracardiac, valvular, and vascular repairs. Accurate tools to aid short-term outcome prediction in pediatric CHS are lacking. Clinical scores, such as the vasoactive-inotrope score and ventilation index, are used to define outcome in clinical studies. MicroRNA-1-3p (miR-1)is expressed by both cardiomyocytes and vascular cells and is regulated by hypoxia. In adult patients, miR-1 increases in the circulation after open-heart cardiac surgery, suggesting its potential as a clinical biomarker. Thus, we investigated whether perioperative circulating miR-1 measurements can help predict post-CHS short-term outcomes in pediatric patients. Methods: Plasma miR-1 was retrospectively measured in a cohort of 199 consecutive pediatric CHS patients (median age 1.2 years). Samples were taken before surgery and at the end of the operation. Plasma miR-1 concentration was measured by reverse transcription-quantitative polymerase chain reaction and expressed as miR-1 copies/μL and as relative expression to spiked-in exogenous cel-miR-39. Results: Baseline plasma miR-1 did not vary across different diagnoses, increased during surgery (204-fold median relative increase, P Conclusions: Our study suggests miR-1 as a novel potential circulating biomarker to predict early postoperative outcome and inform clinical management in pediatric heart surgery.

Studies analyzing the impact of natural antioxidants (NA) on Endothelial Cells (ECs) have dramatically increased during the last years, since a deregulated ECs redox state is at the base of the onset and progression of several cardiovascular diseases. However, whether NA can provide cardiovascular benefits is still a controversial area of debate. Resveratrol (RES), a natural polyphenol found in grapes, is believed to provide cardiovascular benefits by virtue of its antioxidant effect on the endothelium. Here, we report that tissue-attainable doses of resveratrol increased the intracellular oxidative state, thus affecting mitochondrial membrane depolarization and inducing EC death. Cyclosporine A, a mitochondrial permeability transition pore inhibitor, prevented oxidative-mediated cell death, thus implicating mitochondria in resveratrol-induced EC impairment. The specific cytochrome P450 (CYP) 2C9 inhibitor, sulfaphenazole, counteracted both oxidative stress and mitochondrial membrane depolarization, providing EC protection against resveratrol-elicited pro-oxidant effects. Our findings strongly suggest that CYP2C9 mediates resveratrol-induced oxidative stress leading to mitochondria impairment and EC death.

Gestational diabetes mellitus (GDM) is a specific form of diabetes affecting about 7% of all pregnancies. GDM usually reverses after delivery, but altered glucose homeostasis harms the mother. Moreover, since it translates into fetal hyperglycemia, GDM can affect the healthy development of the fetus, with consequences that can be experienced in utero and/or at different stages of post-natal life. In particular, the child from a GDM pregnancy has higher chance of developing type 2 diabetes (T2D) and cardiovascular diseases (CVD). Several clinical trials and epidemiological studies in patients with either type 1 or 2 diabetes show that exposure to prolonged hyperglycemia leaves a long-lasting impression on vascular cells leading to the development of vascular complications which persist after glycemic control is achieved. The same studies also suggested that a good glycemic control can delay the onset of vascular complications in diabetic subjects. The term “epigenetic” refers to heritable changes in the cellular phenotype that do not involve changes in the genotype. There are different kind of epigenetic modifications including methylation and acetylation of histones, and DNA methylation, and these modifications influence gene expression. Although the precise mechanistic insight is still unclear, epigenetic regulation might be involved in the long-lasting effect of diabetes on the vascular system. In this study, we focused our attention on the methyltransferase Enhancer of Zeste Homologue 2 (EZH2), that is part of the Polycomb Repressor Complex 2 (PRC2) and plays an essential role in the epigenetic maintenance of the trimethylation of lysine 27 on histone3 (H3K27me3). It has been recently shown that alternative splicing of the EZH2 locus yields two major transcriptional variants: EZH2α and EZH2β.The two isoforms can each not only regulate distinct targets, but also share gene expression networks, making the regulation of transcription based on the deposition of the H3K27 histone mark more complex than previously anticipated. MicroRNAs (miRNAs) are single-stranded, non-coding RNAs of 19-25 nucleotides that normally bind the 3’-untranslated region of target mRNAs and contribute to the post-transcriptional regulation of gene expression. A link has been shown between EZH2 and some miRNAs. In particular, miR-101 reportedly affects endothelial function and angiogenesis by targeting and inhibiting EZH2 under normoglycemic conditions. The aim of our study was to investigate the impact of GDM on fetal endothelial biology. To this end, we isolated human umbilical vein endothelial cells (HUVECs) from the umbilical cords of GDM and control healthy pregnancies. These cells represent a good cell model allowing for non-invasive analyses of the effect of the uterine environment on the fetal endothelium. The first objective was to investigate the impact of GDM on the functional capacity of HUVECs, with particular focus on apoptosis, migration, proliferation and tube formation. Next, we evaluated the expression of EZH2 (both α and β isoforms) and miR-101 to see whether they affect endothelial function in diabetic conditions. We first found that cultured GDM-HUVECs demonstrated decreased survival and functional capabilities in comparison with HUVECs obtained from healthy non-diabetic mothers. Additionally, we have identified a novel underlying mechanism involving miR-101 and EZH2 in GDM-elicited endothelial dysfunction. In particular, we have found that in HUVECs, EZH2 is both a target of, and a transcriptional repressor for, miR-101. Notably we found that among the two recently identified EZH2 isoforms, EZH2β is the only one which is both a target of GDM and a mediator of some of the GDM-related damaging effects on HUVECs.

Objective— Gestational diabetes mellitus (GDM) produces fetal hyperglycemia with increased lifelong risks for the exposed offspring of cardiovascular and other diseases. Epigenetic mechanisms induce long-term gene expression changes in response to in utero environmental perturbations. Moreover, microRNAs (miRs) control the function of endothelial cells (ECs) under physiological and pathological conditions and can target the epigenetic machinery. We investigated the functional and expressional effect of GDM on human fetal ECs of the umbilical cord vein (HUVECs). We focused on miR-101 and 1 of its targets, enhancer of zester homolog-2 (EZH2), which trimethylates the lysine 27 of histone 3, thus repressing gene transcription. EZH2 exists as isoforms α and β. Approach and Results— HUVECs were prepared from GDM or healthy pregnancies and tested in apoptosis, migration, and Matrigel assays. GDM-HUVECs demonstrated decreased functional capacities, increased miR-101 expression, and reduced EZH2- β and trimethylation of histone H3 on lysine 27 levels. MiR-101 inhibition increased EZH2 expression and improved GDM-HUVEC function. Healthy HUVECs were exposed to high or normal d -glucose concentration for 48 hours and then tested for miR-101 and EZH2 expression. Similar to GDM, high glucose increased miR-101 expression. Chromatin immunoprecipitation using an antibody for EZH2 followed by polymerase chain reaction analyses for miR-101 gene promoter regions showed that both GDM and high glucose concentration reduced EZH2 binding to the miR-101 locus in HUVECs. Moreover, EZH2-β overexpression inhibited miR-101 promoter activity in HUVECs. Conclusions— GDM impairs HUVEC function via miR-101 upregulation. EZH2 is both a transcriptional inhibitor and a target gene of miR-101 in HUVECs, and it contributes to some of the miR-101-induced defects of GDM-HUVECs.

Scientific knowledge of glutamate (GLU) neurobiology is severely hampered by the inadequacy of the available in vivo brain sampling techniques. Due to the crucial role of GLU in central nervous system function and pathology, the development of a reliable sampling device is mandatory. GLU biosensor holds potential to address many of the known issues of in vivo GLU measurement. We report here on the development and test of a labor- and cost-effective microbiosensor, suitable to be applied for measuring brain GLU. A glycerol-based cryopreservation method was also tested. Needle type Pt biosensors were coated with a permselective Nafion–Poly(o-phenylenediamine) layer and cross-linked to l -glutamate oxidase with poly(ethylene glycol) diglycidyl ether. Tested in vitro, the device shows high sensitivity and specificity for GLU, while being poorly influenced by common interfering substances such as ascorbate, dopamine and dihydroxyphenylacetic acid. Further, the cryopreservation procedure kept sensitivity unaltered for 30 days and possibly longer. We conclude that a highly efficient GLU biosensor of minimal dimensions can be consistently and affordably constructed with relative ease. Together with the possibility of cryopreservation this shall foster diffusion and exploitation of GLU biosensors technology.