Asymmetric Synthesis of Spirooxindoles via Nucleophilic Epoxidation Promoted by Bifunctional Organocatalysts

Taking into account the postulated reaction mechanism for the organocatalytic epoxidation of electron-poor olefins developed by our laboratory, we have investigated the key factors able to positively influence the H-bond network installed inside the substrate/catalyst/oxidizing agent. With this aim, we have: (i) tested a few catalysts displaying various effects that noticeably differ in terms of steric hindrance and electron demand; (ii) employed α-alkylidene oxindoles decorated with different substituents on the aromatic ring (11a-g), the exocylic double bond (11h-l), and the amide moiety (11m-v). The observed results suggest that the modification of the electron-withdrawing group (EWG) weakly conditions the overall outcomes, and conversely a strong influence is unambiguously ascribable to either theN-protected orN-unprotected lactam framework. Specifically, when the NH free substrates (11m-u) are employed, an inversion of the stereochemical control is observed, while the introduction of a Boc protecting group affords the desired product12vin excellent enantioselectivity (97:3er)

Genetics and Epigenetics of Bone Remodeling and Metabolic Bone Diseases

Bone metabolism consists of a balance between bone formation and bone resorption, which is mediated by osteoblast and osteoclast activity, respectively. In order to ensure bone plasticity, the bone remodeling process needs to function properly. Mesenchymal stem cells differentiate into the osteoblast lineage by activating different signaling pathways, including transforming growth factor β (TGF-β)/bone morphogenic protein (BMP) and the Wingless/Int-1 (Wnt)/β-catenin pathways. Recent data indicate that bone remodeling processes are also epigenetically regulated by DNA methylation, histone post-translational modifications, and non-coding RNA expressions, such as micro-RNAs, long non-coding RNAs, and circular RNAs. Mutations and dysfunctions in pathways regulating the osteoblast differentiation might influence the bone remodeling process, ultimately leading to a large variety of metabolic bone diseases. In this review, we aim to summarize and describe the genetics and epigenetics of the bone remodeling process. Moreover, the current findings behind the genetics of metabolic bone diseases are also reporte

Prevalence of IgG antibodies against Malawi polyomavirus in patients with autoimmune diseases and lymphoproliferative disorders subjected to bone marrow transplantation

IntroductionHuman polyomaviruses (HPyVs) cause persistent/latent infections in a large fraction of the population. HPyV infections may cause severe diseases in immunocompromised patients. Malawi polyomavirus (MWPyV) is the 10th discovered human polyomavirus (HPyV 10). MWPyV was found in stool samples of healthy children. So far, the few investigations carried out on HPyV 10 did not find an association with human disease.MethodsIn this study, to verify the putative association between MWPyV and human diseases, MWPyV seroprevalence was investigated in patients affected by i) lymphoproliferative disorders (LPDs) and ii) immune system disorders, i.e., autoimmune diseases (ADs), and in iii) healthy subjects. An indirect ELISA, employing virus-like particles (VLPs) to detect serum IgG antibodies against MWPyV/HPyV 10, was carried out. The study also revealed the prevalence of another polyomavirus, Merkel cell polyomavirus (MCPyV).ResultsSera from patients with distinct autoimmune diseases (n = 44; mean age 20 years) had a prevalence of MWPyV antibodies of 68%, while in patients with lymphoproliferative disorders (n = 15; mean age 14 years), subjected to bone marrow transplantation, the prevalence was 47%. In healthy subjects (n = 66; mean age 13 years), the prevalence of MWPyV antibodies was 67%. Our immunological investigation indicates that MWPyV/HPyV 10 seroconversion occurs early in life and MWPyV/HPyV 10 appears to be another polyomavirus ubiquitous in the human population. A significantly lower MWPyV antibody reactivity together with a lower immunological profile was detected in the sera of LPD patients compared with HS2 (*p < 0.05) (Fisher’s exact test). LPD and AD patients have a similar MCPyV seroprevalence compared with healthy subjects.DiscussionMWPyV seroprevalence indicates that this HPyV is not associated with lymphoproliferative and autoimmune diseases. However, the ability to produce high levels of antibodies against MWPyV appears to be impaired in patients with lymphoproliferative disorders. Immunological investigations indicate that MWPyV seroconversion occurs early in life. MCPyV appears to be a ubiquitous polyomavirus, like other HPyVs, in the human population

Merkel cell polyomavirus, a small DNA tumour virus, in humans

Merkel cell polyomavirus (MCPyV) which causes an almost ubiquitous, asymptomatic infection, rarely causes an aggressive skin tumor with limited therapeutic options, i.e., Merkel cell carcinoma (MCC). Despite serum anti-MCPyV IgGs have been detected with a relatively high prevalence in healthy individuals, previous studies reported variable rates. Innovative immunoassays are needed to determine the MCPyV serology in humans. The main aim of the present thesis was to investigate the impact of oncogenic MCPyV infection in the healthy population. To this end, a new indirect ELISA assay using two synthetic peptides mimicking epitopes of MCPyV capsid proteins 1 and 2 (VP1/VP2) was developed and validated to analyze the presence of anti-MCPyV IgGs in sera from healthy individuals, including children (HC, 0-20 yrs), adults (HS, 21-65 yrs), and elderly (ES, 66-100 yrs). Synthetic peptides were initially computationally designed to detect IgGs to MCPyV mimotopes. The assay performance in detecting anti-MCPyV IgGs was afterwards determined on MCPyV-positive/-negative control sera. The assay showed a high performance. Then, the ELISA was extended to HC (n=344), HS (n=510) and ES (n=226) sera with unknown MCPyV serology. Age-/gender-specific MCPyV seroprevalences were examined. The overall prevalence of serum anti-MCPyV IgGs was 40.7% in HC. HC age stratification revealed a significantly lower MCPyV seroprevalence in the 0-5 yrs cohort (13%) compared with the older cohorts. MCPyV serological profile showed that the 0-5 yrs cohort had the lowest Optical densities (ODs) compared to the older cohorts. Higher ODs were found in females than males. The prevalence of anti-MCPyV IgMs was 29.7% in HC, with similar rates in age-stratified HC. The 0-5 yrs cohort had the lowest IgM ODs compared to the older cohorts. The 6-10/11-15 yrs cohorts had the highest ODs compared to the 16-20 yrs cohort. A higher seroprevalence of anti-MCPyV IgG and IgM were found in females than in males. The anti-MCPyV IgG and IgM reactivity patterns in HC were also simultaneously evaluated. IgG and IgM ODs showed an inverse trend, as being increased and decreased, respectively, in the older HC cohort. The prevalence of serum anti-MCPyV IgGs was 61.8% in HS group. Higher ODs were found in females than in males. MCPyV-seroprevalence of ES sera was 63.7%, a proportion higher than the 40.7% observed in HC, and similar to the 61.8% detected in the HS. MCPyV-serology was then examined in HC, HS, and ES, which were stratified in 5-yrs range cohorts. MCPyV seroprevalence was lower in the 0-5 yrs cohort (13%) than in the older cohorts (52.3-72%). Moreover, the 0-5 yrs cohort had the lowest OD values. Higher ODs were found in 6-10, 11-15 and 16-20 yrs cohorts compared to the other cohorts. The results of the present thesis indicate that the assay developed herein can efficiently detect serum anti-MCPyV IgGs. Our data indicate that MCPyV infection is widespread in humans. After seroconversion in pediatric age, MCPyV circulates asymptomatically in the prepuberal, adult and elder populations at a high prevalence, without substantial age-related variations in adulthood and senility. New approaches for MCC treatment are needed. In the second phase of this thesis, the antineoplastic effects of ATRA and decitabine drugs were evaluated in vitro in MCC cell lines, to develop new antitumor therapies with clinical application. The activities of the two drugs were assayed as single agents in MCC cell lines MCC13, MCC26, MKL-1 and Peta to verify their antiproliferative potential. The ATRA IC50-dose was 100 µM and 75 µM in MCC13 and Peta, respectively. ATRA showed no antiproliferative effects on MCC26. The decitabine IC50-dose was 0.5 μM in both MCC13 and MCC26, while it was 50 nM in MKL-1 cells. Both ATRA and decitabine effectively reduced the proliferation of MCC cells in vitro. These preliminary results may improve the development of new approaches for MCC therapy.Il poliomavirus delle cellule di Merkel (MCPyV) causa un'infezione asintomatica nell’uomo. In rari casi, MCPyV induce un tumore cutaneo aggressivo con limitate opzioni terapeutiche, il carcinoma delle cellule di Merkel (CCM). Nonostante le IgG anti-MCPyV siano state rilevate con una prevalenza relativamente alta negli individui sani, dati precedenti indicano tassi di prevalenza variabili. Sono quindi necessari nuovi saggi immunologici per determinare la sierologia MCPyV nell’uomo. Lo scopo di questa tesi era di indagare l'impatto dell'infezione da MCPyV nella popolazione sana. È stato dunque sviluppato e validato un nuovo test ELISA che impiega due peptidi sintetici che mimano gli epitopi delle proteine capsidiche di MCPyV (VP1/2) per valutare la presenza di IgG anti-MCPyV in sieri di soggetti sani, compresi bambini (HC, 0-20 anni), adulti (HS, 21-65 anni) e anziani (ES, 66-100 anni). I peptidi sono stati disegnati in silico per rilevare le IgG verso MCPyV. Il test è stato valutato in termini di performance sui sieri di controllo MCPyV-positivi/-negativi, mostrando prestazioni elevate. L'ELISA è stato esteso a sieri HC (n=344), HS (n=510) ed ES (n=226) con una sierologia per MCPyV ignota. La prevalenza delle IgG anti-MCPyV era del 40.7% negli HC. È stata inoltre valutata la prevalenza di MCPyV specifica per età/genere. La coorte 0-5 anni ha evidenziato una prevalenza di MCPyV più bassa rispetto alle altre coorti. Il profilo sierologico di MCPyV ha mostrato densità ottiche (OD) più basse nella coorte 0-5 anni rispetto alle altre coorti. OD più alte sono state trovate nelle femmine rispetto ai maschi. La prevalenza delle IgM anti-MCPyV nel gruppo HC è risultata essere del 29.7%, con tassi simili negli HC stratificati per età. La coorte 0-5 anni aveva le OD più basse rispetto alle altre coorti. Le coorti 6-10/11-15 anni avevano OD più alte della coorte 16-20 anni. Sono stati valutati simultaneamente i pattern sierici di IgG e IgM anti-MCPyV negli HC. Le OD di IgG e IgM hanno mostrato una tendenza inversa, con rispettivo aumento e diminuzione, nella coorte HC più adulta. La prevalenza delle IgG sieriche di MCPyV era del 61.8% negli HS. Le femmine hanno mostrato OD più alte dei maschi. La prevalenza delle IgG anti-MCPyV negli ES era del 63.7%; una percentuale superiore al 40.7% degli HC e simile al 61.8% degli HS. La sierologia di MCPyV è stata anche studiata in HC, HS ed ES, stratificati in coorti di 5 anni. La prevalenza di MCPyV era inferiore nella coorte 0-5 anni (13%) rispetto alle altre coorti (52.3-72%). Inoltre, la coorte 0-5 anni aveva i valori di OD più bassi. OD più elevate sono state trovate nelle coorti 6-10/11-15/16-20 anni rispetto alle altre coorti. I risultati di questa tesi indicano che l'ELISA indiretto qui sviluppato è affidabile nel rilevare IgG anti-MCPyV sieriche. I nostri dati immunologici rivelano che l'infezione da MCPyV è diffusa nell'uomo. Dopo la sieroconversione in età pediatrica, MCPyV circola senza sintomi nella popolazione prepuberale, adulta e anziana con un'elevata prevalenza, senza variazioni nell'età adulta e nella senilità. Sono necessari nuovi approcci per il trattamento del CCM. In una seconda fase della tesi, gli effetti antineoplastici di ATRA e decitabina sono stati valutati in vitro in linee cellulari di CCM, per sviluppare nuove terapie antitumorali con applicazione clinica. Le attività dei due farmaci sono state analizzate in linee cellulari MCC13, MCC26, MKL-1 e Peta per valutarne il potenziale antiproliferativo. La dose IC50 di ATRA era rispettivamente di 100 µM e 75 µM in MCC13 e Peta. ATRA non ha dato effetti sulle MCC26. La dose IC50 di decitabina era di 0.5 μM sia in MCC13 che in MCC26, mentre era di 50 nM nelle MKL-1. Sia ATRA che decitabina hanno ridotto la proliferazione delle cellule CCM in vitro. Questi risultati preliminari possono aprire la strada a nuovi approcci per la terapia CCM

Long Non-coding RNAs and MicroRNAs Interplay in Osteogenic Differentiation of Mesenchymal Stem Cells

Long non-coding RNAs (lncRNAs) have gained great attention as epigenetic regulators of gene expression in many tissues. Increasing evidence indicates that lncRNAs, together with microRNAs (miRNAs), play a pivotal role in osteogenesis. While miRNA action mechanism relies mainly on miRNA-mRNA interaction, resulting in suppressed expression, lncRNAs affect mRNA functionality through different activities, including interaction with miRNAs. Recent advances in RNA sequencing technology have improved knowledge into the molecular pathways regulated by the interaction of lncRNAs and miRNAs. This review reports on the recent knowledge of lncRNAs and miRNAs roles as key regulators of osteogenic differentiation. Specifically, we described herein the recent discoveries on lncRNA-miRNA crosstalk during the osteogenic differentiation of mesenchymal stem cells (MSCs) derived from bone marrow (BM), as well as from different other anatomical regions. The deep understanding of the connection between miRNAs and lncRNAs during the osteogenic differentiation will strongly improve knowledge into the molecular mechanisms of bone growth and development, ultimately leading to discover innovative diagnostic and therapeutic tools for osteogenic disorders and bone diseases

Probiotics Mechanism of Action on Immune Cells and Beneficial Effects on Human Health

Immune cells and commensal microbes in the human intestine constantly communicate with and react to each other in a stable environment in order to maintain healthy immune activities. Immune system-microbiota cross-talk relies on a complex network of pathways that sustain the balance between immune tolerance and immunogenicity. Probiotic bacteria can interact and stimulate intestinal immune cells and commensal microflora to modulate specific immune functions and immune homeostasis. Growing evidence shows that probiotic bacteria present important health-promoting and immunomodulatory properties. Thus, the use of probiotics might represent a promising approach for improving immune system activities. So far, few studies have been reported on the beneficial immune modulatory effect of probiotics. However, many others, which are mainly focused on their metabolic/nutritional properties, have been published. Therefore, the mechanisms behind the interaction between host immune cells and probiotics have only been partially described. The present review aims to collect and summarize the most recent scientific results and the resulting implications of how probiotic bacteria and immune cells interact to improve immune functions. Hence, a description of the currently known immunomodulatory mechanisms of probiotic bacteria in improving the host immune system is provided

Significantly low levels of IgG antibodies against oncogenic Merkel cell polyomavirus in sera from females affected by spontaneous abortion

Merkel cell polyomavirus (MCPyV) is a small DNA tumor virus being ubiquitous in humans. MCPyV establishes a clinically asymptomatic lifelong infection in healthy immunocompetent individuals. Viral infections are considered to be risk factors for spontaneous abortion (SA), which is the most common adverse complication of pregnancy. The role of MCPyV in SA remains undetermined. Herein, the impact of MCPyV infection in females affected by SA was investigated. Specifically, an indirect ELISA method with two linear synthetic peptides/mimotopes mimicking MCPyV antigens was used to investigate IgG antibodies against MCPyV in sera from 94 females affected by SA (mean [±SD] age 35±[6] years) and from 96 healthy females undergoing voluntary pregnancy interruption, (VI, mean [±SD] age 32±[7] years). MCPyV-seroprevalences and serological profiles were analysed. The overall prevalence of serum IgG antibodies against MCPyV was 34% (33/94) and 37.5% (36/96) in SA and VI females, respectively (P&gt;0.05). Notably, serological profile analyses indicated lower optical densities (ODs) in females with SA compared to those undergoing VI (p&lt;0.05), thus indicating a reduced IgG antibody response in SA females. Circulating IgGs were identified in sera from SA and VI females. Our immunological findings indicate that a relatively reduced fraction of pregnant females carry serum anti-MCPyV IgG antibodies, while SA females presented a more pronounced decrease in IgG antibody response to MCPyV. Although yet to be determined, this immunological decrease might prompt an increase in MCPyV multiplication events in females experiencing abortive events. The role of MCPyV in SA, if present, remains to be determined.Merkel cell polyomavirus (MCPyV) is a small DNA tumor virus ubiquitous in humans. MCPyV establishes a clinically asymptomatic lifelong infection in healthy immunocompetent individuals. Viral infections are considered to be risk factors for spontaneous abortion (SA), which is the most common adverse complication of pregnancy. The role of MCPyV in SA remains undetermined. Herein, the impact of MCPyV infection in females affected by SA was investigated. Specifically, an indirect enzyme-linked immunosorbent assay (ELISA) method with two linear synthetic peptides/mimotopes mimicking MCPyV antigens was used to investigate immunoglobulin G (IgG) antibodies against MCPyV in sera from 94 females affected by SA [mean ± standard deviation (SD) age 35 ± (6) years] and from 96 healthy females undergoing voluntary pregnancy interruption [VI, mean (±SD) age 32 ± (7) years]. MCPyV seroprevalence and serological profiles were analyzed. The overall prevalence of serum IgG antibodies against MCPyV was 35.1% (33/94) and 37.5% (36/96) in SA and VI females, respectively (p > 0.05). Notably, serological profile analyses indicated lower optical densities (ODs) in females with SA compared to those undergoing VI (p < 0.05), thus indicating a reduced IgG antibody response in SA females. Circulating IgGs were identified in sera from SA and VI females. Our immunological findings indicate that a relatively reduced fraction of pregnant females carry serum anti-MCPyV IgG antibodies, while SA females presented a more pronounced decrease in IgG antibody response to MCPyV. Although yet to be determined, this immunological decrease might prompt an increase in MCPyV multiplication events in females experiencing abortive events. The role of MCPyV in SA, if present, remains to be determined

The Role of Purinergic P2X7 Receptor in Inflammation and Cancer: Novel Molecular Insights and Clinical Applications

The purinergic P2X7 receptor (P2X7R) is a transmembrane protein whose expression has been related to a variety of cellular processes, while its dysregulation has been linked to inflammation and cancer. P2X7R is expressed in cancer and immune system cell surfaces. ATP plays a key role in numerous metabolic processes due to its abundance in the tumour microenvironment. P2X7R plays an important role in cancer by interacting with ATP. The unusual property of P2X7R is that stimulation with low doses of ATP causes the opening of a permeable channel for sodium, potassium, and calcium ions, whereas sustained stimulation with high doses of ATP favours the formation of a non-selective pore. The latter effect induces a change in intracellular homeostasis that leads to cell death. This evidence suggests that P2X7R has both pro- and anti-tumour proprieties. P2X7R is increasingly recognised as a regulator of inflammation. In this review, we aimed to describe the most relevant characteristics of P2X7R function, activation, and its ligands, while also summarising the role of P2X7R activation in the context of inflammation and cancer. The currently used therapeutic approaches and clinical trials of P2X7R modulators are also described

Bioactive Materials for Soft Tissue Repair

Over the past decades, age-related pathologies have increased abreast the aging population worldwide. The increased age of the population indicates that new tools, such as biomaterials/scaffolds for damaged tissues, which display high efficiency, effectively and in a limited period of time, for the regeneration of the body’s tissue are needed. Indeed, scaffolds can be used as templates for three-dimensional tissue growth in order to promote the tissue healing stimulating the body’s own regenerative mechanisms. In tissue engineering, several types of biomaterials are employed, such as bioceramics including calcium phosphates, bioactive glasses, and glass–ceramics. These scaffolds seem to have a high potential as biomaterials in regenerative medicine. In addition, in conjunction with other materials, such as polymers, ceramic scaffolds may be used to manufacture composite scaffolds characterized by high biocompatibility, mechanical efficiency and load-bearing capabilities that render these biomaterials suitable for regenerative medicine applications. Usually, bioceramics have been used to repair hard tissues, such as bone and dental defects. More recently, in the field of soft tissue engineering, this form of scaffold has also shown promising applications. Indeed, soft tissues are continuously exposed to damages, such as burns or mechanical traumas, tumors and degenerative pathology, and, thereby, thousands of people need remedial interventions such as biomaterials-based therapies. It is known that scaffolds can affect the ability to bind, proliferate and differentiate cells similar to those of autologous tissues. Therefore, it is important to investigate the interaction between bioceramics and somatic/stem cells derived from soft tissues in order to promote tissue healing. Biomimetic scaffolds are frequently employed as drug-delivery system using several therapeutic molecules to increase their biological performance, leading to ultimate products with innovative functionalities. This review provides an overview of essential requirements for soft tissue engineering biomaterials. Data on recent progresses of porous bioceramics and composites for tissue repair are also presented