In vitro analysis of Porcine Endogenous Retroviruses in different pig cell types.

The shortage of organs for human transplantation is a topic of extreme interest, and xenotransplantation with porcine organs has been recognized as a promising solution. However, the potential spillover linked to infectious agents present in pigs remains a concern. Among these, Pig Endogenous Retroviruses (PERVs), whose proviral DNAs are integrated in the genome of all pig breeds, represent an extremely important biological risk. This study aims to evaluate PERVs distribution in several swine cell lines and samples of domestic and feral pigs. Moreover, the capacity of PERVs to infect human and non-human primate cells and to integrate in the cellular genome was tested by Real-Time PCR and by Reverse Transcriptase assay. Results indicated a widespread diffusion of PERVs both in cell lines and samples analysed: the viral genome was found in all the established cell lines, in 40% of the primary cell lines and in 60% of the tissue samples tested. The assays indicated that the virus can be transmitted from porcine to human cells: in the specific case, infected NSK and NPTr cells allow passage to human 293 and MRC-5 cells with active production of the virus demonstrable via PCR and RT assay. In light of these aspects and also the lack of studies on PERVs, it appears clear that there are still many questions to be clarified, also by means of future studies, before xenotransplantation can be considered microbiologically safe.

Muscle-invasive bladder cancer in elderly and frail people: Is hypofractionated radiotherapy a feasible approach when no other local options are available?

AIM: The study aims to report the feasibility and safety of palliative hypofractionated radiotherapy targeting macroscopic bladder tumors in a monocentric cohort of frail and elderly bladder cancer patients not eligible for curative treatments. METHODS: Patients who underwent hypofractionated radiotherapy to the gross disease or to the tumor bed after transurethral resection of bladder tumor from 2017 to 2021 at the European Institute of Oncology IRCCS, were retrospectively considered. Schedules of treatment were 30 and 25 Gy in 5 fractions (both every other day, and consecutive days). Treatment response was evaluated with radiological investigation and/or cystoscopy. Toxicity assessment was carried out according to RTOG/EORTC v2.0 criteria. RESULTS: A total of 16 patients were included in the study, of these 11 received hypofractionated radiotherapy on the macroscopic target volume and five on the tumor bed after transurethral resection of bladder tumor. No grade (G) >2 acute toxicities were described after treatment for both groups. Only one patient in the group receiving radiotherapy on the macroscopic disease reported G4 GU late toxicity. Ten patients had available follow-up status (median FU time 18 months), of them six had complete response, one had stable disease, and three had progression of disease. The overall response rate and disease control rate were 60% and 70%, respectively. CONCLUSION: Our preliminary data demonstrate that palliative hypofractionated radiotherapy for bladder cancer in a frail and elderly population is technically feasible, with an acceptable toxicity profile. These outcomes emphasize the potential of this approach in a non-radical setting and could help to provide more solid indications in this underrepresented setting of patients.

Investigating Nutritional and Inflammatory Status as Predictive Biomarkers in Oligoreccurent Prostate Cancer-A RADIOSA Trial Preliminary Analysis.

(1) Background: In the RADIOSA phase II randomized clinical trial (NCT03940235), the biology task entails the identification of predictive and prognostic biomarkers in the context of oligorecurrent, castration-sensitive prostate cancer in order to distinguish polymetastatic from oligometastatic disease. This may lay the groundwork for personalized treatments for those patients who could really benefit from metastasis-directed therapies. (2) Methods: Oligorecurrent PCa pts with three or fewer bone or lymph nodal localizations were randomized 1:1 to receive SBRT alone (arm A) or SBRT + 6 months of ADT (arm B). Common serum-derived biomarkers were collected at baseline, and at 3 months after RT. The prognostic nutritional index, an immune and nutrition-based prognostic score, and the controlling nutritional status (CONUT) score, a scoring system for evaluating patient's nutritional status, were calculated in accordance with the body of available literature. As inflammatory indicators, neutrophil-lymphocyte ratio (NLR) and the NLR-albumin ratio (NLRAR) were assessed. Changes in these parameters between baseline and the 3-month timepoint were evaluated both in absolute and relative values. Changes in these parameters between baseline and the 3-month timepoint were evaluated. Significant differences in the trend of these parameters were assessed using the non-parametric Wilcoxon rank-sum test. A network analysis to analyze the relationships between different features stratifying patients according to the arm of study and site of metastases was performed. (3) Results: The current analysis comprised 88 patients (45 arm A, SBRT only, and 43 arm B, SBRT + ADT). When patients were stratified by ADT administration, cholesterol values showed an increasing trend in the group receiving ADT (p = 0.005) which was no longer significant at 1 year. When patients were stratified by site of metastases (52 lymph nodal, 29 bone localizations), the value of NLR was found to be increased in patients with bone localizations (p < 0.05). In addition, the network analysis showed that BMI and NRI are strongly and directly linked for patients at baseline and that this correlation is no longer found at three months. Finally, when patients were divided according to time from surgery to oligorecurrence (enrollment) the patients with a longer time (>6.7 years) showed an increase in CONUT score from baseline. All the other nutritional and inflammatory scores or parameters investigated in the present analysis showed no statistically significant differences at baseline, three months, 1 year, and in absolute change. (4) Conclusions: The nutritional and inflammatory parameters do not seem to represent valuable candidates for possible use in clinical decision making in our cohort of patients and a reliable biological characterization of the oligometastatic state in prostate cancer still seems far from being achieved. Ongoing molecular analysis will show if there is a role of mutational landscape in the definition of the oligometastatic state.

3D Cell Cultures: Evolution of an Ancient Tool for New Applications.

Recently, research is undergoing a drastic change in the application of the animal model as a unique investigation strategy, considering an alternative approach for the development of science for the future. Although conventional monolayer cell cultures represent an established and widely used in vitro method, the lack of tissue architecture and the complexity of such a model fails to inform true biological processes in vivo. Recent advances in cell culture techniques have revolutionized in vitro culture tools for biomedical research by creating powerful three-dimensional (3D) models to recapitulate cell heterogeneity, structure and functions of primary tissues. These models also bridge the gap between traditional two-dimensional (2D) single-layer cultures and animal models. 3D culture systems allow researchers to recreate human organs and diseases in one dish and thus holds great promise for many applications such as regenerative medicine, drug discovery, precision medicine, and cancer research, and gene expression studies. Bioengineering has made an important contribution in the context of 3D systems using scaffolds that help mimic the microenvironments in which cells naturally reside, supporting the mechanical, physical and biochemical requirements for cellular growth and function. We therefore speak of models based on organoids, bioreactors, organ-on-a-chip up to bioprinting and each of these systems provides its own advantages and applications. All of these techniques prove to be excellent candidates for the development of alternative methods for animal testing, as well as revolutionizing cell culture technology. 3D systems will therefore be able to provide new ideas for the study of cellular interactions both in basic and more specialized research, in compliance with the 3R principle. In this review, we provide a comparison of 2D cell culture with 3D cell culture, provide details of some of the different 3D culture techniques currently available by discussing their strengths as well as their potential applications.

Engineered nanoparticles toxicity on adipose tissue derived mesenchymal stem cells: A preliminary investigation.

Nanoscience and nanotechnologies have recently gained importance in several fields, such as industry and medicine. A big issue of the increasing application of nanomaterials is the poor literature regarding their potential toxicity in humans and animals. Recently, adult stem cells have been proposed as putative targets of nanoparticles (NPs). This study aims to investigate the effects of zerovalent-metallic NPs on isolated and amplified equine Adipose tissue derived Mesenchymal Stem Cells (eAdMSCs). Cells were treated with Cobalt (Co-), Iron (Fe-), and Nickel (Ni-) nanoparticles (NPs) at different concentrations and were characterized for the cytotoxic and genotoxic effects of exposure. Treatment with NPs resulted in reduced cell viability and proliferative capability in comparison with untreated cells. However, this did not influence eAdMSCs potency, as treated cells were able to differentiate towards the adipogenic and osteogenic lineages. Ni- and Fe-NPs showed cytoplasmic localization, while Co-NPs entered the nucleus and mitochondria, suggesting a potential genotoxic activity. Regarding p53 expression, it was enhanced in the first 48 h after treatments, with a drastic reduction of expression within 72 h. Higher p53 expression was reported in the case of Co-NP treatment, suggesting the tumorigenic potential of these NPs. Telomerase activity was enhanced by Fe- and Ni-NP treatments in a concentration- and time-dependent way. This was not true for Co-NP treated samples, suggesting a reduced replicative capacity of eAdMSCs upon Co-NP exposure. The present study is a preliminary investigation of the influence exerted by NPs on eAdMSC physiological activity in terms of cytotoxic and genotoxic effects. The present results revealed eAdMSC physiology to be strongly influenced by NPs in a dose-, time- and NP-dependent way.

PARP Inhibitors in Combination with Radiotherapy: To Do or Not to Do?

BACKGROUND: Despite the large use of inhibitors of Poly-ADP ribose polymerase (PARP-I), the feasibility and safety of their combination with radiotherapy (RT) is unclear. AIM: We conducted a literature analysis with the aim to evaluate the efficacy and safety profile of a combination with RT and PARP-I. METHOD: The key issues for the current review were expressed in two questions according to the Population, Intervention, Control, Outcome (PICO) criteria: 1. What is the outcome and 2. What is the toxicity in patients treated with a combination of PARP-I and RT for a newly diagnosed or recurrent tumors? RESULTS: A total of 12 clinical studies met the inclusion criteria including seven single-arm dose-escalation phase I studies, two phase II (two- and three-arms controlled trials) trials, one parallel-arm phase I study, and two phase I/II studies published between 2015 and 2021. RT was performed with photon beams and several schedules according to the clinical situation. The acute toxicity ≥ grade 3 ranged between 25% and >96%, which was divided into hematological or non-hematological adverse events. CONCLUSIONS: despite the heterogeneity of the evaluated patient populations and tumor types, and the limited number of the studies, this review suggests that a combination approach is feasible even though the efficacy profile remains unclear.

Toxicity of carbon ion radiotherapy and immune checkpoint inhibitors in advanced melanoma.

We analyzed CTCAE adverse events of sequential Carbon Ion radiotherapy (CIRT) and immune checkpoint inhibitors (ICIs) in advanced melanoma patients. The frequencies of early and late adverse events (AEs) were 100% and 82% of patients, respectively. The frequency of G3+ AEs was in line with the literature.

Veterinary Regenerative Medicine for Musculoskeletal Disorders: Can Mesenchymal Stem/Stromal Cells and Their Secretome Be the New Frontier?

Regenerative medicine aims to restore the normal function of diseased or damaged cells, tissues, and organs using a set of different approaches, including cell-based therapies. In the veterinary field, regenerative medicine is strongly related to the use of mesenchymal stromal cells (MSCs), which belong to the body repair system and are defined as multipotent progenitor cells, able to self-replicate and to differentiate into different cell types. This review aims to take stock of what is known about the MSCs and their use in the veterinary medicine focusing on clinical reports on dogs and horses in musculoskeletal diseases, a research field extensively reported in the literature data. Finally, a perspective regarding the use of the secretome and/or extracellular vesicles (EVs) in the veterinary field to replace parental MSCs is provided. The pharmaceuticalization of EVs is wished due to the realization of a Good Manufacturing Practice (GMP product suitable for clinical trials.

Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication.

The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field.

LptA assembles into rod-like oligomers involving disorder-to-order transitions.

LptA is a periplasmic protein involved in the transport of lipopolysaccharide (LPS) from the inner membrane (IM) to the outer membrane (OM) of Gram-negative bacteria. Growing evidence supports a model in which LptA assembles into oligomers, forming a physical bridge connecting IM and OM. This work investigates assembly and architecture of LptA oligomers. Circular dichroism and "native" electrospray-ionization ion-mobility mass spectrometry (ESI-IM-MS) are employed to test concentration dependence of LptA structural features and to analyze the morphology of higher-order aggregates. The results show that LptA progressively assembles into rod-like oligomers without fixed stoichiometry, and grows by an n + 1 mechanism up to at least the pentamer. The oligomerization process induces disorder-to-order transitions in the polypeptide chain. Comparison with crystallographic and computational data suggests that these conformational changes likely involve short disordered regions at the N- and C-termini of monomeric LptA. The protein response to thermal denaturation displays strong concentration dependence, indicating that oligomerization increases protein stability. LptA conformational stability can also be enhanced by in vitro LPS binding. The genesis of these fibrillar structures could be relevant for the correct transport of LPS across the bacterial periplasm.

IPEC-J2 cells as reporter system of the anti-inflammatory control actions of interferon-alpha.

Interferon-alpha (IFN-α) shows potent immunomodulatory properties, which underlies its use for low-dose oral treatments of diverse viral infections and immunopathological conditions. The studies on oral administration have been hampered by the lack of recognized in vitro models, reproducing the in vivo control action of IFN-α over inflammatory cytokine responses. Owing to these reasons, the aim of our study was to validate IPEC-J2 (a continuous cell line of porcine intestinal epithelial cells) as a reporter system of the properties of IFN-α. Three different experimental conditions (oxidative stress, inflammatory response, and amplification of lymphoid cell signals) were selected to evaluate the effects of porcine recombinant IFN-α1 (rIFN-α) and 2 natural porcine IFN-α preparations (nIFN-α). The IFNs under study showed significantly different control actions in IPEC-J2 cells. In particular, rIFN-α was shown to down-regulate interleukin (IL)-8, IL-1ß, tumor necrosis factor (TNF)-α, and ß-defensin 1 genes either directly, or indirectly through second messengers released by IFN-α-treated lymphoid cells. With regard to IL-6, only second messengers from IFN-α-treated lymphoid cells could regulate the expression of this cytokine. Our results suggest that IPEC-J2 cells can be a useful tool for investigating the regulatory actions of type I IFNs and the second messengers thereof. The results provided by this model could be conveniently exploited in studies on enteric diseases sustained by infectious or noninfectious stressors.

Characterization of the interferon-α response of pigs to the weaning stress.

The interferon (IFN)-α response of pigs to the stressing event of early weaning was investigated in a field trial. All the animals under study remained healthy and tested negative for common viral infections. However, a low-titered IFN-α response was detected in many sera by a bioassay on Madin-Darby bovine kidney (MDBK) cells on day +6 after weaning. Porcine IFN-α was unambiguously identified by a neutralization assay on a pool of IFN-α-positive sera. By gel filtration chromatography, the antiviral activity of sera on MDBK cells could be traced back to 3 components of apparent molecular mass 27/18/<14 kDa. Additional components of apparent molecular mass 58 and 41 kDa were revealed by ELISA in Nonidet P-40 lysates of peripheral blood mononuclear cells (PBMC). Also, many pigs tested positive in flow cytometry assays on PBMC for intracellular IFN-α. The expression of porcine IFN-α genes was investigated by reverse transcriptase (RT) real-time polymerase chain reaction at days -1, +6, and +12 with regard to weaning in PBMC of 9 piglets. On days -1 and +12, IFN A5, A6, A12, as well as (in fewer pigs) A1, A7, A11, and A2 genes were shown to be expressed. On the contrary, none of the above genes was expressed on day +6, when plenty of pig sera were IFN-α-positive. Our results indicate that weaning causes the release of IFN-α and the transient shut-off of the corresponding gene transcriptions in PBMC. Interestingly, only IFN A9 gene transcription was shown in vitro to be virus induction-dependent.

Components of the E. coli envelope are affected by and can react to protein over-production in the cytoplasm.

BACKGROUND: Protein over-expression in bacteria is still the easiest, cheapest and therefore preferred way to obtain large amounts of proteins for industrial and laboratory scale preparations. Several studies emphasized the importance of understanding cellular and molecular mechanisms triggered by protein over-production in order to obtain higher yield and better quality of the recombinant product. Almost every step leading to a fully functional polypeptide has been investigated, from mRNA stability to the role of molecular chaperones, from aggregation to bottlenecks in the secretory pathway. In this context, we focused on the still poorly addressed relationship between protein production in the cytoplasm and the bacterial envelope, an active and reactive cell compartment that controls interactions with the environment and several major cellular processes. Results available to date show that the accumulation of foreign proteins in the cytoplasm induces changes in the membrane lipids and in the levels of mRNAs for some membrane proteins. However, a direct connection between membrane protein expression levels and soluble/aggregated protein accumulation in the cytoplasm has never been reported. RESULTS: By the use of a combined physiological and proteomic approach, we investigated the effects on the cell membrane of E. coli of the overexpression of two recombinant proteins, the B. cepacia lipase (BCL) and the green fluorescent protein (GFP). Both polypeptides are expressed in the cytoplasm at similar levels but GFP is fully soluble whereas inactive BCL accumulates in inclusion bodies.Growth and viability of the transformed cells were tested in the presence of different drugs. We found that chloramphenycol preferentially inhibited the strain over-producing GFP while SDS was more effective when BCL inclusion bodies accumulated in the cytoplasm. In contrast, both proteins induced a similar response in the membrane proteome, i.e. increased levels of LamB, OmpF, OmpA and TolC. Under all tested conditions, the lipopolysaccharide was not affected, suggesting that a specific rather than a generalized rearrangement of the envelope was induced. CONCLUSION: Taking together physiological and biochemical evidence, our work indicates that the E. coli envelope can sense protein over-expression in the cytoplasm and react by modulating the abundance of some membrane proteins, with possible consequences on the membrane traffic of small solutes, i.e. nutrients, drugs and metabolites. Such a response seems to be independent on the nature of the protein being over-expressed. On the other hand both our data reported herein and previous results indicate that membrane lipids may act as a second stress sensor responsive to the aggregation state of the recombinant protein and further contribute to changes in cellular exchanges with the environment.