NMR-based metabolomic signature: An important tool for the diagnosis and study of pathogenesis of autoimmune hepatitis.

BACKGROUND AND AIMS: Metabolomics is used to predict, diagnose, and monitor metabolic disorders but altered metabolomic signatures have also been reported in diverse diseases, including autoimmune disorders. However, the metabolomic profile in autoimmune hepatitis (AIH) has not been investigated in depth. Therefore, we investigated the metabolomic signature of AIH and its significance as a diagnostic and pathogenetic tool. APPROACH AND RESULTS: Metabolites in plasma samples from 50 patients with AIH at diagnosis, 43 healthy controls, 72 patients with primary biliary cholangitis (PBC), 26 patients with metabolic dysfunction-associated liver disease, and 101 patients with chronic viral hepatitis were determined by 1 H NMR (nuclear magnetic resonance) spectroscopy. Fifty-two metabolites were quantified, and metabolic pathway analysis was performed. Multivariate analysis revealed that AIH could be differentiated from healthy controls and each of the disease controls ( p <0.001). Fifteen metabolites differentiated AIH from disease controls (PBC+chronic viral hepatitis+metabolic dysfunction-associated liver disease) (95% sensitivity and 92% specificity). Ten distinct metabolic pathways were altered in AIH compared to disease controls. The metabolic pathway of branched-chain amino acids (lower valine, leucine, and isoleucine levels and their catabolic intermediates in PBC), methionine (lower methionine, 2-aminobutyrate, and 2-hydroxybutyrate levels in PBC), alanine-aspartate-glutamate (lower metabolites in PBC), and that of metabolites associated with gut microbiota (lower choline, betaine, and dimethylamine levels in PBC) were significantly different between AIH and PBC ( p <0.01). CONCLUSIONS: 1 H NMR spectroscopy could be a promising novel tool to diagnose and study AIH pathogenesis as there is no need for much sample handling, is highly reproducible with high sensitivity and specificity, and low cost.

Autoimmune hepatitis in patients aged 70 years or older: Disease characteristics, treatment response and outcome.

BACKGROUND & AIMS: Autoimmune hepatitis (AIH) affects both sexes and all age groups. However, very few studies have focused specifically on the characteristics and outcome of AIH in patients aged 70 y or older. METHODS: 25/234 patients with well-established AIH and disease onset at ≥70-y (median: 73-y) were analysed and compared to the rest patients (median: 47 y). Treatment response was assessed in all patients from both groups who were eligible for treatment (n = 202). RESULTS: Disease presentation was mainly insidious in both groups (19/25, 76% vs. 134/209, 64.1%; P = .313). At diagnosis, older patients had lower alaninoaminotrasferase (101[433] vs. 199[441] IU/L, P < .05) but were more frequently cirrhotic (12/25, 48% vs. 57/209, 27.3%; P = .03). Importantly, similar rates of on-treatment response (16/18, 89% vs. 154/184, 84%; P = .565), corticosteroid withdrawal (10/16, 62.5% vs. 113/154, 73.4%; P = .355) and complete withdrawal of immunosuppression (1/16, 6.3% vs. 40/154, 26%; P = .122) were achieved in both groups. Treatment-related adverse events were evenly observed between groups (6/18, 33% vs. 54/184, 29%; P = .724). In treated patients, the age ≥70 y was only associated with the overall mortality (HR 8.3 [95% CI: 2.1-36.4], P = .003), but not with the liver-related mortality (HR 3.4 [95% CI: 0.4-30.0], P = .268). CONCLUSION: AIH should be seriously considered in patients ≥70 y with unexplained impaired liver function tests as the disease is not infrequent in this group and seems to bear an increased risk for advanced disease stage at diagnosis. However, if immunosuppression is started promptly, it seems as safe and effective as in younger patients.

Androgen Triggers the Pro-Migratory CXCL12/CXCR4 Axis in AR-Positive Breast Cancer Cell Lines: Underlying Mechanism and Possible Implications for the Use of Aromatase Inhibitors in Breast Cancer.

BACKGROUND/AIMS: Reports regarding the role of androgen in breast cancer (BC) are conflicting. Some studies suggest that androgen could lead to undesirable responses in the presence of certain BC tumor characteristics. We have shown that androgen induces C-X-C motif chemokine 12 (CXCL12) in BC cell lines. Our aim was to identify the mechanisms regulating the phenotypic effects of androgen-induced CXCL12 on Androgen Receptor (AR) positive BC cell lines. METHODS: We analyzed the expression of CXCL12 and its receptors with qPCR and ELISA and the role of Nuclear Receptor Coactivator 1 (NCOA1) in this effect. AR effects on the CXCL12 promoter was studied via Chromatin-immunoprecipitation. We also analyzed publically available data from The Cancer Genome Atlas to verify AR-CXCL12 interactions and to identify the effect or Aromatase Inhibitors (AI) therapy on CXCL12 expression and disease progression in AR positive cases. RESULTS: CXCL12 induction occurs only in AR-positive BC cell lines, possibly via an Androgen Response Element, upstream of the CXCL12 promoter. The steroid receptor co-regulator NCOA1 is critical for this effect. Androgen only induced the motility of p53-mutant BC cells T47D cells via upregulation of CXCR4 expression while they had no effect on wild-type p53 MCF-7 cells. Loss of CXCR4 expression and depletion of CXCL12 abolished the effect of androgen in T47D cells while inhibition of p53 expression in MCF-7 cells made them responsive to androgen and increased their motility in the presence to androgen. Patients with estrogen receptor positive (ER+)/AR+ BC treated with AIs were at increased risk of disease progression compared to ER+/AR+ non-AI treated and ER+/AR- AI treated cases. CONCLUSION: AIs may lead to unfavorable responses in some ER/AR positive BC cases, especially in patients with AR+, p53 mutant tumors.

PNPLA3 I148 M genetic variant in autoimmune hepatitis characterises advanced disease at diagnosis and reduced survival free of cirrhotic events and liver-related mortality.

Background: Autoimmune hepatitis (AIH) is a relatively rare autoimmune disease with a strong genetic background. The patatin-like phospholipase domain-containing protein 3 (PNPLA3) I148 M (rs738409 C/G) variant has been associated with hepatic inflammation and fibrosis in chronic hepatic diseases beyond metabolic dysfunction-associated steatotic liver disease (MASLD). Aim: Our aim was to investigate the significance of PNPLA3 I148 M variant in AIH. Method: Two hundred AIH patients, followed in our centre, were evaluated while 100 healthy subjects served as controls. Genotyping was performed with allelic discrimination end-point polymerase chain reaction (PCR). Results: The I148 M variant was present in 95/200 (47.5 %) AIH patients compared to 47/100 (47 %) healthy controls (p = 1.000). Patients with GG/CG genotypes were more likely to present with decompensated cirrhosis at diagnosis (GG/CG 6.3 % vs. CC 1 %, p = 0.039). Comorbidity with cardiometabolic risk factors and concurrence of MASLD was similar across genotypes. Simple steatosis was present in 37/186 (19.9 %) and steatohepatitis in 14/186 (7.5 %) patients with available liver biopsy without correlation with PNPLA3 genotype. Fibrosis stage and grade of inflammation were not correlated with any genotype. Response to treatment was also independent of the presence of the I148 M variant, even though a longer time was needed to achieve complete biochemical response in those carrying the GG/CG genotypes (p = 0.07). On Kaplan Meier analysis homozygosity for the G allele corelated with reduced survival free of decompensation (p = 0.006), cirrhotic events (decompensation, liver transplantation, hepatocellular carcinoma; p = 0.001) and liver-related death or liver transplantation (p = 0.011) in treated patients. Conclusions: The PNPLA3 I148 M variant in AIH patients is associated with increased risk of advanced disease at diagnosis and reduced survival free of cirrhotic events and liver-related death or liver transplantation, regardless of the presence of MASLD. This signifies a potential role for the PNPLA3 I148 M variant as a new AIH biomarker allowing to identify patients at increased risk of disease progression.

Treatment responses and outcomes in patients with autoimmune hepatitis and concomitant features of non-alcoholic fatty liver disease.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) affect 17-46% of Western countries, making coexistence with other liver diseases inevitable. We investigated the prevalence and clinical significance of NAFLD/NASH or the components of metabolic syndrome (MetS) in a large multicentric cohort of patients with autoimmune hepatitis (AIH). Methods: Data from six academic centres (Greece, Canada, Japan, Germany, The Netherlands, and Spain) were evaluated. The presence of NAFLD/NASH in liver biopsy, MetS components, and clinical and laboratory parameters were recorded. Results: A total of 640 patients (474 females, age 49 [4-87] years; follow-up 78 [1-521] months) were included. NAFLD was present in 146 (22.8%) patients (AIH/non-alcoholic fatty liver [NAFL] 115 [18%], AIH/NASH 31 [4.8%]). AIH/NAFL patients were older (p = 0.017), more frequently overweight or obese (p = 0.002), had hypertension (p = 0.001), and had diabetes (p = 0.016), whereas they less frequently had acute presentation (p = 0.002) and soluble liver antigen/liver pancreas positivity (p <0.05), lower transaminases (p <0.001), ALP (p = 0.028) and IgG (p = 0.004) and higher albumin (p <0.001) than patients with AIH only. Patients with AIH/NASH more frequently had cirrhosis at diagnosis (p = 0.036) and higher IgG (p = 0.009). Response to treatment did not differ between groups. Patients with cirrhosis with AIH/NAFL had higher frequency of decompensation compared with patients with AIH only (p <0.05). Patients with type 2 diabetes mellitus and dyslipidaemia had increased hazard of disease progression (p <0.05 for each). Conclusions: The prevalence of NAFLD in AIH is similar to the general population. Concurrence of NASH in patients with AIH signifies a more severe disease, whereas that of NAFL may indicate a worse prognosis in patients with cirrhosis. T2DM and dyslipidaemia in AIH patients are associated with dismal parameters of outcome. Our findings suggest that NAFLD presence or even components of MetS in patients with AIH may affect prognosis, so closer follow-up of such patients is warranted. Impact and implications: Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) affect many people, making coexistence with other liver diseases inevitable. We investigated the prevalence and clinical significance of NAFLD/NASH or the components of metabolic syndrome (MetS) in patients with autoimmune hepatitis (AIH). NAFLD and NASH presence in patients with AIH is as frequent as in the general population. The concurrence of NASH in patients with AIH seems to signify a more severe disease, whereas that of non-alcoholic fatty liver may indicate a worse prognosis in a specific subgroup of patients who already have cirrhosis at diagnosis. Diabetes or dyslipidaemia in patients with AIH were associated with worse prognosis. Therefore, it seems that closer follow-up of patients with concurrent AIH and NAFLD or AIH and components of MetS is needed.

Acute non-A, non-B, non-C hepatitis differences and similarities between hepatitis E virus infection and autoimmune hepatitis, with phylogenetic analysis of hepatitis E virus in humans and wild boars.

Background: Hepatitis E virus (HEV) infection incidence is increasing in Europe, accounting for the majority of acute hepatitis cases. We investigated the prevalence and clinical characteristics of acute hepatitis E (AHE) in patients with acute non-A/B/C hepatitis from central Greece, their differences from acute autoimmune hepatitis (AIH) patients and the molecular similarity of human strains to local HEV strains in wild boars. Methods: Sera from 20 patients with non-A/B/C acute hepatitis (2015-2017) were tested prospectively for anti-HEV IgM, IgG antibodies and HEV-RNA. Sera from patients diagnosed with acute AIH (2000-2015; n=56) were tested retrospectively. Liver tissue samples from 40 wild boars were tested for HEV-RNA. Positive wild boar and patients' samples were sequenced and phylogenetically analyzed. Results: Twelve of the 76 (16%) patients were diagnosed with AHE: HEV-RNA 11.5x104 (38.7-39.7x106) IU/mL; 11/20 (55%) acute non-A/B/C hepatitis and 1/56 (2%) AIH patients. Patients with AHE were older than those without, predominately men, with higher alanine aminotransferase but lower IgG levels (P=0.005 and P=0.002, respectively), and had high titers of smooth muscle antibodies. Liver biopsies, performed in 6/12 patients with HEV infection, revealed histology compatible with AIH. HEV strains from both patients and wild boars belonged to genotype 3. Conclusions: Approximately one sixth of patients with acute non-A/B/C hepatitis had autochthonous HEV infection with AIH features. Therefore, a careful workup to exclude HEV should be carried out in all acute hepatitis cases before a definite diagnosis of AIH is established. Wild boars seem to be an important reservoir of HEV in Greece.

An Immunocompetent HIV-Negative Elderly Patient with Low-Grade Fever, Generalized Lymphadenopathy, Splenomegaly, and Acute Phase Response: Do Not Forget Castleman Disease.

Multicentric Castleman disease (MCD) is a rare lymphoproliferative disorder that mainly affects middle-aged patients with human immunodeficiency virus (HIV) infection. However, HIV-negative patients can also be affected representing a small proportion of the total MCD cases. Of note, recent studies from China in HIV-negative patients with MCD have suggested that the onset of the disease can be observed in younger age than previously thought. If undiagnosed and untreated, the MCD has a poor prognosis and may progress to lymphoma. We present an 82-year-old immunocompetent male patient who was admitted to our department because of low-grade fever, cachexia, anasarca, hepatosplenomegaly, and generalized lymphadenopathy. Laboratory findings showed anemia and increased markers of inflammation including hyperferritinemia and polyclonal hyperglobulinemia. Infectious causes including HIV were ruled out. Histological examination of a cervical lymph-node revealed lesions supportive of MCD diagnosis. Of note, the outer-zone plasmablasts' nuclei stained positive for human herpesvirus-8 (HHV8). The patient received 4 cycles of cyclophosphamide, vincristine, and dexamethasone with regression of all symptoms. This case underlines that HHV8-associated MCD should be considered as a rare cause of generalized lymphadenopathy even in HIV-negative immunocompetent patients when other causes have been appropriately excluded because a timely diagnosis can be life-saving.

Programmed cell death-1 rs11568821 and interleukin-28B rs12979860 polymorphisms in autoimmune hepatitis.

BACKGROUND: Autoimmune hepatitis (AIH) is a relatively rare chronic liver disease of unknown etiology. The genetic background affects susceptibility, clinical phenotype, and prognosis. The programmed cell death-1 rs11568821 polymorphism (PD1.3) has been associated with susceptibility to autoimmune diseases. The interleukin-28B (IL28B) rs12979860 polymorphism has been associated with steatosis, inflammation, and fibrosis in liver diseases. AIM: Our aim was to investigate for the first time the incidence and clinical significance of PD1.3 and IL28B rs12979860 in AIH. METHODS: Two hundred patients with AIH were evaluated, while 100 healthy subjects were used as controls. Genotyping was performed with in-house allelic discrimination End-Point PCR. RESULTS: The SNP PD1.3/A was present in 36/200 (18%) AIH patients compared to 28/100 (28%) healthy controls (p = 0.065). The AA/GA genotypes were not associated with the mode of presentation of AIH, the histological grade or stage, the presence of cirrhosis, risk of disease progression, response to treatment and survival. The IL28B rs12979860 genotype distribution was CC 79/200 (39.5%), TT 36/200 (18%) and CT 85/200 (42.5%), in similar rates with healthy controls (p = 0.878). Inflammatory activity and fibrosis stage did not differ between CC homozygotes and CT/TT carriers. LDL cholesterol was significantly higher in CC than CT/TT patients (P = 0.027), though no differences was found regarding the presence of steatosis or steatohepatitis. On-treatment response to immunosuppressive treatment was not affected by the IL28B rs12979860 polymorphism. However, CC homozygotes AIH patients achieved treatment withdrawal in significantly higher rates (OR 2.3, 95%CI: 1.1-4.7, P = 0.02) irrespective of the presence of steatosis or steatohepatitis. CONCLUSIONS: The PD1.3 and IL28B rs12979860 variants are unlikely to contribute to AIH susceptibility, disease presentation and prognosis. The IL28B rs12979860 is not associated with the presence of concurrent steatosis or steatohepatitis. However, although on-treatment response rates to immunosuppression were not affected by the IL28B rs12979860 polymorphism, AIH patients with CC homozygosity were more likely to achieve complete treatment withdrawal. This novel finding needs validation and further clarification from larger multicenter studies.

Severe Pharyngodynia Followed by Migratory Polyarthritis and High Fever in Young Immigrants: Remember That Rheumatic Fever Is Still Relevant in 2020!

Acute rheumatic fever (ARF) is the immune-mediated sequelae of untreated group-A streptococcal infection. In this regard, rheumatic heart disease is the most prominent manifestation with devastating long-term complications. In the postantibiotic era, ARF is extremely rare in high-income countries; thus, its diagnosis might escape the clinicians' notice. However, its incidence remains high not only in certain low- and middle-income regions with poor public health systems but also in socioeconomically vulnerable populations residing in high-income countries. Herein, we report two cases of ARF in young immigrant adults in order to highlight the need for increased clinical suspicion to establish a prompt and timely diagnosis of ARF and describe in detail its differential diagnosis and approach to treatment.

Glycogenic hepatopathy as a cause of severe deranged liver enzymes in a young patient with type 1 diabetes mellitus.

Glycogenic hepatopathy (GH) is a rare complication of poorly controlled type 1 diabetes mellitus (T1DM). We present a 19-year-old woman with T1DM and autoimmune thyroiditis who admitted to our department because of abrupt onset intermittent abdominal pain in the right upper quadrant accompanied by laboratory evidence of acute anicteric hepatitis. Physical examination revealed significant hepatomegaly but the common imagining studies were negative. Following exclusion of common causes of acute hepatitis and because of the presence of smooth muscle antibodies in a young female patient with already established two autoimmune diseases, a liver biopsy was performed in order to exclude the potential presence of autoimmune hepatitis. However, liver histology showed typical findings of GH. Intense treatment targeting strict glycemic control resulted in normalisation of liver biochemistry. This case underlines that GH should be considered as a rare cause of acute hepatitis in T1DM patients with poor glycemic control.

Patients with Haemoglobinopathies and Chronic Hepatitis C: A Real Difficult to Treat Population in 2016?

BACKGROUND & OBJECTIVES: In the past, patients with haemoglobinopathies were at high risk of acquiring hepatitis C virus (HCV) due to multiple transfusions before HCV screening. In these patients, the coexistence of haemochromatosis and chronic hepatitis C (CHC) often leads to more severe liver disease. We assessed the HCV prevalence, clinical characteristics and outcome in this setting with particular attention to the response to treatment including therapies with the new direct acting antivirals (DAAs). METHODS: The medical records of 81 consecutive patients followed the last 15 years were reviewed retrospectively. RESULTS: 43/81 (53%) patients were anti-HCV positive including 31/43 (72.1%) with CHC (HCV-RNA positive; age 25±7 years; 45.2% with genotype 1b; 19.4% cirrhotics; baseline ferritin 887 ng/ml; range: 81-10.820). Thirty patients received IFN-based therapy with or without ribavirin with sustained virological response (SVR) in 14/30 (46.7%). Eleven patients (9 non-responders to IFN-based therapies, one in relapse and one naïve) received treatment with DAAs (SVR: 100%). 3/11 patients increased their transfusion needs while 1/11 reported mild arthralgias. No drug-drug interactions between DAAs and chelation agents were observed as attested by the stability of ferritin levels during treatment. CONCLUSIONS: More than 1/3 of patients with haemoglobinopathies suffered from CHC. Response rates to IFN-based treatment seem to be similar to other patients with CHC, while most importantly, treatment with DAAs was excellent and safe even in difficult to treat patients (most null responders with severe fibrosis) suggesting that this group of HCV patients should no longer be regarded as a difficult to treat.

Geoepidemiology, clinical manifestations and outcome of primary biliary cholangitis in Greece.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is a disease with rising prevalence and considerable geographical variation. To describe the prevalence, spatial and time distribution, baseline characteristics, response to treatment, outcome and the validity of GLOBE score in a large cohort of Greek PBC patients as an independent validation of this score has not been done so far. METHODS: The last 16years, 482 PBC patients (86.5% females) were evaluated and analysed retrospectively, using a prospectively collected database. Special attention was paid to the assessment of treatment response according to GLOBE score. RESULTS: Age at initial evaluation was 56.3±13.7years. Among 432 Thessaly residents, prevalence was 582/million (non-homogeneous distribution). Nineteen districts showed a prevalence >800/million. Symptomatic disease onset could be identified in 91 patients, with a significant peak during spring (P=0.03). At diagnosis, 43.6% were asymptomatic and 16.2% cirrhotic. Male sex (P=0.02), older age (P<0.001), alcohol consumption (P<0.01) and concomitant liver disease (P<0.001) were negative prognostic factors for cirrhosis. During a median [interquartile range, range] follow-up of 5.1 (7.8, 15.7) years, 62 patients died or underwent liver transplantation. Patients with GLOBE score>0.30 had significantly worse prognosis (P<0.001) with 5-, 10-, and 15-year survival rates of 84%, 50% and 42%. CONCLUSIONS: There is increased PBC prevalence in Thessaly with remarkable geographic clustering and seasonal variability. PBC is diagnosed at early stages although males had a more advanced disease. GLOBE score applies perfectly in Greek patients and this will likely help detecting patients that may benefit from new therapies.

Decreased serum DNase1-activity in patients with autoimmune liver diseases.

Deoxyribonuclease1 (DNase1) is involved in chromatin degradation of apoptotic cells. Its deficiency results in accumulation of self-DNA, which in turn may induce inflammation and autoimmunity. We assessed for the first time serum DNase1-activity in a large consecutive cohort of treatment-naïve patients with autoimmune liver diseases (ALD). DNase1-activity was determined by single radial enzyme-diffusion (SRED) at diagnosis of 224 patients with autoimmune hepatitis (AIH), 249 with primary biliary cirrhosis (PBC) and 36 with primary sclerosing cholangitis (PSC). Sera from 146 patients with chronic hepatitis B or C, 140 with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) and 114 healthy individuals served as disease and healthy controls. Available serum samples during remission from 50 AIH and 39 PBC patients were also investigated by paired analyzes. DNase1-activity was significantly lower in AIH, PBC and PSC compared to viral hepatitis (p < 0.02, p < 0.001, p = 0.03), NAFLD/NASH (p < 0.001) and healthy (p < 0.001). No significant difference was found in between each specific ALD. In AIH, DNAse1-activity was positively correlated with aspartate aminotransferase (AST) (p < 0.02), bilirubin (p < 0.01) and increased IgG (>1400 mg/dl; p < 0.05); in PBC, with AST (p < 0.01), alanine aminotransferase (ALT) (p < 0.03) and anti-mitochondrial antibodies (AMA) (p = 0.008). In PSC, DNase1-activity was inversely associated with alkaline phosphatase (ALP) (p < 0.05). In AIH, complete responders were characterized by increased baseline DNase1-activity compared to partial responders, relapsers and non-responders (p < 0.02), whereas it was significantly increased after achievement of remission (p < 0.001). Serum DNase1-activity is significantly decreased in ALD patients, indicating its potential implication in their pathogenesis. Furthermore, DNase1-activity could be used as a new surrogate biomarker for predicting response to AIH treatment.

Accurate prediction of severe allergic reactions by a small set of environmental parameters (NDVI, temperature).

Severe allergic reactions of unknown etiology,necessitating a hospital visit, have an important impact in the life of affected individuals and impose a major economic burden to societies. The prediction of clinically severe allergic reactions would be of great importance, but current attempts have been limited by the lack of a well-founded applicable methodology and the wide spatiotemporal distribution of allergic reactions. The valid prediction of severe allergies (and especially those needing hospital treatment) in a region, could alert health authorities and implicated individuals to take appropriate preemptive measures. In the present report we have collecterd visits for serious allergic reactions of unknown etiology from two major hospitals in the island of Crete, for two distinct time periods (validation and test sets). We have used the Normalized Difference Vegetation Index (NDVI), a satellite-based, freely available measurement, which is an indicator of live green vegetation at a given geographic area, and a set of meteorological data to develop a model capable of describing and predicting severe allergic reaction frequency. Our analysis has retained NDVI and temperature as accurate identifiers and predictors of increased hospital severe allergic reactions visits. Our approach may contribute towards the development of satellite-based modules, for the prediction of severe allergic reactions in specific, well-defined geographical areas. It could also probably be used for the prediction of other environment related diseases and conditions.

Early membrane initiated transcriptional effects of estrogens in breast cancer cells: First pharmacological evidence for a novel membrane estrogen receptor element (ERx).

The complexity of estrogen actions mainly relies to the presence of different identified receptors (ERα, ERß, their isoforms, and GPR30/GPER) and their discrete cellular distribution. Depending on the localization of the receptor that mediates estrogen effects, nuclear and extra-nuclear actions have been described. The latter can trigger a number of signaling events leading also to transcriptional modifications. In an attempt to clarify the nature of the receptor(s) involved in the membrane initiated effect of estrogens on gene expression, we performed a whole transcriptome analysis of breast cancer cell lines with different receptor profiles (T47D, MCF7, MDA-MB-231, SK-BR-3). A pharmacological approach was conducted with the use of estradiol (E(2)) or membrane-impermeable E(2)-BSA in the absence or presence of a specific ERα-ß or GPR30/GPER antagonist. Our results clearly show that in addition to the ERα isoforms and/or GPR30/GPER that mainly mediate the transcriptional effect of E(2)-BSA, there is a specific transcriptional signature (found in T47D and MCF-7 cells) suggesting the presence of an unidentified membrane ER element (ERx). Analysis of its signature and phenotypic verification revealed that important cell function such as apoptosis, transcriptional regulation, and growth factor signaling are associated with ERx.