PCSK9 ablation attenuates Aß pathology, neuroinflammation and cognitive dysfunctions in 5XFAD mice.

BACKGROUND: Increasing evidence highlights the importance of novel players in Alzheimer's disease (AD) pathophysiology, including alterations of lipid metabolism and neuroinflammation. Indeed, a potential involvement of Proprotein convertase subtilisin/kexin type 9 (PCSK9) in AD has been recently postulated. Here, we first investigated the effects of PCSK9 on neuroinflammation in vitro. Then, we examined the impact of a genetic ablation of PCSK9 on cognitive performance in a severe mouse model of AD. Finally, in the same animals we evaluated the effect of PCSK9 loss on Aß pathology, neuroinflammation, and brain lipids. METHODS: For in vitro studies, U373 human astrocytoma cells were treated with Aß fibrils and human recombinant PCSK9. mRNA expression of the proinflammatory cytokines and inflammasome-related genes were evaluated by q-PCR, while MCP-1 secretion was measured by ELISA. For in vivo studies, the cognitive performance of a newly generated mouse line - obtained by crossing 5XFADHet with PCSK9KO mice - was tested by the Morris water maze test. After sacrifice, immunohistochemical analyses were performed to evaluate Aß plaque deposition, distribution and composition, BACE1 immunoreactivity, as well as microglia and astrocyte reactivity. Cholesterol and hydroxysterols levels in mouse brains were quantified by fluorometric and LC-MS/MS analyses, respectively. Statistical comparisons were performed according to one- or two-way ANOVA, two-way repeated measure ANOVA or Chi-square test. RESULTS: In vitro, PCSK9 significantly increased IL6, IL1B and TNFΑ mRNA levels in Aß fibrils-treated U373 cells, without influencing inflammasome gene expression, except for an increase in NLRC4 mRNA levels. In vivo, PCSK9 ablation in 5XFAD mice significantly improved the performance at the Morris water maze test; these changes were accompanied by a reduced corticohippocampal Aß burden without affecting plaque spatial/regional distribution and composition or global BACE1 expression. Furthermore, PCSK9 loss in 5XFAD mice induced decreased microgliosis and astrocyte reactivity in several brain regions. Conversely, knocking out PCSK9 had minimal impact on brain cholesterol and hydroxysterol levels. CONCLUSIONS: In vitro studies showed a pro-inflammatory effect of PCSK9. Consistently, in vivo data indicated a protective role of PCSK9 ablation against cognitive impairments, associated with improved Aß pathology and attenuated neuroinflammation in a severe mouse model of AD. PCSK9 may thus be considered a novel pharmacological target for the treatment of AD.

PCSK9 Affects Astrocyte Cholesterol Metabolism and Reduces Neuron Cholesterol Supplying In Vitro: Potential Implications in Alzheimer's Disease.

The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) involvement in Alzheimer's disease (AD) is poorly investigated. We evaluated the in vitro PCSK9 modulation of astrocyte cholesterol metabolism and neuronal cholesterol supplying, which is fundamental for neuronal functions. Moreover, we investigated PCSK9 neurotoxic effects. In human astrocytoma cells, PCSK9 reduced cholesterol content (−20%; p < 0.05), with a greater effect in presence of beta amyloid peptide (Aß) (−37%; p < 0.01). PCSK9 increased cholesterol synthesis and reduced the uptake of apoE-HDL-derived cholesterol (−36%; p < 0.0001), as well as the LDL receptor (LDLR) and the apoE receptor 2 (ApoER2) expression (−66% and −31%, respectively; p < 0.01). PCSK9 did not modulate ABCA1- and ABCG1-cholesterol efflux, ABCA1 levels, or membrane cholesterol. Conversely, ABCA1 expression and activity, as well as membrane cholesterol, were reduced by Aß (p < 0.05). In human neuronal cells, PCSK9 reduced apoE-HDL-derived cholesterol uptake (−41%; p < 0.001) and LDLR/apoER2 expression (p < 0.05). Reduced cholesterol internalization occurred also in PCSK9-overexpressing neurons exposed to an astrocyte-conditioned medium (−39%; p < 0.001). PCSK9 reduced neuronal cholesterol content overall (−29%; p < 0.05) and increased the Aß-induced neurotoxicity (p < 0.0001). Our data revealed an interfering effect of PCSK9, in cooperation with Aß, on brain cholesterol metabolism leading to neuronal cholesterol reduction, a potentially deleterious effect. PCSK9 also exerted a neurotoxic effect, and thus represents a potential pharmacological target in AD.

Influence of vehicular emissions on the levels of polycyclic aromatic hydrocarbons (PAHs) in urban and industrial areas of La Plata, Argentina.

Polycyclic aromatic hydrocarbons (PAHs) are considered potentially toxic, even carcinogenic, because of their affection to public health and the environment. It is necessary to know their ambient levels and the origin of these pollutants in order to mitigate them. A concerning scenario is the one in which commercial/administrative, industrial, and residential activities coexist. In this context, Gran La Plata (Argentina) presents such characteristics, in addition to the presence of one of the most important petrochemical complexes in the country and intense vehicular traffic. The source apportionment of PAH emission in the region, associated to 10-µm and 2.5-µm particulate matter fractions, was studied. First, different missing value imputation methods were evaluated for PAH databases. GSimp presented a better performance, with mean concentrations of ∑PAHs of 65.8 ± 40.2 ng m-3 in PM10 and 39.5 ± 18.0 ng m-3 in PM2.5. For both fractions, it was found that the highest contribution was associated with low molecular weight PAHs (3 rings), with higher concentrations of anthracene. Emission sources were identified by using principal component analysis (PCA) together with multiple linear regression (MLR) and diagnostic ratios of PAHs. The results showed that the main emission source is associated with vehicular traffic in both fractions. Classification by discriminant analysis showed that emissions can be identified by region and that fluoranthene, benzo(a)anthracene, and anthracene in PM10 and anthracene and phenanthrene in PM2.5 are a characteristic of emissions from the petrochemical complex.

Is there any therapeutic role of pelvic and para-aortic lymphadenectomy in apparent early stage epithelial ovarian cancer?

OBJECTIVE: The therapeutic role of pelvic and para-aortic lymphadenectomy in surgical staging of apparent early-stage epithelial ovarian cancer (eEOC) is still under debate. The aim of this study was to evaluate the potential therapeutic role of systematic lymphadenectomy in patients with eEOC. METHODS: Multi-center retrospective cohort study, comparing women with apparent eEOC who underwent comprehensive bilateral pelvic and para-aortic lymphadenectomy (defined as ≥20 lymph nodes) versus patients receiving no lymphadenectomy or lymph node sampling, from 05/1985 to 12/2016. Patients with bulky nodes at CT-scan and those without complete intra-peritoneal surgical staging were excluded. Only patients who received at least 3 cycles of platinum-based adjuvant chemotherapy were included. RESULTS: Out of 2559 patients with FIGO stage IA-IIIA1 ovarian cancer, 639 (25.0%) met inclusion criteria. 360 (56.3%) underwent comprehensive lymphadenectomy, 150 (23.5%) lymph node sampling and 129 (20.2%) no lymphadenectomy. Patients who underwent comprehensive lymphadenectomy were younger (p < 0.001), experienced a higher number of severe post-operative complications (p = 0.008) and had a longer time to start chemotherapy (p = 0.034). There was no difference in intra-operative complications. Median follow-up was 63 months (range, 5-342). The 5-year disease-free survival (DFS) was 79.7% vs. 76.5% vs. 68.3% (p = 0.006), and 5-year overall survival (OS) was 92.3% vs. 94.5% vs. 89.8% (p = 0.165) in women who received comprehensive lymphadenectomy vs. lymph node sampling vs. no lymphadenectomy, respectively. Lymphadenectomy represented an independent factor for DFS improvement, HR 0.52 (95%CI 0.37-0.73) (p < 0.001). CONCLUSION: Pelvic and para-aortic lymphadenectomy in surgical staging of eEOC improves DFS for the price of increasing post-operative complications and time to chemotherapy but does not affect OS. Better understanding of tumor biology may help to identify those patients in whom lymphadenectomy should still play a role.

Combining positron emission tomography/computed tomography, radiomics, and sentinel lymph node mapping for nodal staging of endometrial cancer patients.

OBJECTIVE: To evaluate the combination of positron emission tomography/computed tomography (PET/CT) and sentinel lymph node (SLN) biopsy in women with apparent early-stage endometrial carcinoma. The correlation between radiomics features extracted from PET images of the primary tumor and the presence of nodal metastases was also analyzed. METHODS: From November 2006 to March 2019, 167 patients with endometrial cancer were included. All women underwent PET/CT and surgical staging: 60/167 underwent systematic lymphadenectomy (Group 1) while, more recently, 107/167 underwent SLN biopsy (Group 2) with technetium-99m +blue dye or indocyanine green. Histology was used as standard reference. PET endometrial lesions were segmented (n=98); 167 radiomics features were computed inside tumor contours using standard Image Biomarker Standardization Initiative (IBSI) methods. Radiomics features associated with lymph node metastases were identified (Mann-Whitney test) in the training group (A); receiver operating characteristic (ROC) curves, area under the curve (AUC) values were computed and optimal cut-off (Youden index) were assessed in the test group (B). RESULTS: In Group 1, eight patients had nodal metastases (13%): seven correctly ridentified by PET/CT true-positive with one false-negative case. In Group 2, 27 patients (25%) had nodal metastases: 13 true-positive and 14 false-negative. Sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of PET/CT for pelvic nodal metastases were 87%, 94%, 93%, 70%, and 98% in Group 1 and 48%, 97%, 85%, 87%, and 85% in Group 2, respectively. On radiomics analysis a significant association was found between the presence of lymph node metastases and 64 features. Volume-density, a measurement of shape irregularity, was the most predictive feature (p=0001, AUC=0,77, cut-off 0.35). When testing cut-off in Group B to discriminate metastatic tumors, PET false-negative findings were reduced from 14 to 8 (-43%). CONCLUSIONS: PET/CT demonstrated high specificity in detecting nodal metastases. SLN and histologic ultrastaging increased false-negative PET/CT findings, reducing the sensitivity of the technique. PET radiomics features of the primary tumor seem promising for predicting the presence of nodal metastases not detected by visual analysis.

Ovarian function, fertility, and menopause occurrence after fertility-sparing surgery and chemotherapy for ovarian neoplasms.

BACKGROUND: The effect of chemotherapy exposure (CE) on ovarian function in young women with ovarian neoplasms undergoing fertility-sparing treatment (FST) remains unclear. We investigated whether CE is correlated with the outcomes (1) during-treatment and (2) post-treatment amenorrhea, (3) conception rate, (4) pregnancy outcome, and (5) spontaneous menopausal age. PATIENTS AND METHODS: Eligibility criteria were patients with a diagnosis of epithelial (EOC) or nonepithelial (no-EOC) invasive ovarian neoplasm, premenopausal age, undergoing FST ±â€¯CE, histopathology confirmation, and adequate follow-up. The groups' outcomes were compared by logistic and linear regression analysis. RESULTS: A total of 548 patients diagnosed during 1980 and 2014 were included, 198 in the EOC group and 350 in the no-EOC group, and 44% received chemotherapy, with a median follow-up of 15.9 years. In no-EOC patients, CE conferred a higher risk for Outcomes 1 (adjusted OR [aOR] 27; 95% CI 12 to 61; P < .0001) and 2 (aOR 5.42; 95% CI 1 to 24; P = .0256) and was associated with a younger menopausal age (adjusted ß -5.52; 95% CI -10.53 to -0.52; P = .0313). Overall, 57% of patients attempted pregnancy, with a conception rate of 89%. In EOC patients, no association between CE and a decreased fertility was demonstrated (aOR, 3.05; 95% CI 0.72 to 12.88; P = .1298). CONCLUSIONS: CE in no-EOC was associated with an increased risk of during-treatment amenorrhea, post-treatment amenorrhea, and earlier spontaneous menopausal age; CE in EOC was not associated with any item at study. Patients undergoing FST had reassuringly high conception rates and low premature ovarian failure rates; however, in pretreatment counseling, the risks of this approach in such young population should be discussed.

Mechanisms of Hydrogen Sulfide against the Progression of Severe Alzheimer's Disease in Transgenic Mice at Different Ages.

Backgroud: Alzheimer disease is an age-related severe neurodegenerative pathology. The level of the third endogenous gas, hydrogen sulfide (H2S), is decreased in the brain of Alzheimer's disease (AD) patients compared with the brain of the age-matched normal individuals; also, plasma H2S levels are negatively correlated with the severity of AD. Recently, we have demonstrated that systemic H2S injections are neuroprotective in an early phase of preclinical AD. OBJECTIVES: This study focuses on the possible neuroprotection of a chronic treatment with an H2S donor and sulfurous water (rich of H2S) in a severe transgenic 3×Tg-AD mice model. METHOD: 3×Tg-AD mice at 2 different ages (6 and 12 months) were daily treated intraperitoneally with an H2S donor and sulfurous water (rich of H2S) for 3 months consecutively. We investigated the cognitive ability, brain morphological alterations, amyloid/tau cascade, excitotoxic, inflammatory and apoptotic responses. RESULTS: Three months of treatments with H2S significantly protected against impairment in learning and memory in a severe 3×Tg-AD mice model, at both ages studied, and reduced the size of Amyloid ß plaques with preservation of the morphological picture. This neuroprotection appeared mainly in the cortex and hippocampus, associated with reduction in activity of c-jun N-terminal kinases, extracellular signal-regulated kinases and p38, which have an established role not only in the phosphorylation of tau protein but also in the inflammatory and excitotoxic response. CONCLUSION: Our findings indicate that appropriate treatments with various sources of H2S, might represent an innovative approach to counteract early and severe AD progression in humans.

Laparoscopic Sentinel Node Mapping in Cervical and Endometrial Malignancies: A Case-Control Study Comparing Two Near-Infrared Fluorescence Systems.

STUDY OBJECTIVE: The goal of this study was to evaluate the intraoperative and perioperative surgical outcomes of 2 different florescence systems commonly used for sentinel lymph node (SLN) mapping in women with early-stage cervical cancer or endometrial cancer. DESIGN: Case-control study (Canadian Task Force classification II-2). SETTING: The Gynecology Oncology Surgical Unit of the San Gerardo Hospital, Italy. PATIENTS: Thirty-four consecutive women with early stage-cervical cancer (stage IA-1B1) or apparent confined stage I endometrial cancer were included in the study. INTERVENTIONS: Between October 2016 and May 2017, 34 patients underwent laparoscopic surgery with SLN mapping using indocyanine green dye: 22 women were mapped with the Storz 1S system (Karl Storz Endoscopy, Tuttlingen, Germany; Group A), whereas 12 women underwent planned surgery with the Novadaq PinPoint system (Novadaq, Mississauga, Ontario, Canada; Group B). MEASUREMENT AND MAIN RESULTS: We compared the surgical and perioperative outcomes of Group A and Group B. Patients in Group B had a shorter duration of the SLN mapping time than those in Group A (p = .0003). The median number of SLNs removed was 2 (range, 0-5) in Group A and 2 (range, 1-3) in Group B (p = .501). Bilateral mapping was 77.3% in Group A and 83.3% in Group B (p = .334), respectively. No differences were recorded in terms of body mass index, length of hospital stay, type of tumor, bilateral mapping, or number of lymph nodes removed. Body mass index was found to have no impact on the duration of the mapping (p = .353). CONCLUSION: From our preliminary experience we can conclude that both fluorescence systems are valid and applicable for SLN detection in the case of early-stage cervical or endometrial cancer. The PinPoint system seems to allow surgeons easier and faster identification of the SLNs, particularly in endometrial cancer patients.

The impact on survival of two different staging strategies in apparent early stage endometrial cancer comparing sentinel lymph nodes mapping algorithm and selective lymphadenectomy: An Italian retrospective analysis of two reference centers.

OBJECTIVE: The role of lymphadenectomy in endometrial cancer is still uncertain. We aimed to evaluate the survival outcomes of two different strategies in apparent uterine confined disease by comparing sentinel lymph node (SLN) mapping and selective lymphadenectomy (LD). METHODS: We retrospectively reviewed women with preoperative stage I endometrial cancer underwent surgical staging with either SLN mapping, or LD in two Italian centers. RESULTS: Eight hundred and two women underwent surgical staging for preoperative stage I endometrial cancer were revised (145 Monza; 657 Rome). All patients underwent peritoneal washing, simple hysterectomy with bilateral salpingo-oophorectomy and nodal staging including SLN mapping, or LD. Overall 8229 lymph nodes were removed (1595 in Monza, 6634 in Rome). Pelvic lymphadenectomy was performed in 33.1% and 52.4% in Monza and Rome, respectively (p<0.001). Patients with positive pelvic LN were 16.7% and 7.3%, in SLN and LD groups, respectively (p=0.002). Disease-free survival (DFS) curves did not showed a statistically significant difference between centers and strategies adopted (SLN mapping, LD, SLN+LD) with a HR of 0.87 (95% CI 0.63-2.16; p=0.475). CONCLUSIONS: Survival outcomes were similar for both strategies. The SLN strategy allowed to identify a higher rate of stage IIIC1 disease even with a lower median number of lymph node removed in SLN group. Applying a SLN algorithm does not impair the prognosis of endometrial cancer patients. The clinical impact and management of low volume metastasis in high-risk patients should be further clarify.

Effects of COX1-2/5-LOX blockade in Alzheimer transgenic 3xTg-AD mice.

OBJECTIVE AND DESIGN: Alzheimer's disease (AD) is associated with amyloid plaques (Aß) and hyperphosphorylated tau protein tangles in the brain. We investigated the possible neuroprotective role of flavocoxid, a dual inhibitor of cyclooxygenases-1/2 (COX-1/2) and 5-Lipoxygenase (5-LOX), in triple-transgenic (3xTg-AD) mice. SUBJECTS: Mice were 3 months at the beginning of the study. TREATMENT: Animals received once daily for 3-month saline solution or flavocoxid (20 mg/kg/ip). METHODS: Morris water maze was used to assess learning and memory. Histology was performed to evidence Aß plaques and neuronal loss, while inflammatory proteins were determined by western blot analysis. RESULTS: Saline-treated 3xTg-AD mice showed an impairment in spatial learning and memory (assessed at 6 months of age), and increased expression of inflammatory and apoptotic molecules. Treatment of 3xTg-AD mice with flavocoxid reduced: (1) learning and memory loss; (2) the increased eicosanoid production and the phosphorylation level of amyloid precursor protein (APP-pThr668), Aß 1-42, p-tau (pThr181), pERK, and the activation of the NLRP3 inflammasome; (3) Aß plaques; and (4) neuronal loss, compared to saline-treated animals. CONCLUSIONS: Pharmacological blockade of both COX-1/2 and 5-LOX was able to counteract the progression of AD by targeting pathophysiological mechanisms up- and downstream of Aß and tau.

Long-term results of fertility-sparing treatment compared with standard radical surgery for early-stage epithelial ovarian cancer.

BACKGROUND: The objective of this study is to evaluate the safety of fertility-sparing surgery (FSS) for early-stage epithelial ovarian cancer (EOC). METHODS: A retrospective analysis was performed to identify patients treated for early-stage EOC and to compare the clinical outcomes of patients treated with FSS and radical surgery (RS). RESULTS: A total of 1031 patients were treated at two Institutions, 242 with FSS (group A) and 789 with RS (group B). Median duration of follow-up was 11.9 years. At univariate analyses, FSS was associated with decreased risk of relapse (P=0.002) and of tumour-related death (P=0.001). Multivariate analysis did not confirm the independent positive role of FSS neither on relapse-free interval (RFI) nor on cancer-specific survival (CSS). Tumour grade was associated with shorter RFI (P<0.001) and shorter CSS (P=0.001). The type of treatment did not influence CSS or RFI in any grade group. We also found a significant association between low-grade tumours and younger age. CONCLUSIONS: Fertility-sparing surgery is an adequate treatment for patients with stage I EOC. The clinical outcome of patients with G3 tumours, which is confirmed to be the most important prognostic factor, is not determined by the type of treatment received.

Impact of Indocyanine Green for Sentinel Lymph Node Mapping in Early Stage Endometrial and Cervical Cancer: Comparison with Conventional Radiotracer (99m)Tc and/or Blue Dye.

PURPOSE: To compare the detection rate (DR) and bilateral optimal mapping (OM) of sentinel lymph nodes (SLNs) in women with endometrial and cervical cancer using indocyanine green (ICG) versus the standard technetium-99m radiocolloid ((99m)Tc) radiotracer plus methylene or isosulfan blue, or blue dye alone. METHODS: From October 2010 to May 2015, 163 women with stage I endometrial or cervical cancer (118 endometrial and 45 cervical cancer) underwent SLN mapping with (99m)Tc with blue dye, blue dye alone, or ICG. DR and bilateral OM of ICG were compared respectively with the results obtained using the standard (99m)Tc radiotracer with blue dye, or blue dye alone. RESULTS: SLN mapping with (99m)Tc radiotracer with blue dye was performed on 77 of 163 women, 38 with blue dye only and 48 with ICG. The overall DR of SLN mapping was 97, 89, and 100 % for (99m)Tc with blue dye, blue dye alone, and ICG, respectively. The bilateral OM rate for ICG was 85 %-significantly higher than the 58 % obtained with (99m)Tc with blue dye (p = 0.003) and the 54 % for blue dye (p = 0.001). Thirty-one women (19 %) had positive SLNs. Sensitivity and negative predictive value of SLN were 100 % for all techniques. CONCLUSIONS: SLNs mapping using ICG demonstrated higher DR compared to other modalities. In addition, ICG was significantly superior to (99m)Tc with blue dye in terms of bilateral OM in women with early stage endometrial and cervical cancer. The higher number of bilateral OM may consequently reduce the overall number of complete lymphadenectomies, reducing the duration and additional costs of surgical treatment.

Quality of Care for Cervical and Endometrial Cancer Patients: The Impact of Different Techniques of Sentinel Lymph Node Mapping on Patient Satisfaction.

OBJECTIVES: The purpose of this study was to assess the quality of care in patients who underwent sentinel lymph node (SLN) mapping for endometrial and cervical cancer staging, and evaluate the impact of different techniques on patient satisfaction, i.e. radiotracer Tc99m versus indocyanine green (ICG) or methylene blue injection. METHOD: Women with preoperative stage I endometrial cancer or stage I (1A2-1B1) cervical cancer who underwent surgical staging, including SLN mapping, were considered for this study. Patient satisfaction was assessed using the European Organisation for Research and Treatment of Cancer IN-PATSAT32 questionnaire. Women were classified into two groups according to the different nodal mapping techniques: intracervical preoperative injection of Tc99m nanocolloid + intraoperative blue dye (Group 1) versus intraoperative cervical injection of ICG or blue dye (Group 2). Differences in patient satisfaction scores between the groups were analyzed. RESULTS: Of the 178 eligible women, 143 were included in the study (endometrial cancer n = 106, cervical cancer n = 37): 57 underwent SLN mapping with Tc99m and blue dye (Group 1), and 86 women were mapped intraoperatively with blue dye alone or ICG (Group 2). Analysis of IN-PATSAT32 questionnaire scores showed a higher patient satisfaction score for patients in Group 2 (p = 0.001), which was independent of the physician and surgical outcomes evaluated. The scores were statistically better for Group 2, and also in rating doctors (p = 0.0001), nurses (p = 0.006), and care and services organizations (p = 0.001). CONCLUSIONS: Cervical and endometrial cancer patients who underwent SLN mapping by ICG or blue dye perceived a better quality of care when compared with those patients who underwent the combined radiocolloid and blue dye technique.

NDP-α-MSH induces intense neurogenesis and cognitive recovery in Alzheimer transgenic mice through activation of melanocortin MC4 receptors.

Melanocortins exert neuroprotection in a variety of experimental neurodegenerative disorders, including Alzheimer's disease (AD). Further, in previous research we showed that these endogenous peptides stimulate neurogenesis in an acute neurodegenerative disorder such as ischemic stroke. In the present research, we investigated the potential neurogenic effect of melanocortins in AD using APPSwe transgenic mice (Tg2576). To this purpose, 24week-old animals were prepared for 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells on days 1-11 of the study. Treatment of Tg2576 mice with nanomolar doses of the melanocortin analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH), administered once daily from day 1 to 50, improved brain histology and cognitive functions relative to saline-treated Tg2576 animals. No signs of toxicity were observed. Immunohistochemical examination of the hippocampus at the end of the study (day 50) showed that NDP-α-MSH-treated Tg2576 mice had a greater number of BrdU immunoreactive cells colocalized with NeuN (an indicator of mature neurons) and Zif268 (an indicator of functionally integrated neurons) in the dentate gyrus, relative to saline-treated Tg2576 animals; no newly formed astrocytes were found. Animal pretreatment with the selective melanocortin MC4 receptor antagonist HS024 before each NDP-α-MSH administration prevented all the beneficial effects of the peptide. The present data indicate that MC4 receptor stimulation by a melanocortin prevents cognitive decline in experimental AD, this effect being associated not only with neuroprotection but also with an intense neurogenesis. MC4 receptor agonists could be innovative and safe candidates to counteract AD progression in humans.

Outcome of patients with stage I immature teratoma after surveillance or adjuvant chemotherapy.

Objective: Immature teratomas are rare malignant ovarian germ cell tumours, typically diagnosed in young women, where fertility-sparing surgery is the treatment of choice. The role of adjuvant chemotherapy in stage I disease remains controversial. We evaluated the impact of surveillance versus chemotherapy on the recurrence rate in stage I immature teratomas. Methods: We collected a single centre retrospective series of patients with stage I immature teratomas treated with fertility-sparing surgery at San Gerardo Hospital, Monza, Italy, between 1980 and 2019. Potential risk factors for recurrence were investigated by multivariate logistic regression. Results: Of the 74 patients included, 12% (9/74) received chemotherapy, while 88% (65/74) underwent surveillance. Median follow-up was 188 months. No difference in recurrence was found in stage IA/IB and IC immature teratomas [10% (6/60) vs. 28.6% (4/14) (P=0.087)], grade 1, grade 2, and grade 3 [7.1% (2/28) vs. 14.3% (4/28) vs. 22.2% (4/18) (p=0.39)], and surveillance versus chemotherapy groups [13.9% (9/65) vs. 11.1% (1/9)) (p = 1.00)]. In univariate analysis, the postoperative approach had no impact on recurrence. The 5-year disease-free survival was 87% and 90% in the surveillance and chemotherapy groups, respectively; the overall survival was 100% in both cohorts. Conclusions: Our results support the feasibility of surveillance in stage I immature teratomas. Adjuvant chemotherapy may be reserved for relapses. However, the potential benefit of chemotherapy should be discussed, especially for high-risk tumours. Prospective series are warranted to confirm our findings. What is already known on this topic: To date, no consensus has been reached regarding the role of adjuvant chemotherapy in stage I immature teratomas of the ovary. Some studies suggest that only surveillance is an acceptable choice. However, guidelines are not conclusive on this topic. What this study adds: No difference in terms of recurrence was observed between the surveillance and the adjuvant chemotherapy group. All patients who relapsed were successfully cured with no disease-related deaths. How this study might affect research practice or policy: Adjuvant chemotherapy should be appropriately discussed with patients. However, it may be reserved for relapse according to our data.

Identification of 4-amino-2-Pyridones as new potent PCSK9 inhibitors: From phenotypic hit discovery to in vivo tolerability.

Among the strategies to overcome the underperformance of statins in cardiovascular diseases (CVDs), the development of drugs targeting the Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9) is considered one of the most promising. However, only anti-PCSK9 biological drugs have been approved to date, and orally available small-molecules for the treatment of hypercholesterolemic conditions are still missing on the market. In the present work, we describe the application of a phenotypic approach to the identification and optimization of 4-amino-2-pyridone derivatives as a new chemotype with anti-PCSK9 activity. Starting from an in-house collection of compounds, functional assays on HepG2 cells followed by a chemistry-driven hit optimization campaign, led to the potent anti-PCSK9 candidate 5c. This compound, at 5 µM, totally blocked PCSK9 secretion from HepG2 cells, significantly increased LDL receptor (LDLR) expression, and acted cooperatively with simvastatin by reducing its induction of PCSK9 expression. Finally, compound 5c also proved to be well tolerated in C57BL/6J mice at the tested concentration (40 mg/kg) with no sign of toxicity or behavior modifications.

Melanocortin receptor 4 as a new target in melanoma therapy: Anticancer activity of the inhibitor ML00253764 alone and in association with B-raf inhibitor vemurafenib.

The aim of our study is to investigate in vitro and in vivo MC4R as a novel target in melanoma using the selective antagonist ML00253764 (ML) alone and in combination with vemurafenib, a B-rafV600E inhibitor. The human melanoma B-raf mutated A-2058 and WM 266-4 cell lines were used. An MC4R null A-2058 cell line was generated using a CRISPR/Cas9 system. MC4R protein expression was analysed by western blotting, immunohistochemistry, and immunofluorescence. Proliferation and apoptotic assays were performed with ML00253764, whereas the synergism with vemurafenib was evaluated by the combination index (CI) and Loewe methods. ERK1/2 phosphorylation and BCL-XL expression were quantified by western blot. In vivo experiments were performed in Athymic Nude-Foxn1nu male mice, injecting subcutaneously melanoma cells, and treating animals with ML, vemurafenib and their concomitant combination. Comet and cytome assays were performed. Our results show that human melanoma cell lines A-2058 and WM 266-4, and melanoma human tissue, express functional MC4R receptors on their surface. MC4R receptors on melanoma cells can be inhibited by the selective antagonist ML, causing antiproliferative and proapoptotic activity through the inhibition of phosphorylation of ERK1/2 and a reduction of BCL-XL. The concomitant combination of vemurafenib and ML caused a synergistic effect on melanoma cells in vitro and inhibited in vivo tumor growth in a preclinical model, without causing mouse weight loss or genotoxicity. Our original research contributes to the landscape of pharmacological treatments for melanoma, providing MC4R antagonists as drugs that can be added to established therapies.

Melanocortins as potential therapeutic agents in severe hypoxic conditions.

Melanocortin peptides with the adrenocorticotropin/melanocyte-stimulating hormone (ACTH/MSH) sequences and synthetic analogs have protective and life-saving effects in experimental conditions of circulatory shock, myocardial ischemia, ischemic stroke, traumatic brain injury, respiratory arrest, renal ischemia, intestinal ischemia and testicular ischemia, as well as in experimental heart transplantation. Moreover, melanocortins improve functional recovery and stimulate neurogenesis in experimental models of cerebral ischemia. These beneficial effects of ACTH/MSH-like peptides are mostly mediated by brain melanocortin MC(3)/MC(4) receptors, whose activation triggers protective pathways that counteract the main ischemia/reperfusion-related mechanisms of damage. Induction of signaling pathways and other molecular regulators of neural stem/progenitor cell proliferation, differentiation and integration seems to be the key mechanism of neurogenesis stimulation. Synthesis of stable and highly selective agonists at MC(3) and MC(4) receptors could provide the potential for development of a new class of drugs for a novel approach to management of severe ischemic diseases.

Hydrogen sulfide slows down progression of experimental Alzheimer's disease by targeting multiple pathophysiological mechanisms.

It has been previously reported that brain hydrogen sulfide (H2S) synthesis is severely decreased in Alzheimer's disease (AD) patients, and plasma H2S levels are negatively correlated with the severity of AD. Here we extensively investigated whether treatment with a H2S donor and spa-waters rich in H2S induces neuroprotection and slows down progression of AD. Studies with sodium hydrosulfide (a H2S donor) and Tabiano's spa-water were carried out in three experimental models of AD. Short-term and long-term treatments with sodium hydrosulfide and/or Tabiano's spa-water significantly protected against impairment in learning and memory in rat models of AD induced by brain injection of ß-amyloid1-40 (Aß) or streptozotocin, and in an AD mouse model harboring human transgenes APPSwe, PS1M146V and tauP301L (3xTg-AD mice). The improvement in behavioral performance was associated with hippocampus was size of Aß plaques and preservation of the morphological picture, as found in AD rats. Further, lowered concentration/phosphorylation levels of proteins thought to be the central events in AD pathophysiology, namely amyloid precursor protein, presenilin-1, Aß1-42 and tau phosphorylated at Thr181, Ser396 and Ser202, were detected in 3xTg-AD mice treated with spa-water. The excitotoxicity-triggered oxidative and nitrosative stress was counteracted in 3xTg-AD mice, as indicated by the decreased levels of malondialdehyde and nitrites in the cerebral cortex. Hippocampus reduced activity of c-jun N-terminal kinases, extracellular signal-regulated kinases and p38, which have an established role not only in phosphorylation of tau protein but also in inflammation and apoptosis, was also found. Consistently, decrease in tumor necrosis factor-α level, up-regulation of Bcl-2, and down-regulation of BAX and the downstream executioner caspase-3, also occurred in the hippocampus of 3xTg-AD mice after treatment with Tabiano's spa-water, thus suggesting that it is also able to modulate inflammation and apoptosis. Our findings indicate that appropriate treatments with H2S donors and Tabiano's spa-waters, and may be other spa-waters rich in H2S content, might represent an innovative approach to slow down AD progression in humans by targeting multiple pathophysiological mechanisms.