Deciphering inflammation and immunomodulation in host-parasite interface: caco-2 cells and human intestinal organoids response to Anisakis’ messengers of pathogenicity

Background: Anisakiasis is a zoonosis caused by consumption of raw fish parasitized with Anisakis spp third stage larvae (L3). Even if humans are accidental hosts, only one L3 is sufficient to induce mild to severe symptoms. In fact, larval migration through the gastrointestinal tract, the excreted/secreted products and extracellular vesicles (EVs) can progressively determine allergic reactions, erosive ulcerous lesions, granulomas and chronic inflammation (Audicana et al., 2008 Clin Microbiol Rev. 21,360). Furthermore, reports regarding patients affected by gastric and intestinal tumors in co-occurrence with anisakiasis are increasing from endemic countries, suggesting exposure as a risk factor (Sonoda et al., 2015 Surg Today. 45, 1321; Murata et al., 2018 BMC Infect Dis. 4;619; Kita et al., 2019 Int J Surg Case Rep. 60, 209). Despite its relevance for public health, investigations on host inflammatory and immune response during anisakiasis are still very scarce. To date, studies aimed to understanding the messengers of pathogenicity, based on in vitro models able to recount the microenvironment in which the parasite acts during infection in humans, are at their infancy (Speciale et al., 2017 Parasitol Res. 116, 2471; Carballeda-Sangiao et al., 2020 PLoS Negl Trop Dis. 14, e0008462). Objective: the general aim of this PhD thesis is to investigate Anisakis-human interactions, focusing on human host inflammatory response and recreating the microenvironment in which Anisakis act. To achieve this goal, the project has been structured in two sections: i) the gene expression and the amounts of the pro-inflammatory cytokines (IL-6, IL-8) were analyzed to investigate the inflammatory pathway in in-vitro human epithelial colorectal adenocarcinoma cells (Caco-2) exposed to the live L3, the crude extract (CE) and the extracellular vesicles (EVs), as representative of the mechanical action of larval motility, whole body of senescent larvae and inflammation silencing, respectively. ii) expanding the knowledge on Anisakis EVs effects on human interface, using a bi or three-dimensional cutting-edge model capable of exhibiting the architecture and functionality of the organ of origin: the human intestinal organoids (HIO). In particular, the gene expression of three cytokines of interest was analyzed (Il33, Il1β, Il8). Methods: a total of 855 L3 were collected from fishes from selected areas (FAO37) and species identification was carried out through PCR-RFLP. CE were analyzed using Qubit and EVs were isolated through commercial kit and characterized using western blot and Nanoparticle tracking analyses. The gene expression (Caco-2 cells check-points: 1h, 6h, 24h; HIO checkpoint: 48h) and amounts of the pro-inflammatory cytokines (IL-6, IL-8) were analyzed by qRT-PCR and ELISA, respectively. Results: ELISA tests on Caco-2+L3 showed a progressive decrease of IL-6 (P<0.01) and IL-8 if compared to controls. Interestingly, no signals were obtained for IL-6 in Caco-2+EVs (P<0.01) and IL-8 was downregulated (P<0.01). Contrariwise, CE induced a strongly increased secretion of IL-6 (P<0.01) and a decreasing trend was observed for IL-8. Real-time PCR results on Caco-2+L3, CE and EVs suggested a detectable early effect on cytokines expression (1h). RT-PCR carried out on HIO exposed to EVs revealed a decreasing trend in Il33 gene expression and a slight increase in Il1β, a dynamic possibly linked to helminth infection chronicity, while Il8 gene expression seemed to be not affected. Conclusion: This study represents the first attempt to decipher the Anisakis EVs effects on human host (for both the models used) and results obtained showed an intricate host-parasite interplay, characterized by an early phase where the active L3 and its released EVs modulate the immune response to find a long-lasting niche to survive, and a second phase where L3 senescence may induce host immune response activation, leading to the granuloma formation

TP53 isoforms and effect of genetic polymorphisms on the internal promoter activity

P53 tumour suppressor protein plays a key role in maintaining genomic integrity. For 25 years, the p53 was considered the only protein expressed by TP53 gene. Since 2002, several groups have identified and described the existence of up to 10 p53 isoforms. It is now clear that the patterns of p53 expression are much more complex than previously recognized and that these isoforms have the potential to act either synergistically or antagonistically, depending on their structure and mechanism of production. This thesis focuses on the ΔNp53 Isoforms Δ133p53 and Δ40p53 to elucidate their specific properties on p53 suppressive activity as well as their expression in different normal and tumour tissues. We confirmed that ΔNp53 are dominant-negative toward full-length p53 (FL-p53) inhibiting p53-mediated apoptosis, thereby modulating the FL-p53 transcriptional activity and the cell fate outcome in response to stress. Moreover, isoform ΔN133p53 is produced by an internal promoter region (IPR) encompassing introns three and four. Interestingly, case-control studies showed that some polymorphisms within the IPR are associated with the susceptibility to several types of cancer. We sequenced the IPR in 47 human cell lines and eight polymorphisms were found (-/PIN3; IVS3 -29C>A; Pro36Pro; Arg72Pro; IVS4 -283C>T; IVS4 -125T>C; IVS4 -117A>G, IVS4 -91A>G). The haplotypes were reconstructed and their functional effect was examined with a reporter gene expression assay transfecting HeLa and 293T human cell lines. Our study suggests a role for the different haplotypes of the IPR in affecting the expression of the Δ133p53 isoform in both these types of cells. Quantitative PCR experiments performed on human normal tissues appeared to confirm the findings obtained in vitro. These results could help to explain the regulation of Δ133p53 expression and why some polymorphisms in TP53 are associated with the risk of cancer for humans

Increased age and male sex are independently associated with higher frequency of blood–cerebrospinal fluid barrier dysfunction using the albumin quotient

Background: The cerebrospinal fluid (CSF)/serum quotient of albumin (QAlb) is the most used biomarker for the evaluation of blood–cerebrospinal fluid barrier (B-CSF-B) permeability. For years QAlb was considered only as an age-related parameter but recently it has also been associated to sex. The aim of the present study was to explore the impact of sex in the determination of B-CSF-B dysfunction. Methods: The analysis was retrospectively conducted on subjects consecutively admitted to the neurological ward. CSF and serum albumin levels were measured by immunonephelometry and pathological QAlb thresholds were considered: 6.5 under 40 years, 8.0 in the age 40–60 and 9.0 over 60 years. Results: 1209 subjects were included in the study. 718 females and 491 males (age: 15–88 years): 24.6% of patients had a diagnosis of multiple sclerosis, 23.2% suffered from other inflammatory neurological diseases, 24.6% were affected by non-inflammatory neurological diseases, and for 27.6% of patients the final neurological diagnosis could not be traced. Dysfunctional B-CSF-B was detected more frequently (44 vs. 20.1%, p < 0.0001) and median QAlb value were higher (7.18 vs. 4.87, p < 0.0001) in males than in females in the overall study population and in all disease sub- groups. QAlb and age were positively correlated both in female (p < 0.0001) and male (p < 0.0001) patients, however the slopes of the two regression lines were not significantly different (p = 0.7149), while the difference between the elevations was extremely significant (p < 0.0001) with a gap of 2.2 units between the two sexes. Finally, in a multivari- able linear regression analysis increased age and male sex were independently associated with higher QAlb in the overall study population (both p < 0.001) and after stratification by age and disease group. Conclusions: Accordingly, identification and validation of sex-targeted QAlb thresholds should be considered as a novel tool in an effort to achieve more precision in the medical approach

Self-healing CD30- T-clonal proliferation of the tongue: report of an extremely rare case

The etiology of traumatic ulcerative granulomas with stromal eosinophilia (TUGSE) is not clear, traumatic irritation having advocated as the most likely cause. TUGSEs are typically self-limiting slow-healing lesions of the oral mucosa with unclear pathogenesis, commonly manifesting as a rapidly developing, long-lasting ulcer

Biological Aspects, Advancements and Techno-Economical Evaluation of Biological Methanation for the Recycling and Valorization of CO2

Nowadays, sustainable and renewable energy production is a global priority. Over the past decade, several Power-to-X (PtX) technologies have been proposed to store and convert the surplus of renewable energies into chemical bonds of chemicals produced by different processes. CO2 is a major contributor to climate change, yet it is also an undervalued source of carbon that could be recycled and represents an opportunity to generate renewable energy. In this context, PtX technologies would allow for CO2 valorization into renewable fuels while reducing greenhouse gas (GHG) emissions. With this work we want to provide an up-to-date overview of biomethanation as a PtX technology by considering the biological aspects and the main parameters affecting its application and scalability at an industrial level. Particular attention will be paid to the concept of CO2-streams valorization and to the integration of the process with renewable energies. Aspects related to new promising technologies such as in situ, ex situ, hybrid biomethanation and the concept of underground methanation will be discussed, also in connection with recent application cases. Furthermore, the technical and economic feasibility will be critically analyzed to highlight current options and limitations for implementing a sustainable process

Intraoperative β-Detecting probe for radio-guided surgery in tumour resection

The development of the β− based radio-guided surgery aims to extend the technique to those tumours where surgery is the only possible treatment and the assessment of the resection would most profit from the low background around the lesion, as for brain tumours. Feasibility studies on meningioma and gliomas already estimated the potentiality of this new treatment. To validate the technique, a prototype of the intraoperative probe detecting β− decays and specific phantoms simulating tumour remnant patterns embedded in healthy tissue have been realized. The response of the probe in this simulated environment is tested with dedicated procedures. This document discusses the innovative aspects of the method, the status of the developed intraoperative β− detecting probe and the results of the preclinical tests

Stress granules induced by oxidative stress in cultured fibroblast from TDP-43 mutant ALS patients

Stress granules (SGs) are transient cytoplasmic aggregates that rapidly form when cells are exposed to stress and consist of large messenger ribonucleoprotein (mRNPs) complexes. The SGs seem to function as storage depots for translation silenced complex and are implicated in stress-induced inhibition of global protein synthesis. Protein aggregation, has been observed in several neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS). The protein TDP-43 (TAR DNA-Binding Protein-43), encoded by one of the ALS-causative gene (TARDBP), is a major constituent of pathological inclusions in this disease and it is seems to be implicated in the regulation of SGs. Therefore we investigated the different characteristics of SGs in human cultured fibroblasts from ALS patients carrying TARDBPA382T mutation (group 1) versus healthy subjects (group 2). The cells were exposed to stressful conditions using sodium arsenite (SA) at different concentrations (0.5 mM, 1 mM) and exposure times (30 min, 1h). Preliminary results showed, after 30 minutes, small and sporadic cytoplasmic inclusions immunostained for TIA-1(T-cell internal antigen-1), an early marker for SGs, in both groups of cells. After 1h, the TIA-1 immunostained granules were bright, copious and scattered into the cytosol. Interestingly, we observed a significantly higher number of cells exhibiting SGs in fibroblasts from healthy controls (66%) compared to ALS patients (34%). In parallel, we identified the RNA binding protein HuR-1 (Human antigen R) in a fraction of Tia-1 positive SGs, as well as TDP- 43 localized into the nucleus of all the cells. These data raise the possibility that TDP-43 may modulate the stress granule formation, contributing to the cellular response to acute stress. Moreover the TDP-43 may regulate gene expression as well as cellular recovery and survival, and consequently its mutation may contribute to the neurodegeneration

Poly(L-glutamic acid)-co-poly(ethylene glycol) block copolymers for protein conjugation

Poly(L-glutamic acid)-co-poly(ethylene glycol) block copolymers (PLE-PEG) are here investigated as polymers for conjugation to therapeutic proteins such as granulocyte colony stimulating factor (G-CSF) and human growth hormone (hGH). PLE-PEG block copolymers are able to stabilize and protect proteins from degradation and to prolong their residence time in the blood stream, features that are made possible thanks to PEG's intrinsic properties and the simultaneous presence of the biodegradable anionic PLE moiety. When PLE-PEG copolymers are selectively tethered to the N-terminus of G-CSF and hGH, they yield homogeneous monoconjugates that preserve the protein's secondary structure. During the current study the pharmacokinetics of PLE10-PEG20k-G-CSF and PLE20-PEG20k-G-CSF derivatives and their ability to induce granulopoiesis were, respectively, assessed in Sprague-Dawley rats and in C57BL6 mice. Our results show that the bioavailability and bioactivity of the derivatives are comparable to or better than those of PEG20k-Nter-G-CSF (commercially known as Pegfilgrastim). The therapeutic effects of PLE10-PEG20k-hGH and PLE20-PEG20k-hGH derivatives tested in hypophysectomized rats demonstrate that the presence of a negatively charged PLE block enhances the biological properties of the conjugates additionally with respect to PEG20k-Nter-hGH

“Innovative high pressure/high temperature, multi-sensing bioreactors system for microbial risk assessment in underground hydrogen storage”

This study addresses the microbial risks associated with Underground Hydrogen Storage (UHS), a critical component in the transition towards renewable energy systems, by employing an innovative multi-reactor system (Bio-xplorer) to simulate UHS conditions in two Italian reservoirs. The microbiological risk assessment (MRA) of Reservoir A and B was evaluated by subjecting them to gas mixtures of 10 % H2 and 90 % CH4, and 99 % H2 and 1 % CO2, respectively. In Reservoir A, the stability of pressure and temperature, the negligible optical density, and lack of microbial metabolites suggested a low risk of microbial activation. Molecular analyses confirmed the absence of sulphate- reducing bacteria (SRB) and limited growth of hydrogenotrophic methanogens (HM). Similarly, in Reservoir B, the absence of SRB and limited occurrence of HM indicated a low microbiological risk. Overall, the present work supports the safe and efficient implementation of UHS, a promising mitigation technique for climate change, using an innovative tool for MRA

Towards a Radio-guided Surgery with β−\beta^{-} Decays: Uptake of a somatostatin analogue (DOTATOC) in Meningioma and High Grade Glioma

A novel radio guided surgery (RGS) technique for cerebral tumors using $\beta^{-}$ radiation is being developed. Checking the availability of a radio-tracer that can deliver a $\beta^{-}$ emitter to the tumor is a fundamental step in the deployment of such technique. This paper reports a study of the uptake of 90Y labeled (DOTATOC) in the meningioma and the high grade glioma (HGG) and a feasibility study of the RGS technique in these cases.Comment: 21 pages, 5 figure