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Environmental and anthropogenic factors may significantly affect the diffusion of wild animals, enhancing the interface of human–wildlife interactions and driving the spread of pathogens and vector-borne diseases between animals and humans. However, in the last decade, the involvement of citizens in scientific research (the so-called citizen science approach, henceforth abbreviated as CS) provided a network of large-scale and cost-effective surveillance programmes of wildlife populations and their related arthropod species. Therefore, this review aims to illustrate different methods and tools used in CS studies, by arguing the main advantages and considering the limitations of this approach. The CS approach has proven to be an effective method for establishing density and distribution of several wild animal species, in urban, peri-urban and rural environments, as well a source of information regarding vector–host associations between arthropods and wildlife. Extensive efforts are recommended to motivate citizens to be involved in scientific projects to improve both their and our knowledge of the ecology and diseases of wildlife. Following the One Health paradigm, collaborative and multidisciplinary models for the surveillance of wildlife and related arthropod species should be further developed by harnessing the potentiality of the CS approach.

IntroductionFollowing the increase of wild boar (Sus scrofa) populations in Europe, a potential risk of emerging infections by vector-borne pathogens may occur. Despite this, the circulation of piroplasmid species in these ungulates is still a neglected topic, particularly in the Mediterranean basin. Therefore, this study aimed to investigate the presence of Babesia/Theileria spp. in wild boars from southern Italy to assess the epidemiological role of these ungulates in the circulation of piroplasmids.MethodsBy using a citizen science approach among hunters and veterinarians, wild boar spleen samples were collected in the Campania region (southern Italy) between 2016 and 2022. A combined semi-nested PCR/sequencing analysis targeting the V4 hyper-variable region of 18S rRNA was run to detect Babesia/Theileria spp. DNA.ResultsOut of 243 boars, 15 (i.e., 6.2, 95% CI: 3.4–9.9) tested positive to Babesia/Theileria spp., Babesia vulpes (n = 13, 5.3, 95% CI: 3.1–8.9) the most prevalent, followed by Babesia capreoli (n = 2, 0.8, 95% CI: 0.2–2.9). Three different B. vulpes sequence types were identified (i.e., ST1, ST2, ST3), with the most representative as ST1 (60%), and a single B. capreoli sequence type. No statistically significant difference (p > 0.05) were found between the presence of the pathogens and boar age, sex, province and sample collection year.DiscussionData demonstrate for the first time the occurrence of B. vulpes and B. capreoli in wild boars, which may play a role in the biological cycle of piroplasmids. We emphasize the importance of monitoring these ungulates to prevent potential foci of infection. The engagement of hunters in epidemiological scientifically based surveys can constitute a technically sound control strategy of piroplasmids in a One Health perspective.

As a result of the increase of game meat intended for human consumption through Europe, a plethora of food-borne diseases, including trichinellosis, may occur in consumers, posing a relevant public health threat.Thus, this study aims to a citizen science approach to monitor the occurrence of Trichinella spp. in wild boar meat intended for human consumption, evaluating the risk of infection for consumers.Following the European Regulation 2015/1375 (laying down specific rules on official controls for Trichinella in meat), from 2015 to 2021, hunters (n = 478) were involved to collect diaphragm pillar samples of wild boars from mainland southern Italy, which were tested for Trichinella spp. L1 larvae via HCl-pepsin digestion and Multiplex PCR.Overall, 139,160 animals were collected (average of 19,880 per year), being 14 (i.e., 0.01%) tested positive to Trichinella britovi by the combined biochemical and molecular approach. An average larval burden of 28.4 L1 per gram of meat was found (minimum 3.2 - maximum 132.6). A statistically significant difference was found in the prevalence according to hunting seasons (p

Abstract Hepatitis C virus (HCV) is a major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma in humans. Humans were long considered the only hosts of Hepacivirus. Recently HCV-like sequences have been found in several animal species. Hepaciviruses are considered species-specific but a wider host range and a zoonotic role has been hypothesized. We report the first detection of bovine hepacivirus (BovHepV) sequences in wild boars. A total of 310 wild boars hunted in Campania region were investigated with a pan-hepacivirus nested-PCR protocol for the NS3 gene. Hepacivirus RNA was detected in 5.8% of the animals. Sequence and phylogenetic analysis showed high homology with BovHepV subtype F, with nucleotide identity of 99%. The positive wild boars were georeferenced, revealing high density of livestock farms, with no clear distinction between animal husbandry and hunting areas. These findings might suggest the ability of BovHepV to cross the host-species barrier and infect wild boars.

Systematic wildlife surveillance is important to aid the prevention of zoonotic infections that jeopardize human health and undermine biodiversity. Toxoplasma gondii is an opportunistic zoonotic protozoan that can infect all endothermic vertebrates, causing severe disease in immunocompromised humans and cases of congenital transmission. Humans can be infected by ingestion of raw meat containing bradyzoites or water contaminated by oocysts. In our study, we assessed the potential circulation of Toxoplasma gondii in wild mammals by performing surveillance in the Campania region (southern Italy) and surveyed its presence from 2020 to 2022 within the framework of the Regional Plans for Wildlife Surveillance. In detail, 211 individuals belonging to five wild mammals (wolf, fox, wild boar, badger, and roe deer) underwent necropsy and the organs were analyzed by real-time PCR for the detection of the parasite. Toxoplasma gondii was found in 21.8% (46/211) of the subjects examined. No statistically significant differences were noticed between the prevalence and the host’s trophic level or age, rejecting the hypotheses that Toxoplasma gondii will have a higher prevalence in top predators and adult individuals, respectively. Our work emphasized the high circulation of Toxoplasma gondii in wildlife and remarked on the critical role of anthropized areas where domestic cats and wildlife may come into contact, urging a systematic surveillance.

Abstract Pseudorabies virus (PRV) or suid herpesvirus 1 (SHV‐1) is the causative agent of Aujeszky's disease, a highly contagious viral infection which causes neurological fatal illness in mammals other than suids. Here we report a case of a young wolf (Canis lupus) of around 2 years found dead by a hunter in the province of Avellino, Campania Region. Necropsy showed pathological findings consistent with encephalitis and gastroenteritis. Organs were analysed by microbiological and molecular investigations following standard procedures to ascertain the possible cause of death. Real‐time PCR revealed the presence of PRV in the brain and of canine parvovirus 2b in organs like intestine, liver, brain, kidney and pancreas. Death probably occurred very shortly after SHV‐1 infection in an animal already weakened by parvovirosis.

Wild boars can be infected with several foodborne pathogens which may be transmitted to humans through the consumption of their meat, but currently, data of their prevalence are still limited. The present study aimed to evaluate the presence of enteric pathogens in wild boar meat samples killed in the Campania region. Twentyeight wild boar meat samples were analyzed for the detection of Salmonella spp, Y. enterocolitica, Campylobacter spp., and Shiga- Toxigenic E. coli. Salmonella spp. was detected and isolated in ten samples and after serotyping S. Veneziana, S. Kasenyi, S. Coeln, S. Manhattan, S. Thompson, and S. Stanleyville were identified. Twenty-one meat samples were found to be contaminated with Y. enterocolitica; in 6 samples the ystA and ystB genes were detected simultaneously, while in 15 only the ystB gene, which characterizes the bacteria belonging to the biotype 1A, was present. Shiga-Toxin producing E. coli was detected in 12 while Campylobacter spp was never detected. In conclusion, due to the high occurrence of pathogenic bacteria detected, the present research shows that wild boars are important reservoirs for foodborne zoonoses which may be transmitted to livestock and humans. This confirms the importance of controls throughout the wild boar supply chain. In the Campania region, checks are guaranteed by the Veterinarians who work within the “management and control plan for wild boar in the Campania region” which has the twofold objective of containing the increasingly invasive presence of this animal and guaranteeing greater safety, traceability, and transparency in the consumption of meat.

Ovine papillomaviruses (OaPVs) were detected and quantified, for the first time, using droplet digital polymerase chain reaction (ddPCR) and real-time quantitative PCR (qPCR) via blood samples of 165 clinically healthy sheep. OaPV DNA was detected in 126 blood samples (~76.4%). DdPCR detected OaPV DNA in 124 samples; in only two additional samples positive for real-time qPCR, ddPCR failed to detect the presence of any OaPVs. In 70 of the positive samples (~55.6%), a single OaPV infection was observed, 12 of which were caused by OaPV1 (~17.1%) and 14 by OaPV2 (20%). OaPV3 was responsible for 19 single infections (~27.1%), and OaPV4 for 25 single infections (~35.7%). Multiple OaPV coinfections were observed in 56 (~44.4%) positive samples. OaPV coinfections caused by two genotypes were observed in 31 positive samples (~55.4%), with dual OaPV3/OaPV4 infection being the most prevalent as seen in 11 blood samples. In addition, five OaPV1/OaPV4, four OaPV1/OaPV2, four OaPV2/OaPV3, four OaPV1/OaPV3, and three OaPV2/OaPV4 dual coinfections were also detected. OaPV coinfections by triple and quadruple genotypes were detected in 24 (~42.8%) and only one (~1.8%) of coinfected blood samples, respectively. Multiple infections caused by OaPV1/OaPV3/OaPV4 genotypes were the most prevalent, as observed in 12 (50%) blood samples harboring triple OaPV infections. This study showed that ddPCR is the most sensitive and accurate assay for OaPV detection and quantification thus outperforming real-time qPCR in terms of sensitivity and specificity. Therefore, ddPCR may represent the molecular diagnostic tool of choice, ultimately providing useful insights into OaPV molecular epidemiology and field surveillance.

A case of Mycobacterium bovis infection is described in a death adult female wild boar in the province of Avellino, Campania Region (Southern Italy). The carcass was sent to the Istituto Zooprofilattico Sperimentale del Mezzogiorno (IZSM) of Portici, Naples, Italy, where postmortem examination was performed. At necropsy, a disseminated granulomatous infection was observed, with involvement of various lymph node districts, spleen and lungs. Therefore, all lymph nodes were collected, together with spleen and lung lesions, in order to carry out bacteriological and molecular analyses that confirmed an uncommon disseminated Mycobacterium bovis infection. Subsequently, an analysis of the spoligotype, performed by the National Reference Center of Mycobacterium bovis in Brescia (Northern Italy), resulted in the spoligotype SB0134, previously identified in bovine outbreaks in the same area where the wild boar was found.

This study aims to investigate the presence of canine distemper virus (CDV) infection in 949 autochthonous or illegally imported dogs from Southern Italy, over a period of eight years (2014–2021). CDV RNA was detected in 6.8% (65/949) of the animals tested, with no detection of CDV in dogs sampled in 2020–2021. The frequency of CDV detection was higher in imported dogs (19/103, 18.3%) with respect to stray (27/365, 7.4%) and household dogs (19/481, 3.9%). On sequence and phylogenetic analyses of selected strains, the analyzed viruses belonged to the Arctic clade, which has already been reported in Italy and in Europe. The results of our study may suggest a reduction of CDV circulation in Southern Italy, while at the same time highlighting the need for strict controls on dog importation, in order to prevent the introduction of viruses from endemic countries.

Cystic Echinococcosis (CE) caused by Echinococcus granulosus sensu lato (s.l.) is one of the most important parasitic zoonotic diseases in the world and it represents an important public health and socio-economic concern. In the Mediterranean basin, CE is widespread and it is endemic in Italy, with major prevalence in southern areas. Several studies have investigated CE in domestic pigs, however, such data in wild boars are scant. In the last decades the wild boar population in Italy has increased and this ungulate could play an important role in the spreading of CE in the wild. Here we report on the prevalence and fertility rate of hydatid cysts in wild boars that were shot during two hunting seasons (2016–2017) in the Campania region of southern Italy.For each animal, a detailed inspection of the carcass and organs (lungs, liver and spleen) was performed and when cysts were found, their number, morphology and fertility were determined by visual and microscopic examination. Cysts were classified morphologically as fertile, sterile, caseous and calcified. Protoscoleces and germinal layers were collected from individual cysts and DNA was extracted to identify different strains/genotypes of E. granulosus s.l.Out of a total of 2108 wild boars 93 (4.4%) were found positive for CE. Infected animals were 45 males and 48 females, aged between 1 and 8 years. The average number of cysts per wild boar was 1.3 (min 1 - max 13). The total number of cysts collected was 123, of which 118 (95.9%) in the liver, 4 (3.3%) in the lungs and 1 (0.8%) in the spleen. Of all analyzed cysts, 70 (56.9%) were fertile and 53 (43.1%) sterile/acephalous. The presence of fertile cysts in 19.4% of CE-positive animals is noteworthy. Overall, molecular diagnosis showed 19 wild boars infected with the pig strain (G7). Keywords: Cystic echinococcosis, Wild boar, Genotyping, Drive hunting, Southern Italy

In recent years, porcine circovirus (PCV) infection has been documented as an important and emerging health concern for livestock and wildlife worldwide. The purpose of the present study was to assess the molecular prevalence of PCV-2 and PCV-3 and to clarify the epidemiological role of wild boars in the circulation of this virus in Campania, Southern Italy. For this purpose, samples from several organs were collected during the hunting season 2017–2018 from 148 wild boars in the Campania region. Quantitative real-time PCR was used for the detection and quantification of PCV-2 and PCV-3 genomes. The combined prevalence of PCV-2 and PCV-3 was 74.32% in the wild boars tested. The proportions of wild boars positive for PCV-2 or PCV-3, or coinfected, were 47.30%, 49.32%, and 22.30%, respectively. No link was detected between PCV positivity and location, but gender was a risk factor for the disease (female; p < 0.0001; OR 0.29). Furthermore, our study provides a snapshot of PCV-2 and PCV-3 circulation in wild boars in the Campania region: our findings can help us to better understand the role of wildlife in PCV circulation.

Aujeszky’s disease (AD, pseudorabies) is a viral disease of suids caused by Suid Herpesvirus 1 (SHV-1) also referred as Aujeszky’s disease virus (ADV) or Pseudorabies virus (ADV). Domestic pig and Wild boar (Sus scrofa) are the natural host, but many species can be infected with ADV. The aim of our study was to evaluate seroprevalence of AD in wild boar hunted in the Campania Region, during the 2016–2017 hunting season. A total of 503 serum samples from wild boars hunted in the provinces of Campania Region (Southern Italy) were collected and were tested for antibody against ADV using an AD, blocking ELISA assay. A Seroprevalence of 23.85% (120/503, 95% Confidence Interval (CI): 20.15–27.55) was found. Gender was not significantly associated with of ADV seropositivity (p > 0.05), while the presence of ADV antibodies was statistically associated with age (>36-month, p < 0.0001) and location (Avellino, p = 0.0161). Our prevalence values are like those obtained in 2010 in our laboratory (30.7%), demonstrating a constant circulation of ADV in the area.

Babesiosis is an emerging tick-borne disease caused by apicomplexan parasites with widespread geographical distribution and various wildlife species as reservoir hosts. The aims of this study were to investigate the prevalence and assess the role of free-ranging canids and mustelids in the maintenance of Babesia spp. in southern Italy. PCR analysis of splenic samples targeting the 18S rRNA gene revealed the presence of Babesia spp. in 36 of 82 (43.9%) red foxes (Vulpes vulpes) including 29 (58%) from Campania region and seven (21.8%) from Calabria region, in seven of 13 (53.8%) Eurasian badgers (Meles meles), and in one of 13 (7.7%) gray wolves (Canis lupus). Samples from other host species including 9 Eurasian otters (Lutra lutra), 1 stone marten (Martes foina), 1 least weasel (Mustela nivalis), and 1 European polecat (Mustela putorius) tested Babesia spp. negative. Sequence analysis of the 18S rRNA gene demonstrated the presence of B. vulpes in the red fox and two sequence types of badger-associated Babesia spp. in the Eurasian badger. The Babesia sp. sequence detected in the gray wolf was identical to a badger-associated Babesia sp. This study shows that the number of Babesia spp. infecting free-ranging carnivores in Italy is higher than currently believed, and suggests that these hosts may play an important role in the maintenance of the sylvatic cycle of these parasites. It is the first report of badger-associated Babesia spp. in Italy and in a gray wolf.

Wild animals are potential vectors of antibiotic-resistant bacteria in the environment. The present study aimed to investigate the occurrence of antimicrobial resistance among Salmonella serovars isolated from wildlife and the environment in Italy. A total of 164 Salmonella isolates were analyzed, and six different subspecies and 64 serovars were detected. High proportions of Salmonella isolates proved resistant to streptomycin (34.1%), followed by trimethoprim-sulfamethoxazole (23.2%), tetracycline (17.7%), ciprofloxacin (14.63%) and ampicillin (11.59%). By source, the lowest level of resistance was observed in Salmonella serovars isolated from a water environment, while antimicrobial resistance was frequent in strains collected from shellfish, reptiles and birds. Multidrug-resistant strains were recovered from seafood (n = 11), mammals (n = 3) and water (n = 1). Three S. Typhimurium monophasic variant strains showed asimultaneous resistance to ampicillin, streptomycin, tetracycline and trimethoprim-sulfamethoxazole, which represents a recognized alert resistance profile for this serovar. These data indicate the environmental dissemination of resistant strains due to anthropogenic activities, which, in southern Italy, probably have a higher impact on marine ecosystems than on terrestrial ones. Moreover, as most of the animals considered in the present study are usually consumed by humans, the presence of resistant bacteria in them is a matter of great concern.

Canine monocytic ehrlichiosis and rickettsiosis are zoonotic tick-borne diseases of canids caused by the intracellular obligate bacteria Ehrlichia canis and Rickettsia species respectively. In this study, we investigated using standard and real-time PCR and sequencing, the occurrence and molecular characterization of E. canis and Rickettsia species in the Eurasian otter (Lutra lutra) from the southern Italian population. Samples were screened by using molecular assays also for Neospora caninum, Toxoplasma gondii, Clamydophyla spp., Coxiella burnetii, Leishmania spp., Cryptosporidium spp., and Giardia spp. detection, and helminths were studied by traditional methods. Out of six carcasses tested, three were positive for E. canis and co-infection with Rickettsia sp. occurred in one of those. Sequences of the 16S rRNA E. canis gene were identical to each other but differed from most of those previously found in red foxes (Vulpes vulpes) and wolves (Canis lupus) from southern Italy. Helminths included just cystacanths of Sphaerirostris spp. from the intestine of two Eurasian otters and the nematode Angiostrongylus vasorum from the lungs of a single Eurasian otter. None of the samples was positive for the other investigated selected pathogens. This study is the first report on the evidence of infection by rickettsial pathogens in the Eurasian otter. The present result prompts some inquiries into the pathogenic role of those bacteria for the isolated sub-populations of the endangered Eurasian otter in southern Italy.

Abstract Osteosarcoma (OS) is the most prevalent and fatal type of bone tumor. It is characterized by great heterogeneity of genomic aberrations, mutated genes, and cell types contribution, making therapy and patients management particularly challenging. A unifying picture of molecular mechanisms underlying the disease could help to transform those challenges into opportunities. This review deeply explores the occurrence in OS of large-scale RNA regulatory networks, denominated “competing endogenous RNA network” (ceRNET), wherein different RNA biotypes, such as long non-coding RNAs, circular RNAs and mRNAs can functionally interact each other by competitively binding to shared microRNAs. Here, we discuss how the unbalancing of any network component can derail the entire circuit, driving OS onset and progression by impacting on cell proliferation, migration, invasion, tumor growth and metastasis, and even chemotherapeutic resistance, as distilled from many studies. Intriguingly, the aberrant expression of the networks components in OS cells can be triggered also by the surroundings, through cytokines and vesicles, with their bioactive cargo of proteins and non-coding RNAs, highlighting the relevance of tumor microenvironment. A comprehensive picture of RNA regulatory networks underlying OS could pave the way for the development of innovative RNA-targeted and RNA-based therapies and new diagnostic tools, also in the perspective of precision oncology.

Abstract Cells that are exposed to harmful genetic damage, either from internal or external sources, may undergo senescence if they are unable to repair their DNA. Senescence, characterized by a state of irreversible growth arrest, can spread to neighboring cells through a process known as the senescence-associated secretory phenotype (SASP). This phenomenon contributes to both aging and the development of cancer. The SASP comprises a variety of factors that regulate numerous functions, including the induction of secondary senescence, modulation of immune system activity, remodeling of the extracellular matrix, alteration of tissue structure, and promotion of cancer progression. Identifying key factors within the SASP is crucial for understanding the underlying mechanisms of senescence and developing effective strategies to counteract cellular senescence. Our research has specifically focused on investigating the role of IGFBP5, a component of the SASP observed in various experimental models and conditions. Through our studies, we have demonstrated that IGFBP5 actively contributes to promoting senescence and can induce senescence in neighboring cells. We have gained valuable insights into the mechanisms through which IGFBP5 exerts its pro-senescence effects. These mechanisms include its release following genotoxic stress, involvement in signaling pathways mediated by reactive oxygen species and prostaglandins, internalization via specialized structures called caveolae, and interaction with a specific protein known as RARα. By uncovering these mechanisms, we have advanced our understanding of the intricate role of IGFBP5 in the senescence process. The significance of IGFBP5 as a pro-aging factor stems from an in vivo study we conducted on patients undergoing Computer Tomography analysis. In these patients, we observed an elevation in circulating IGFBP5 levels in response to radiation-induced organismal stress. Globally, our findings highlight the potential of IGFBP5 as a promising therapeutic target for age-related diseases and cancer.

We compared the helminth communities of 5 owl species from Calabria (Italy) and evaluated the effect of phylogenetic and ecological factors on community structure. Two host taxonomic scales were considered, i.e., owl species, and owls vs. birds of prey. The latter scale was dealt with by comparing the data here obtained with that of birds of prey from the same locality and with those published previously on owls and birds of prey from Galicia (Spain). A total of 19 helminth taxa were found in owls from Calabria. Statistical comparison showed only marginal differences between scops owls (Otus scops) and little owls (Athene noctua) and tawny owls (Strix aluco). It would indicate that all owl species are exposed to a common pool of 'owl generalist' helminth taxa, with quantitative differences being determined by differences in diet within a range of prey relatively narrow. In contrast, birds of prey from the same region exhibited strong differences because they feed on different and wider spectra of prey. In Calabria, owls can be separated as a whole from birds of prey with regard to the structure of their helminth communities while in Galicia helminths of owls represent a subset of those of birds of prey. This difference is related to the occurrence in Calabria, but not Galicia, of a pool of 'owl specialist' species. The wide geographical occurrence of these taxa suggest that local conditions may determine fundamental differences in the composition of local communities. Finally, in both Calabria and Galicia, helminth communities from owls were species-poor compared to those from sympatric birds of prey. However, birds of prey appear to share a greater pool of specific helmith taxa derived from cospeciation processes, and a greater potential exchange of parasites between them than with owls because of phylogenetic closeness.

Cells and extracts derived from adipose tissue are gaining increasing attention not only in plastic surgery and for aesthetic purposes but also in regenerative medicine. The ability of hyaluronan (HA) to support human adipose stromal cell (hASC) viability and differentiation has been investigated. However, the compatibility of adipose tissue with HA-based formulation in terms of biophysical and rheological properties has not been fully addressed, although it is a key feature for tissue integration and in vivo performance. In this study, the biophysical and biochemical properties of highly concentrated (45 mg/mL) high/low-molecular-weight HA hybrid cooperative complex were assessed with a further focus on the potential application in adipose tissue augmentation/regeneration. Specifically, HA hybrid complex rheological behavior was observed in combination with different adipose tissue ratios, and hyaluronidase-catalyzed degradation was compared to that of a high-molecular-weight HA (HHA). Moreover, the HA hybrid complex’s ability to induce in vitro hASCs differentiation towards adipose phenotype was evaluated in comparison to HHA, performing Oil Red O staining and analyzing gene/protein expression of PPAR-γ, adiponectin, and leptin. Both treatments supported hASCs differentiation, with the HA hybrid complex showing better results. These outcomes may open new frontiers in regenerative medicine, supporting the injection of highly concentrated hybrid formulations in fat compartments, eventually enhancing residing staminal cell differentiation and improving cell/growth factor persistence towards tissue regeneration districts.

Abstract DNA damage resulting from genotoxic injury can initiate cellular senescence, a state characterized by alterations in cellular metabolism, lysosomal activity, and the secretion of factors collectively known as the senescence-associated secretory phenotype (SASP). Senescence can have beneficial effects on our bodies, such as anti-cancer properties, wound healing, and tissue development, which are attributed to the SASP produced by senescent cells in their intermediate stages. However, senescence can also promote cancer and aging, primarily due to the pro-inflammatory activity of SASP. Studying senescence is complex due to various factors involved. Genotoxic stimuli cause random damage to cellular macromolecules, leading to variations in the senescent phenotype from cell to cell, despite a shared program. Furthermore, senescence is a dynamic process that cannot be analyzed as a static endpoint, adding further complexity. Investigating SASP is particularly intriguing as it reveals how a senescence process triggered in a few cells can spread to many others, resulting in either positive or negative consequences for health. In our study, we conducted a meta-analysis of the protein content of SASP obtained from different research groups, including our own. We categorized the collected omic data based on: i) cell type, ii) harmful agent, and iii) senescence stage (early and late senescence). By employing Gene Ontology and Network analysis on the omic data, we identified common and specific features of different senescent phenotypes. This research has the potential to pave the way for the development of new senotherapeutic drugs aimed at combating the negative consequences associated with the senescence process. Video Abstract

Mesenchymal stromal cells (MSCs) are a heterogeneous population containing multipotent adult stem cells with a multi-lineage differentiation capacity, which differentiated into mesodermal derivatives. MSCs are employed for therapeutic purposes and several investigations have demonstrated that the positive effects of MSC transplants are due to the capacity of MSCs to modulate tissue homeostasis and repair via the activity of their secretome. Indeed, the MSC-derived secretomes are now an alternative strategy to cell transplantation due to their anti-inflammatory, anti-apoptotic, and regenerative effects. The cellular senescence is a dynamic process that leads to permanent cell cycle arrest, loss of healthy cells’ physiological functions and acquiring new activities, which are mainly accrued through the release of many factors, indicated as senescence-associated secretory phenotype (SASP). The senescence occurring in stem cells, such as those present in MSCs, may have detrimental effects on health since it can undermine tissue homeostasis and repair. The analysis of MSC secretome is important either for the MSC transplants and for the therapeutic use of secretome. Indeed, the secretome of MSCs, which is the main mechanism of their therapeutic activity, loses its beneficial functions and acquire negative pro-inflammatory and pro-aging activities when MSCs become senescent. When MSCs or their derivatives are planned to be used for therapeutic purposes, great attention must be paid to these changes. In this review, we analyzed changes occurring in MSC secretome following the switch from healthy to senescence status.

Although adult stem cells may be useful for studying tissue-specific diseases, they cannot be used as a general model for investigating human illnesses given their limited differentiation potential. Multilineage-differentiating stress-enduring (Muse) stem cells, a SSEA3(+) cell population isolated from mesenchymal stromal cells, fat, and skin fibroblasts, may be able to overcome that restriction. The Muse cells present in fibroblast cultures obtained from biopsies of patients’ skin may be differentiated into cells of interest for analyzing diseases. We isolated Muse stem cells from patients with an intellectual disability (ID) and mutations in the IQSEC2 gene (i.e., BRAG1 gene) and induced in vitro neuroglial differentiation to study cell commitment and the differentiation of neural lineages. The neuroglial differentiation of Muse cells revealed that IQSEC2 mutations may alter the self-renewal and lineage specification of stem cells. We observed a decrease in the percentage of SOX2 (+) neural stem cells and neural progenitors (i.e., SOX2+ and NESTIN+) in cultures obtained from Muse cells with the mutated IQSEC2 gene. The alteration in the number of stem cells and progenitors produced a bias toward the astrocytes’ differentiation. Our research demonstrates that Muse stem cells may represent a new cell-based disease model.

Abstract Background The term mesenchymal stromal cells (MSCs) designates an assorted cell population comprised of stem cells, progenitor cells, fibroblasts, and stromal cells. MSCs contribute to the homeostatic maintenance of many organs through paracrine and long-distance signaling. Tissue environment, in both physiological and pathological conditions, may affect the intercellular communication of MSCs. Methods We performed a secretome analysis of MSCs isolated from subcutaneous adipose tissue (sWAT) and visceral adipose tissue (vWAT), and from bone marrow (BM), of normal and obese mice. Results The MSCs isolated from tissues of healthy mice share a common core of released factors: components of cytoskeletal and extracellular structures; regulators of basic cellular functions, such as protein synthesis and degradation; modulators of endoplasmic reticulum stress; and counteracting oxidative stress. It can be hypothesized that MSC secretome beneficially affects target cells by the horizontal transfer of many released factors. Each type of MSC may exert specific signaling functions, which could be determined by looking at the many factors that are exclusively released from every MSC type. The vWAT-MSCs release factors that play a role in detoxification activity in response to toxic substances and drugs. The sWAT-MSC secretome contains proteins involved in in chondrogenesis, osteogenesis, and angiogenesis. Analysis of BM-MSC secretome revealed that these cells exert a signaling function by remodeling extracellular matrix structures, such as those containing glycosaminoglycans. Obesity status profoundly modified the secretome content of MSCs, impairing the above-described activity and promoting the release of inflammatory factors. Conclusion We demonstrated that the content of MSC secretomes depends on tissue microenvironment and that pathological condition may profoundly alter its composition. Video abstract

Two different types of adipose depots can be observed in mammals: white adipose tissue (WAT) and brown adipose tissue (BAT). The primary role of WAT is to deposit surplus energy in the form of triglycerides, along with many metabolic and hormonal activities; as thermogenic tissue, BAT has the distinct characteristic of using energy and glucose consumption as a strategy to maintain the core body temperature. Under specific stimuli—such as exercise, cold exposure, and drug treatment—white adipocytes can utilize their extraordinary flexibility to transdifferentiate into brown-like cells, called beige adipocytes, thereby acquiring new morphological and physiological characteristics. For this reason, the process is identified as the ‘browning of WAT’. We evaluated the ability of some drugs, including GW501516, sildenafil, and rosiglitazone, to induce the browning process of adult white adipocytes obtained from differentiated mesenchymal stromal cells (MSCs). In addition, we broadened our investigation by evaluating the potential browning capacity of IRISIN, a myokine that is stimulated by muscular exercises. Our data indicate that IRISIN was effective in promoting the browning of white adipocytes, which acquire increased expression of UCP1, increased mitochondrial mass, and modification in metabolism, as suggested by an increase of mitochondrial oxygen consumption, primarily in presence of glucose as a nutrient. These promising browning agents represent an appealing focus in the therapeutic approaches to counteracting metabolic diseases and their associated obesity.

Objective: In the present study we aim to evaluate whether some of the proteins previously identified in the secretome of senescent bone marrow (BM)- and adipose (A)-mesenchymal stromal cells (MSCs) could be involved in regulation of senescence phenomena. Materials and Methods: Among the identified proteins, we selected Filamin B (FLNB) and Aminopeptidase N (CD13) that were exclusively found in the secretome of senescent cells. We silenced their mRNA expression in BM-MSCs by means of RNA interference technology and induced acute senescence by hydrogen peroxide treatment. Our goal was to evaluate if FLNB or CD13 silencing may affect onset of senescence. Results: Our preliminary data showed that CD13 protein could be a key factor involved in the regulation and determination of both primary and paracrine induced senescence in human BM-MSCs. On the other hand, Filamin B seems not to be involved in the determination of primary senescence whereas its presence could be part of the mechanism that regulates the senescence induction in human BM-MSCs by paracrine signaling. Conclusion: Our study provides a useful base for identifying the complex extracellular protein networks involved in the regulation of MSC cellular senescence.

Mesenchymal stromal cells (MSCs) are currently used for cartilage cell therapy because of their well proven capacity to differentiate in chondrocytes. The advantage of MSC-based therapy is the possibility of producing a high number of chondrocytes for implants. The transplant procedure, however, has some limitations, since MSCs may produce non-functional chondrocytes. This limit has been challenged by cultivating MSC in media with hydrogels containing hyaluronic acid (HA), extractive chondroitin sulfate (CS), or bio-fermentative unsulphated chondroitin (BC) alone or in combination. Nevertheless, a clear study of the effect of glycosaminoglycans (GAGs) on chondrocyte differentiation is still lacking, especially for the newly obtained unsulfated chondroitin of biotechnological origin. Are these GAGs playing a role in the commitment of stem cells to chondrocyte progenitors and in the differentiation of progenitors to mature chondrocytes? Alternatively, do they have a role only in one of these biological processes? We evaluated the role of HA, CS, and – above all – BC in cell commitment and chondrocyte differentiation of MSCs by supplementing these GAGs in different phases of in vitro cultivation. Our data provided evidence that a combination of HA and CS or of HA and BC supplemented during the terminal in vitro differentiation and not during cell commitment of MSCs improved chondrocytes differentiation without the presence of fibrosis (reduced expression of Type I collagen). This result suggests that a careful evaluation of extracellular cues for chondrocyte differentiation is fundamental to obtaining a proper maturation process.

Senescent cells secrete several molecules, collectively named senescence-associated secretory phenotype (SASP). In the SASP of cells that became senescent following several in vitro chemical and physical stress, we identified the IGFBP-4 protein that can be considered a general stress mediator. This factor appeared to play a key role in senescence-paracrine signaling. We provided evidences showing that genotoxic injury, such as low dose irradiation, may promote an IGFBP-4 release in bloodstream both in mice irradiated with 100 mGy X-ray and in human subjects that received Computer Tomography. Increased level of circulating IGFBP-4 may be responsible of pro-aging effect. We found a significant increase of senescent cells in the lungs, heart, and kidneys of mice that were intraperitoneally injected with IGFBP-4 twice a week for two months. We then analyzed how genotoxic stressors may promote the release of IGFBP-4 and the molecular pathways associated with the induction of senescence by this protein.

Managing medical procedures for children with problematic disorders is a challenging approach, especially in the case of blood withdrawal for autism spectrum disorder-affected children. Peripheral blood mononuclear cells (PBMC) represent an important cellular model to study immune responses and drug toxicity. The monocytic cells, a fraction of PBMC, are strongly involved in some pathophysiological processes, such as inflammation and immune system changes. Here, we propose a simple, reliable protocol for obtaining peripheral blood-derived mononuclear cells from small volumes of blood samples.

Rett syndrome: Mutation affects neural stem cells In Rett syndrome, neural stem cells lose some of their “stem cell like” properties, impairing brain functions. Patients with this rare neurological condition, almost exclusively girls, show impaired movement and speech beginning at 6–18 months of age. Mutations in the MECP2 gene are known to be involved, but the specifics are poorly understood. Umberto Galderisi at Temple University in Philadelphia and co-workers in Italy used a mouse model to trace how MECP2 mutations affect neural stem cells. They found that the mutated cells lost key stem cell abilities, including the capacity to renew themselves by dividing, and the ability to differentiate, or turn into other cell types. The cells were also highly susceptible to DNA damage and unable to repair it. These results improve our understanding of Rett syndrome and may help develop new treatments.

Several investigations on senescence and its causative role in aging have underscored the importance of developing senotherapeutics, a field focused on killing senescent cells and/or preventing their accumulation within tissues. Using polyphenols in counteracting senescence may facilitate the development of senotherapeutics given their presence in the human diet, their confirmed tolerability and absence of severe side effects, and their role in preventing senescence and inducing the death of senescent cells. Against that background, we evaluated the effect of piceatannol, a natural polyphenol, on the senescence of mesenchymal stromal cells (MSCs), which play a key role in the body’s homeostasis. Among our results, piceatannol reduced the number of senescent cells both after genotoxic stress that induced acute senescence and in senescent replicative cultures. Such senotherapeutics activity, moreover, promoted the recovery of cell proliferation and the stemness properties of MSCs. Altogether, our findings demonstrate piceatannol’s effectiveness in counteracting senescence by targeting its associated pathways and detecting and affecting P53-dependent and P53-independent senescence. Our study thus suggests that, given piceatannol’s various mechanisms to accomplish its pleiotropic activities, it may be able to counteract any senescent phenotypes.

Although mice models rank among the most widely used tools for understanding human genetics, biology, and diseases, differences between orthologous genes among species as close as mammals are possible, particularly in orthologous gene pairs in which one or more paralogous (i.e., duplicated) genes appear in the genomes of the species. Duplicated genes can possess overlapping functions and compensate for each other. The retinoblastoma gene family demonstrates typical composite functionality in its three member genes (i.e., RB1, RB2/P130, and P107), all of which participate in controlling the cell cycle and associated phenomena, including proliferation, quiescence, apoptosis, senescence, and cell differentiation. We analyzed the role of the retinoblastoma gene family in regulating senescence in mice and humans. Silencing experiments with each member of the gene family in mesenchymal stromal cells (MSCs) and fibroblasts from mouse and human tissues demonstrated that RB1 may be indispensable for senescence in mouse cells, but not in human ones, as an example of species specificity. Furthermore, although RB2/P130 seems to be implicated in maintaining human cell senescence, the function of RB1 within any given species might differ by cell type, as an example of cell specificity. For instance, silencing RB1 in mouse fibroblasts induced a reduced senescence not observed in mouse MSCs. Our findings could be useful as a general paradigm of cautions to take when inferring the role of human genes analyzed in animal studies and when examining the role of the retinoblastoma gene family in detail.

During their life span, cells have two possible states: a non-cycling, quiescent state (G0) and a cycling, activated state. Cells may enter a reversible G0 state of quiescence or, alternatively, they may undergo an irreversible G0 state. The latter may be a physiological differentiation or, following a stress event, a senescent status. Discrimination among the several G0 states represents a significant investigation, since quiescence, differentiation, and senescence are progressive phenomena with intermediate transitional stages. We used the expression of Ki67, RPS6, and beta-galactosidase to identify healthy cells that progressively enter and leave quiescence through G0-entry, G0 and G0-alert states. We then evaluated how cells may enter senescence following a genotoxic stressful event. We identified an initial stress stage with the expression of beta-galactosidase and Ki67 proliferation marker. Cells may recover from stress events or become senescent passing through early and late senescence states. Discrimination between quiescence and senescence was based on the expression of RPS6, a marker of active protein synthesis that is present in senescent cells but absent in quiescent cells. Even taking into account that fixed G0 states do not exist, our molecular algorithm may represent a method for identifying turning points of G0 transitional states that continuously change.

The cells present in the stromal compartment of many tissues are a heterogeneous population containing stem cells, progenitor cells, fibroblasts, and other stromal cells. A SSEA3(+) cell subpopulation isolated from human stromal compartments showed stem cell properties. These cells, known as multilineage-differentiating stress-enduring (MUSE) cells, are capable of resisting stress and possess an excellent ability to repair DNA damage. We isolated MUSE cells from different mouse stromal compartments, such as those present in bone marrow, subcutaneous white adipose tissue, and ear connective tissue. These cells showed overlapping in vitro biological properties. The mouse MUSE cells were positive for stemness markers such as SOX2, OCT3/4, and NANOG. They also expressed TERT, the catalytic telomerase subunit. The mouse MUSE cells showed spontaneous commitment to differentiation in meso/ecto/endodermal derivatives. The demonstration that multilineage stem cells can be isolated from an animal model, such as the mouse, could offer a valid alternative to the use of other stem cells for disease studies and envisage of cellular therapies.

The bioactive form of vitamin D, 1,25-dihydroxyvitamin D (1,25D3), exerts immunomodulatory actions resulting in neuroprotective effects potentially useful against neurodegenerative and autoimmune diseases. In fact, vitamin D deficiency status has been correlated with painful manifestations associated with different pathological conditions. In this study, we have investigated the effects of vitamin D deficiency on microglia cells, as they represent the main immune cells responsible for early defense at central nervous system (CNS), including chronic pain states. For this purpose, we have employed a model of low vitamin D intake during gestation to evaluate possible changes in primary microglia cells obtained from postnatal day(P)2-3 pups. Afterwards, pain measurement and microglia morphological analysis in the spinal cord level and in brain regions involved in the integration of pain perception were performed in the parents subjected to vitamin D restriction. In cultured microglia, we detected a reactive—activated and proliferative—phenotype associated with intracellular reactive oxygen species (ROS) generation. Oxidative stress was closely correlated with the extent of DNA damage and increased β-galactosidase (B-gal) activity. Interestingly, the incubation with 25D3 or 1,25D3 or palmitoylethanolamide, an endogenous ligand of peroxisome proliferator-activated-receptor-alpha (PPAR-α), reduced most of these effects. Morphological analysis of ex-vivo microglia obtained from vitamin-D-deficient adult mice revealed an increased number of activated microglia in the spinal cord, while in the brain microglia appeared in a dystrophic phenotype. Remarkably, activated (spinal) or dystrophic (brain) microglia were detected in a prominent manner in females. Our data indicate that vitamin D deficiency produces profound modifications in microglia, suggesting a possible role of these cells in the sensorial dysfunctions associated with hypovitaminosis D.

Brown-like adipocytes can be induced in white fat depots by a different environmental or drug stimuli, known as “browning” or “beiging”. These brite adipocytes express thermogenin UCP1 protein and show different metabolic advantages, such as the ability to acquire a thermogenic phenotype corresponding to standard brown adipocytes that counteracts obesity. In this research, we evaluated the effects of several browning agents during white adipocyte differentiation of bone marrow-derived mesenchymal stromal cells (MSCs). Our in vitro findings identified two compounds that may warrant further in vivo investigation as possible anti-obesity drugs. We found that rosiglitazone and sildenafil are the most promising drug candidates for a browning treatment of obesity. These drugs are already available on the market for treating diabetes and erectile dysfunction, respectively. Thus, their off-label use may be contemplated, but it must be emphasized that some severe side effects are associated with use of these drugs.

Understanding the mechanisms by which mesenchymal stromal cells (MSCs) interact with the physical properties (e.g. topography, charge, ζ-potential, and contact angle) of polymeric surfaces is essential to design new biomaterials capable of regulating stem cell behavior. The present study investigated the ability of two polymers (pHM1 and pHM3) with different positive surface charge densities to modulate the differentiation of MSCs into osteoblast-like phenotype via cell-cell ephrinB2/EphB4 signaling. Although pHM1 promoted the phosphorylation of EphB4, leading to cell differentiation, pHM3, characterized by a high positive surface charge density, had no significant effect on EphB4 activation or MSCs differentiation. When the MSCs were cultured on pHM1 in the presence of a forward signaling blocking peptide, the osteoblast differentiation was compromised. Our results demonstrated that the ephrinB2/EphB4 interaction was required for MSCs differentiation into an osteoblast-like phenotype and that the presence of a high positive surface charge density altered this interaction.

Stem cell biology represents a challenging research area with a huge potential translational approach. This review focuses on the most recent findings on stem cell basics and clinics in several fields of research, as final outcome of the 10th conference held by Stem Cell Research Italy (SCR Italy) in Naples, Italy in June 2019. Current state-of-the-art and novel findings on stem cell research are discussed, bringing together basic and applied research with the newest insights in stem cell therapy.

The study explored the anti-hypertrophic effect of the melanocortin MC5R stimulation in H9c2 cardiac myocytes exposed to high glucose. This has been done by using α-MSH and selective MC5R agonists and assessing the expression of GLUT4 and GLUT1 transporters, miR-133 and urotensin receptor levels as a marker of cardiac hypertrophy. The study shows for the first time an up-regulation of MC5R expression levels in H9c2 cardiomyocytes exposed to high glucose medium (33 mM D-glucose) for 48 h, compared to cells grown in normal glucose medium (5.5 mM D-glucose). Moreover, H9c2 cells exposed to high glucose showed a significant reduction in cell viability (-40%), a significant increase in total protein per cell number (+109%), and an increase of the urotensin receptor expression levels as an evidence of cells hypertrophy. The pharmacological stimulation of MC5R with α-MSH (90 pM)of the high glucose exposed H9c2 cells increased the cell survival (+50,8%) and reduced the total protein per cell number (-28,2%) with respect to high glucose alone, confirming a reduction of the hypertrophic state as per cell area measurement. Similarly, PG-901 (selective agonist, 10-10 M) significantly increased cell viability (+61,0 %) and reduced total protein per cell number (-40,2%), compared to cells exposed to high glucose alone. Interestingly, the MC5R agonist reduced the GLUT1/GLUT4 glucose transporters ratio on the cell membranes exhibited by the hypertrophic H9c2 cells and increased the intracellular PI3K activity, mediated by a decrease of the levels of the miRNA miR-133a. The beneficial effects of MC5R agonism on the cardiac hypertrophy caused by high glucose was also observed also by echocardiographic evaluations of rats made diabetics with streptozotocin (65 mg/kg i.p.). Therefore, the melanocortin MC5R could be a new target for the treatment of high glucose-induced hypertrophy of the cardiac H9c2 cells.

Chromatin modifiers play a crucial role in maintaining cell identity through modulation of gene expression patterns. Their deregulation can have profound effects on cell fate and functions. Among epigenetic regulators, the MECP2 protein is particularly attractive. Mutations in the Mecp2 gene are responsible for more than 90% of cases of Rett syndrome (RTT), a progressive neurodevelopmental disorder. As a chromatin modulator, MECP2 can have a key role in the government of stem cell biology. Previously, we showed that deregulated MECP2 expression triggers senescence in mesenchymal stromal cells (MSCs) from (RTT) patients. Over the last few decades, it has emerged that senescent cells show alterations in the metabolic state. Metabolic changes related to stem cell senescence are particularly detrimental, since they contribute to the exhaustion of stem cell compartments, which in turn determine the falling in tissue renewal and functionality. Herein, we dissect the role of impaired MECP2 function in triggering senescence along with other senescence-related aspects, such as metabolism, in MSCs from a mouse model of RTT. We found that MECP2 deficiencies lead to senescence and impaired mitochondrial energy production. Our results support the idea that an alteration in mitochondria metabolic functions could play an important role in the pathogenesis of RTT.