In vitro study of the UV-filter homosalate effects on rat and human thyroid cells.

Homosalate is a UV-B filter, commonly used in sunscreens and personal-care products. Homosalate was shown to exert estrogenic and anti-androgenic effects in animal models, while few data are available on the effects of Homosalate on thyroid cells. The aim of this study was to evaluate if Homosalate exposure could exert adverse effect on thyroid cells in vitro. FRTL-5 and NHT were treated with increasing concentration of Homosalate for 24-48-72 h. Cell viability was assessed by WST-1. Cell proliferation was evaluated by cristal violet. Micronucleus staining was performed to assess genotoxicity. mRNA levels of thyroid-related genes (TSHR, TPO, TG, NIS, and PAX8) were evaluated by RT-PCR. Changes in ROS production by FRTL-5 and NHT were assessed with H2DCFDA. Homosalate significantly reduced cell viability after 72 h in FRTL-5 starting from the concentration 250 µM, while in NHT, Homosalate exposure significantly reduced cell viability after 48 and 72 h only at highest concentration (2000 µM). Cell proliferation was not modified by Homosalate at any concentration and time-point. Homosalate significantly up-regulated mRNA expression levels of TPO and Tg genes in FRTL-5, while a significant increase only in Tg mRNA expression was observed in NHT. No changes in ROS production was found in both cell types. The present study suggest that the effects of Homosalate exposure may differ according to the type of cell tested. The in vitro exposure of thyroid cells to Homosalate produces: i) cytotoxicity at high concentrations or after long time of incubation, ii) genotoxicity only in rat thyroid cells at the highest concentration, iii) upregulation of Tg mRNA in both thyroid cell types and of TPO mRNA in rat thyroid cells, iv) no changes in cell proliferation or oxidative stress. Further studies on the effects of Homosalate on thyroid cells should be encouraged.

Treatment with proton pump inhibitors is associated with secondary bacterial infections and sepsis in patients with COVID-19: a retrospective analysis of their joint impact on in-hospital prognosis.

Introduction and objectives. Secondary bacterial infections (SBIs) contribute to worse in-hospital outcomes in patients with Coronavirus disease 2019 (COVID-19). Treatment with proton pump inhibitors (PPIs) is associated with an increased risk of bacterial infections in different clinical settings. However, the association between PPI treatment prior to hospital admission and the occurrence of either SBIs or secondary bacterial sepsis (SBS) as well as their joint impact on clinical outcomes of patients hospitalized for COVID-19 are not clarified.Patients and methods. We retrospectively analyzed preadmission PPI use, in-hospital occurrence of SBIs and SBS, and in-hospital outcomes of a cohort of patients hospitalized for COVID-19.Results. Among 1087 patients, 447 (41%) were on PPI treatment prior to hospital admission. During the hospital stay, 197 (18%) and 223 (20%) patients were diagnosed with SBIs and SBS, respectively. The composite endpoint of intensive care unit (ICU) admission/in-hospital death was met by 214 (20%) patients. Preadmission PPI treatment was independently associated with up to a 2.1-fold and 1.7-fold increased risk of SBIs and SBS, respectively. The occurrence of SBS was independently associated with up to a 2.2-fold increased risk of ICU admission/in-hospital death. A significant preadmission PPI treatment x SBS interaction emerged in predicting ICU admission/in-hospital death (F = 5.221, pinteraction = 0.023).Conclusions. PPI treatment prior to hospital admission for COVID-19 is associated with an increased risk of SBIs and SBS. In addition, it interacts with SBS in predicting in-hospital prognosis. An appropriate use of PPIs may attenuate the risk of adverse clinical outcomes during hospitalization for COVID-19.

Inflamm-ageing: How cytokines and nutrition shape the trajectory of ageing.

Population ageing is increasing in prevalence in most developed countries. Ageing is the decline of functional properties at the cellular, tissue, and organ level. Biochemical changes that occur in all organisms that experience biological ageing are referred to as the "Hallmarks of ageing". Inflammation is a common denominator of the hallmarks of ageing, being mechanistically involved in most age-related health consequences. Inflamm-ageing refers to age-related changes in the inflammatory and immune systems which somehow drive the ageing process towards healthy or unhealthy ageing. Current evidences, support that, reversing the age-related pro-inflammatory status of inflamm-ageing, is able to modulate most hallmarks of ageing. Inflamm-ageing is associated with increased levels of pro-inflammatory molecules (e.g. cytokines, chemokines), ultimately producing a chronic low-grade inflammatory state typically observed in older individuals. It is commonly accepted that, the balance between pro- and anti-inflammatory cytokines/chemokines is one of the factors determining whether healthy or unhealthy ageing occurs. Malnutrition and nutritional imbalances, are highly prevalent in the elderly, playing a role in driving the balance of pro- and anti-inflammatory immunoactive molecules. In particular, malnutrition is a major risk factor for sarcopenia, a phenomenon characterized by loss of muscle mass, which is often referred to as the biological basis for frailty. Given the close relationship between malnutrition and sarcopenia, there is also evidence for a link between malnutrition and frailty. Indeed, changes in cytokine/chemokine levels in elderly patients with malnutrition were demonstrated. The demonstration that specific cytokines play a role in modulating appetite and nutrient sensing and taste reception, provided further evidence for the existence of a link between inflamm-ageing, nutrition and cytokines in shaping the trajectory of ageing. The present review will overview current evidence supporting the role of specific circulating cytokines and chemokines in the relationship between ageing, inflammation, and malnutrition.

Do PFCAs drive the establishment of thyroid cancer microenvironment? Effects of C6O4, PFOA and PFHxA exposure in two models of human thyroid cells in primary culture.

BACKGROUND: Exposure to environmental pollutants is suspected to be one of the potential causes accounting for the increase in thyroid cancer (TC) incidence worldwide. Among the ubiquitous pollutants, per-polyfluoroalkyl substances (PFASs), were demonstrated to exert thyroid disrupting effects. Perfluoroalkyl carboxylates (PFCAs) represent a subgroup of PFAS and include perfluoro carboxylic acids (PFOA and PFHxA) and perfluoropolyether carboxylic acid (C6O4). The potential relationship between exposure to PFCAs and TC was not yet fully elucidated. This in vitro study investigated whether certain PFCAs (C6O4, PFOA, and PFHxA) can influence the composition of TC microenvironment. METHODS: Two models of normal thyroid cells in primary cultures: Adherent (A-NHT) and Spheroids (S-NHT) were employed. A-NHT and S-NHT were exposed to C6O4, PFOA or PFHxA (0; 0.01; 0.1, 1; 10; 100; 1000 ng/mL) to assess viability (WST-1 and AV/PI assay), evaluate spherification index (SI) and volume specifically in S-NHT. CXCL8 and CCL2 (mRNA and protein), and EMT-related genes were assessed in both models after exposure to PFCAs. RESULTS: PFHxA reduced the viability of both A-NHT and S-NHT. None of the PFCAs interfered with the volume or spherification process in S-NHT. CXCL8 and CCL2 mRNA and protein levels were differently up-regulated by each PFCAs, being PFOA and PFHxA the stronger inducers. Moreover, among the tested PFCAs, PFHxA induced a more consistent increase in the mRNA levels of EMT-related genes. CONCLUSIONS: This is the first evaluation of the effects of exposure to PFCAs on factors potentially involved in establishing the TC microenvironment. PFHxA modulated the TC microenvironment at three levels: cell viability, pro-tumorigenic chemokines, and EMT-genes. The results provide further evidence of the pro-tumorigenic effect of PFOA. On the other hand, a marginal effect was observed for C6O4 on pro-tumorigenic chemokines.

Per-polyfluoroalkyl substances (PFAS) as thyroid disruptors: is there evidence for multi-transgenerational effects?

INTRODUCTION: The environmental spread of pollutants has led to a persistent exposure of living beings to multiple chemicals, by now become ubiquitous in the surrounding environment. Environmental exposure to these substances has been reported to cause multi- and/or transgenerational health effects. Per- and Polyfluorinated Substances (PFAS) raise great concern, given their known effects both as endocrine disruptors and potential carcinogens. The multi/trans-generational effects of different endocrine disruptors have been investigated by several studies, and harmful effects observed also for PFAS. AREAS COVERED: This review examines the current data on the multi-trans-generational effects of PFAS, with a focus on their impact on the thyroid axis. The aim is to determine if there is evidence of potential multi-trans-generational effects of PFAS on the thyroid and/or if more research is needed. EXPERT OPINION: PFAS exposure impacts thyroid homeostasis and can cross the placental barrier. In addition PFAS have shown multi-transgenerational effects in laboratory experiences and animal models, but thyroid disruptive effects of PFAS were also investigated only in a small number of these studies. Efforts are needed to study the adverse effects of PFAS, as not all PFAS are regulated and removal strategies are still being developed.

Canagliflozin reduces thyroid cancer cells migration in vitro by inhibiting CXCL8 and CCL2: An additional anti-tumor effect of the drug.

PURPOSE: Canagliflozin exert anti-cancer effects in several types of cancer including thyroid cancer (TC). However, whether it could modulate chemokines secreted in TC microenvironment is still unknown. The aim of the present study is to evaluate whether Canagliflozin could inhibit pro-tumorigenic chemokines CXCL8 and CCL2 and/or the TC cell migration induced by them. EXPERIMENTAL DESIGN: TC cell lines, TPC-1 and 8505C, HUVEC and normal thyroid cells NHT were treated with increasing concentrations of Canagliflozin. Viability was assessed by WST-1 and colony formation/proliferation by cristal violet. Chemokines were measured in cell supernatants by ELISA. mRNAs were evaluated by RT-PCR. TC migration (trans-well) and HUVEC proliferation (cristal violet) were assessed by treating cells with Canagliflozin alone or in combination with CXCL8 or CCL2. RESULTS: Canagliflozin reduced TC, HUVEC and NHT cells viability. The ability to form colonies of TC and the HUVEC proliferation (basal and CXCL8 or CCL2-induced) was also inhibited. mRNA and the secretion of CXCL8 was reduced in all cell types. The secretion of CCL2 was reduced by Canagliflozin in all cell types whereas its mRNA levels were reduced only in TPC-1. IL-6 was reduced in all cell types, while CXCL10 increased. More interestingly the CXCL8 and CCL2-induced TC cell migration as well as HUVEC proliferation was inhibited by Canagliflozin in both cell types. CONCLUSION: Canagliflozin exerts anti-cancer effects not only by reducing TC viability or colonies formation, but also by modulating two pro-tumorigenic chemokines resulting in reduced TC cells migration. These results expand the spectrum of canagliflozin-promoted anti-cancer effects.

Drug repositioning in thyroid cancer treatment: the intriguing case of anti-diabetic drugs.

Cancer represents the main cause of death worldwide. Thyroid cancer (TC) shows an overall good rate of survival, however there is a percentage of patients that do not respond or are refractory to common therapies. Thus new therapeutics strategies are required. In the past decade, drug repositioning become very important in the field of cancer therapy. This approach shows several advantages including the saving of: i) time, ii) costs, iii) de novo studies regarding the safety (just characterized) of a drug. Regarding TC, few studies considered the potential repositioning of drugs. On the other hand, certain anti-diabetic drugs, were the focus of interesting studies on TC therapy, in view of the fact that they exhibited potential anti-tumor effects. Among these anti-diabetic compounds, not all were judjed as appropriate for repositioning, in view of well documented side effects. However, just to give few examples biguanides, DPP-4-inhibitors and Thiazolidinediones were found to exert strong anti-cancer effects in TC. Indeed, their effects spaced from induction of citotoxicity and inhibition of metastatic spread, to induction of de-differentiation of TC cells and modulation of TC microenvironment. Thus, the multifacial anti-cancer effect of these compounds would make the basis also for combinatory strategies. The present review is aimed at discuss data from studies regarding the anti-cancer effects of several anti-diabetic drugs recently showed in TC in view of their potential repositioning. Specific examples of anti-diabetic repositionable drugs for TC treatment will also be provided.

Human induced pluripotent stem cells (hiPSC), enveloped in elastin-like recombinamers for cell therapy of type 1 diabetes mellitus (T1D): preliminary data.

Introduction: Therapeutic application and study of type 1 diabetes disease could benefit from the use of functional ß islet-like cells derived from human induced pluripotent stem cells (hiPSCs). Considerable efforts have been made to develop increasingly effective hiPSC differentiation protocols, although critical issues related to cost, the percentage of differentiated cells that are obtained, and reproducibility remain open. In addition, transplantation of hiPSC would require immunoprotection within encapsulation devices, to make the construct invisible to the host's immune system and consequently avoid the recipient's general pharmacologic immunosuppression. Methods: For this work, a microencapsulation system based on the use of "human elastin-like recombinamers" (ELRs) was tested to envelop hiPSC. Special attention was devoted to in vitro and in vivo characterization of the hiPSCs upon coating with ERLs. Results and Discussion: We observed that ELRs coating did not interfere with viability and function and other biological properties of differentiated hiPSCs, while in vivo, ELRs seemed to afford immunoprotection to the cell grafts in preliminary in vivo study. The construct ability to correct hyperglycemia in vivo is in actual progress.

Lipoprotein(a) Does Not Predict Thrombotic Events and In-Hospital Outcomes in Patients with COVID-19.

The prothrombotic and proinflammatory properties of lipoprotein(a) (Lp(a)) have been hypothesized to play a role in the pathogenesis of severe COVID-19; however, the prognostic impact of Lp(a) on the clinical course of COVID-19 remains controversial. This study aimed to investigate whether Lp(a) may be associated with biomarkers of thrombo-inflammation and the occurrence of thrombotic events or adverse clinical outcomes in patients hospitalized for COVID-19. We consecutively enrolled a cohort of patients hospitalized for COVID-19 and collected blood samples for Lp(a) assessment at hospital admission. A prothrombotic state was evaluated through D-dimer levels, whereas a proinflammatory state was evaluated through C-reactive protein (CRP), procalcitonin, and white blood cell (WBC) levels. Thrombotic events were marked by the diagnosis of deep or superficial vein thrombosis (DVT or SVT), pulmonary embolism (PE), stroke, transient ischemic attack (TIA), acute coronary syndrome (ACS), and critical limb ischemia (CLI). The composite clinical end point of intensive care unit (ICU) admission/in-hospital death was used to evaluate adverse clinical outcomes. Among 564 patients (290 (51%) men, mean age of 74 ± 17 years) the median Lp(a) value at hospital admission was 13 (10-27) mg/dL. During hospitalization, 64 (11%) patients were diagnosed with at least one thrombotic event and 83 (15%) patients met the composite clinical end point. Lp(a), as either a continuous or categorical variable, was not associated with D-dimer, CRP, procalcitonin, and WBC levels (p > 0.05 for all correlation analyses). In addition, Lp(a) was not associated with a risk of thrombotic events (p > 0.05 for multi-adjusted odds ratios) nor with a risk of adverse clinical outcomes (p > 0.05 for multi-adjusted hazard ratios). In conclusion, Lp(a) does not influence biomarkers of plasma thrombotic activity and systemic inflammation nor has any impact on thrombotic events and adverse clinical outcomes in patients hospitalized for COVID-19.

PFOA, PFHxA and C6O4 differently modulate the expression of CXCL8 in normal thyroid cells and in thyroid cancer cell lines.

Industrial chemical PFAS are persistent pollutants. Long chain PFAS were taken out of production due to their risk for human health, however, new congeners PFAS have been introduced. The in vitro effects of the long-chain PFOA, the short-chain PFHxA and the new-generation C6O4 were evaluated in normal and in thyroid cancer cell lines in terms of cell viability and proliferation, and secretion of a pro-tumorigenic chemokine (CXCL8), both at the mRNA and at the protein level. The Nthy-ory 3-1 normal-thyroid cell line, the TPC-1 and the 8505C (RET/PTC rearranged and BRAFV600e mutated, respectively) thyroid-cancer cell lines were exposed to increasing concentrations of each PFAS in a time-course. We evaluated viability using WST-1 (confirmed by AnnexinV/PI) and proliferation using the cristal-violet test. To evaluate CXCL8 mRNA we used RT-PCR and measured CXCL8 in the supernatants by ELISA. The exposure to none PFAS did not affect thyroid cells viability (except for a reduction of 8505C cells viability after 144 h) or proliferation. Individual PFAS differently modulated CXCL8 mRNA and protein level. PFOA increased CXCL8 both at mRNA and protein level in the three cell lines; PFHxA increased CXCL8 mRNA in the three cell lines, but increased the protein only in TPC-1 cells; C6O4 increased the CXCL8 mRNA only in thyroid cancer cell lines, but never increased the CXCL8 protein. The results of the present study indicate that the in vitro exposure to different PFAS may modulate both at the mRNA and secreted protein levels of CXCL8 in normal and cancer thyroid cells. Strikingly different effects emerged according to the specific cell type and to the targeted analyte (CXCL8 mRNA or protein).

Emerging of a new CD3+CD31HCD184+ tang cell phenothype in Sjögren's syndrome induced by microencapsulated human umbilical cord matrix-derived multipotent stromal cells.

Background: Sjögren's syndrome (SS) is an autoimmune disease hallmarked by infiltration and destruction of exocrine glands. Currently, there is no therapy that warrants full recovery of the affected tissues. Umbilical cord-derived multipotent stromal cells, microincapsulated in an endotoxin-free alginate gel (CpS-hUCMS), were shown to modulate the inflammatory activity of PBMCs in SS patients in vitro, through release of soluble factors (TGFß1, IDO1, IL6, PGE2, VEGF). These observations led us to set up the present study, aimed at defining the in vitro effects of CpS-hUCMS on pro- and anti-inflammatory lymphocyte subsets involved in the pathogenesis of SS. Methods and results: Peripheral blood mononuclear cells (PBMCs) upon collection from SS patients and matched healthy donors, were placed in co-culture with CpS-hUCMS for five days. Cellular proliferation and T- (Tang, Treg) and B- (Breg, CD19+) lymphocyte subsets were studied by flow cytometry, while Multiplex, Real-Time PCR, and Western Blotting techniques were employed for the analysis of transcriptome and secretome. IFNγ pre-treated hUCMS were assessed with a viability assay and Western Blotting analysis before co-culture. After five days co-culture, CpS-hUCMS induced multiple effects on PBMCs, with special regard to decrease of lymphocyte proliferation, increase of regulatory B cells and induction of an angiogenic T cell population with high expression of the surface marker CD31, that had never been described before in the literature. Conclusion: We preliminarily showed that CpS-hUCMS can influence multiple pro- and anti-inflammatory pathways that are deranged in SS. In particular, Breg raised and a new Tang phenothype CD3+CD31HCD184+ emerged. These results may considerably expand our knowledge on multipotent stromal cell properties and may open new therapeutic avenues for the management of this disease, by designing ad hoc clinical studies.

In vitro study of glyphosate effects on thyroid cells.

Glyphosate is a pesticide, which contaminates the environment and exposes workers and general population to its residues present in foods and waters. In soil, Glyphosate is degraded in metabolites, amino-methyl-phosphonic acid (AMPA) being the main one. Glyphosate is considered a potential cancerogenic and endocrine-disruptor agent, however its adverse effects on the thyroid were evaluated only in animal models and in vitro data are still lacking. Aim of this study was to investigate whether exposure to Glyphosate could exert adverse effects on thyroid cells in vitro. Two models (adherent-2D and spheroid-3D) derived from the same cell strain Fisher-rat-thyroid-cell line-5 (FRTL-5) were employed. After exposure to Glyphosate at increasing concentrations (0.0, 0.1-0.25- 0.5-1.0-2.0-10.0 mM) we evaluated cell viability by WST-1 (adherent and spheroids), results being confirmed by propidium-iodide staining (only for spheroids). Proliferation of adherent cells was assessed by crystal violet and trypan-blue assays, the increasing volume of spheroids was taken as a measure of proliferation. We also evaluated the ability of cells to form spheroids after Glyphosate exposure. We assessed changes of reactive-oxygen-species (ROS) by the cell-permeant H2DCFDA. Glyphosate-induced changes of mRNAs encoding for thyroid-related genes (TSHR, TPO, TG, NIS, TTF-1 and PAX8) were evaluated by RT-PCR. Glyphosate reduced cell viability and proliferation in both models, even if at different concentrations. Glyphosate at the highest concentration reduced the ability of FRTL-5 to form spheroids. An increased ROS production was found in both models after exposure to Glyphosate. Finally, Glyphosate increased the mRNA levels of some thyroid related genes (TSHR, TPO, TG and TTF-1) in both models, while it increased the mRNAs of PAX8 and NIS only in the adherent model. The present study supports an adverse effect of Glyphosate on cultured thyroid cells. Glyphosate reduced cell viability and proliferation and increased ROS production in thyroid cells.

Which factors influence the success rate of egg donation programmes with imported vitrified oocytes?

RESEARCH QUESTION: Which factors influence the success rate of egg donation programmes with imported vitrified oocytes? DESIGN: Observational longitudinal cohort study of 431 oocyte donation cycles conducted between January 2015 and February 2019. A total of 398 couples underwent an IVF cycle with imported donated vitrified eggs. All consecutive oocyte donation cycles conducted at the Centre for Reproductive Medicine of the European Hospital in Rome, Italy, were included. RESULTS: A univariable analysis was conducted. Among the demographic characteristics studied, body mass index (BMI) was significantly able to influence outcome. In 49% of unsuccessful cycles, men were overweight, compared with 39.4% in cycles with a positive beta-HCG test (P = 0.03). The relationship between female or donor BMI and treatment outcome, however, was not statistically significant. The day on which the embryo is transferred affects the outcome of the cycle: transferring on day 2 versus day 5 reduces the probability of clinical pregnancy. The relationship between male BMI classes and semen parameters was studied: obese men have a significantly lower sperm concentration than normal-weight men (P = 0.006 after Bonferroni correction). No statistically significant differences were found in semen volume (P = 0.722), sperm morphology (P = 0.100) and motility (P = 0.179) in obese men compared with normal weight men. CONCLUSION: In addition to the number of oocytes available after warming, male BMI, semen characteristics and blastocyst transfer are critical parameters able to influence the reproductive outcomes of egg donation programmes with imported vitrified oocytes.

Vitamin D Reduces Thyroid Cancer Cells Migration Independently From the Modulation of CCL2 and CXCL8 Chemokines Secretion.

Background: Vitamin D3 is largely involved in the regulation of calcium homeostasis. More recently, it was demonstrated that vitamin D exerts several beneficial effects against cancer progression through several mechanisms, including the reduction of cancer cells proliferation and migration. CXCL8 and CCL2 are two chemokines secreted by thyroid tumor cells. In the thyroid tumor microenvironment, these chemokines exert several pro-tumorigenic effects including the one to increase the metastatic potential. The aim of the present study was to investigate if vitamin D could modulate both thyroid cancer cell migration and their ability to secrete CCL2 and CXCL8. Methods: TPC-1 (RET/PTC rearranged) and 8505C (BRAFV600e mutated) thyroid cancer cell lines were treated with increasing concentrations of 1,25-OH-vitamin D3 (0-1,000 nM). Cell viability was assessed by WST-1 assay, cell migration was evaluated by transwell-migration chamber system, and CCL2 and CXCL8 levels were measured in the cell culture supernatants by ELISA. Results: Vitamin D did not affect cell viability but reduced, in a dose-dependent and significant manner, thyroid cancer cell migration (ANOVAs p < 0.05 for both TPC-1 and 8505C). Vitamin D differently modulated the secretion of CCL2 and CXCL8, by significantly inhibiting the secretion of CCL2 in both thyroid cancer cell lines and inhibiting the secretion of CXCL8 only in TPC-1 (ANOVAs p < 0.05). Conclusions: Vitamin D treatment of thyroid cancer cell lines reduces cell migration independently from the inhibition of the secretion of pro-tumorigenic chemokines. Future studies specifically designed at clarifying the pathways involved in the different inhibitory effects of vitamin D on CCL2 and CXCL8 in thyroid cancer cells appear worthwhile.

Microencapsulated Wharton Jelly-derived adult mesenchymal stem cells as a potential new therapeutic tool for patients with COVID-19 disease: an in vitro study.

BACKGROUND: The recent newly appeared Coronavirus disease (COVID-19), caused by an enveloped RNA virus named "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)", is associated with severe respiratory morbidity and mortality. Recent studies have shown that lymphopenia and a cytokine mass release represent important pathogenic features, with clinical evidence of dyspnea and hypoxemia, often leading to acute respiratory distress syndrome (ARDS), in severely ill patients, with a high death toll. Currently, stem cells are actively being investigated for their potential use in many "untreatable" diseases. In this regard and in particular, Mesenchymal Stem Cells (MSC), due to their intrinsic features, including either ability to impact on regulation of the immune system, or association with both anti-viral and anti-inflammatory properties, or potential for differentiation into several cell lineages, have become a promising tool for cell and molecular-based therapies. On this background, we wished to explore whether human umbilical cord-derived mesenchymal stem cells (hUCMS) would represent a potential viable therapeutic approach for the management of critically ill COVID19 patients. METHODS: We tested the hUCMS effects on peripheral blood mononuclear cell (PBMCs) retrieved from patients with COVID19 (Ethical Committee CEAS Umbria, Italy CER N°3658/20 7, May, 2020), both as free cell monolayers and after envelopment in sodium alginate microcapsules. Both cell systems, after priming with IFN-γ, proved able to produce several immunomodulatory molecules such as IDO1 and HLAG5, although only the microencapsulated hUCMS were associated with massive and dose-dependent production of these factors. RESULTS: The microencapsulated hUCMS improved allo-suppression in mixed lymphocytes reactions (MLRs), while also blunting T helper 1 and T helper 17 responses, that are involved with the cytokine storm and greatly contribute to the patient death. Moreover, we observed that both free and microencapsulated hUCMS permitted 5 days survival of in vitro culture maintained PBMCs extracted from very ill patients. CONCLUSION: We have provided evidence that microencapsulated hUCMS in vitro, seem to represent a powerful tool to impact on several immune pathways, clearly deranged in COVID19 patients. Further study is necessary to begin in vivo assessment of this experimental system, upon determining both, the most appropriate time of the disease onset for intervention, and cell dosage/patient of our experimental product.

Co-microencapsulation of human umbilical cord-derived mesenchymal stem and pancreatic islet-derived insulin producing cells in experimental type 1 diabetes.

INTRODUCTION: Post-partum umbilical cord Wharton Jelly-derived adult mesenchymal stem cells (hUCMS) hold anti-inflammatory and immunosuppressive properties. Human pancreatic islet-derived progenitor cells (hIDC) may de-differentiate, and subsequently re-differentiate into insulin producing cells. The two cell types share common molecules that facilitate their synergistic interaction and possibly crosstalk, likely useful for the cell therapy of type 1 diabetes (T1D). MATERIALS AND METHODS: Upon microencapsulation in sodium alginate (AG), hUCMS and hIDC were able to form cell co-aggregates that looked well integrated and viable. We then grafted microencapsulated hUCMS/hIDC co-aggregates into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, and observed an acquired ability of cells to produce and store hormones. Finally, we transplanted these biohybrid constructs into NOD mice with recent onset, spontaneous overt diabetes, observing a decline of blood glucose levels. RESULTS: In vitro, we have shown that hUCMS inhibited proliferation of allogeneic polymorphonuclear blood cells from patients with T1D, while promoting expansion of FoxP3+ Tregs. Reversal of hyperglycemia in diabetic NODs seems to suggest that hUCMS and hIDC, upon co-microencapsulation, anatomically and functionally synergized to accomplish two goals: maintain tracer insulin output by hIDC, while exploting the immunoregulatory properties of hUCMS. CONCLUSION: We have gathered preliminary evidence that the two adult stem cell types within AG microcapsules, may synergistically promote tracer insulin production, while "freezing" the autoimmune disease process, and help reversal of the recent onset hyperglycemia in a spontaneous, autoimmune rodent model of diabetes, the NOD mouse, with no need for pharmacologic immunosuppression.

Microencapsulated G3C Hybridoma Cell Graft Delays the Onset of Spontaneous Diabetes in NOD Mice by an Expansion of Gitr+ Treg Cells.

As an alternative to lifelong insulin supplementation, potentiation of immune tolerance in patients with type 1 diabetes could prevent the autoimmune destruction of pancreatic islet ß-cells. This study was aimed to assess whether the G3c monoclonal antibody (mAb), which triggers the glucocorticoid-induced TNFR-related (Gitr) costimulatory receptor, promotes the expansion of regulatory T cells (Tregs) in SV129 (wild-type) and diabetic-prone NOD mice. The delivery of the G3c mAb via G3C hybridoma cells enveloped in alginate-based microcapsules (G3C/cps) for 3 weeks induced Foxp3+ Treg-cell expansion in the spleen of wild-type mice but not in Gitr-/- mice. G3C/cps also induced the expansion of nonconventional Cd4+Cd25-/lowFoxp3lowGitrint/high (GITR single-positive [sp]) Tregs. Both Cd4+Cd25+GitrhighFoxp3+ and GITRsp Tregs (including also antigen-specific cells) were expanded in the spleen and pancreas of G3C/cps-treated NOD mice, and the number of intact islets was higher in G3C/cps-treated than in empty cps-treated and untreated animals. Consequently, all but two G3C/cps-treated mice did not develop diabetes and all but one survived until the end of the 24-week study. In conclusion, long-term Gitr triggering induces Treg expansion, thereby delaying/preventing diabetes development in NOD mice. This therapeutic approach may have promising clinical potential for the treatment of inflammatory and autoimmune diseases.