mRNA Technology and Mucosal Immunization.

Current mRNA vaccines are mainly administered via intramuscular injection, which induces good systemic immunity but limited mucosal immunity. Achieving mucosal immunity through mRNA vaccination could diminish pathogen replication at the entry site and reduce interhuman transmission. However, delivering mRNA vaccines to mucosae faces challenges like mRNA degradation, poor entry into cells, and reactogenicity. Encapsulating mRNA in extracellular vesicles may protect the mRNA and reduce reactogenicity, making mucosal mRNA vaccines possible. Plant-derived extracellular vesicles from edible fruits have been investigated as mRNA carriers. Studies in animals show that mRNA vehiculated in orange-derived extracellular vesicles can elicit both systemic and mucosal immune responses when administered by the oral, nasal, or intramuscular routes. Once lyophilized, these products show remarkable stability. The optimization of mRNA to improve translation efficiency, immunogenicity, reactogenicity, and stability can be obtained through adjustments of the 5'cap region, poly-A tail, codons selection, and the use of nucleoside analogues. Recent studies have also proposed self-amplifying RNA vaccines containing an RNA polymerase as well as circular mRNA constructs. Data from parenterally primed animals demonstrate the efficacy of nasal immunization with non-adjuvanted protein, and studies in humans indicate that the combination of a parenteral vaccine with the natural exposure of mucosae to the same antigen provides protection and reduces transmission. Hence, mucosal mRNA vaccination would be beneficial at least in organisms pre-treated with parenteral vaccines. This practice could have wide applications for the treatment of infectious diseases.

Edible Plant-Derived Extracellular Vesicles for Oral mRNA Vaccine Delivery.

Nucleic acid delivery through extracellular vesicles (EVs) is a well-preserved evolutionary mechanism in all life kingdoms including eukaryotes, prokaryotes, and plants. EVs naturally allow horizontal transfer of native as well as exogenous functional mRNAs, which once incorporated in EVs are protected from enzymatic degradation. This observation has prompted researchers to investigate whether EVs from different sources, including plants, could be used for vaccine delivery. Several studies using human or bacterial EVs expressing mRNA or recombinant SARS-CoV-2 proteins showed induction of a humoral and cell mediated immune response. Moreover, EV-based vaccines presenting the natural configuration of viral antigens have demonstrated advantages in conferring long-lasting immunization and lower toxicity than synthetic nanoparticles. Edible plant-derived EVs were shown to be an alternative to human EVs for vaccine delivery, especially via oral administration. EVs obtained from orange juice (oEVs) loaded with SARS-CoV-2 mRNAs protected their cargo from enzymatic degradation, were stable at room temperature for one year, and were able to trigger a SARS-CoV-2 immune response in mice. Lyophilized oEVs containing the S1 mRNA administered to rats via gavage induced a specific humoral immune response with generation of blocking antibodies, including IgA and Th1 lymphocyte activation. In conclusion, mRNA-containing oEVs could be used for developing new oral vaccines due to optimal mucosal absorption, resistance to stress conditions, and ability to stimulate a humoral and cellular immune response.

Absence of IL-6 Receptor Blockade Effect on the Outcomes of Transplant Glomerulopathy in the Absence of Anti-HLA Donor-specific Antibodies.

Background: Transplant glomerulopathy (TG) is the hallmark of chronic antibody-mediated rejection but often occurs without anti-HLA donor-specific antibodies (DSAs) in the assumption that other DSAs may be the effectors of the tissue injury. Recently, we reported a positive effect of interleukin-6 (IL-6) receptor blocker tocilizumab (TCZ) in TG/DSA+. In the present study, we investigate the effect of TCZ in a cohort of TG cases without detectable anti-HLA DSAs. Methods: Single-center retrospective analysis of TG cases without anti-HLA DSAs (TG/DSA) treated with TCZ for chronic antibody-mediated rejection as first-line therapy evaluated through clinical, protocol biopsies, and gene expression analyses was included. Results: Differently from TG/DSA+, TG/DSA- showed a progressive reduction in the estimated glomerular filtration rate at 12 mo and after that with no significant modification in microvascular inflammation or C4d+. No upregulation in tight junction protein-1, aldo-keto reductase family 1 member C3, and calcium/calmodulin-dependent serine protein kinase, documented in TG/DSA+, was noted in post-TCZ biopsies. The reduction of microvascular inflammation was associated with natural killer-cell reduction in TG/DSA+, whereas TG/DSA- tends to maintain or increase periglomerular/interstitial infiltration. Conclusions: In the absence of anti-HLA DSAs, TG behavior seems not to be modified by IL-6 receptor blockade. These results are at variance with observational studies and previous trials with IL-6 inhibitors in TG associated with anti-HLA DSAs. These data may fuel the hypothesis of different mechanisms underlying TGs (including the potentially different roles of natural killer cells) and suggest carefully selecting patients with TG for clinical trials or off-label treatment based on their antidonor serologic status.

High-volume hemofiltration does not protect human kidney endothelial and tubular epithelial cells from septic plasma-induced injury.

High volume hemofiltration (HVHF) could remove from plasma inflammatory mediators involved in sepsis-associated acute kidney injury (SA-AKI). The IVOIRE trial did not show improvements of outcome and organ dysfunction using HVHF. The aim of this study was to evaluate in vitro the biological effects of plasma of patients treated by HVHF or standard volume hemofiltration (SVHF). We evaluated leukocyte adhesion, apoptosis and functional alterations of endothelial cells (EC) and tubular epithelial cells (TEC). In vitro data were correlated with plasma levels of TNF-α, Fas-Ligand (FasL), CD40-Ligand (CD40L), von Willebrand Factor (vWF) and endothelial-derived microparticles. An experimental model of in vitro hemofiltration using LPS-activated blood was established to assess cytokine mass adsorption during HVHF or SVHF. Plasma concentrations of TNF-ɑ, FasL, CD40L and von Willebrand Factor (vWF) were elevated at the start (d1h0) of both HVHF and SVHF, significantly decreased after 6 h (d1h6), remained stable after 12 h (d1h12) and then newly increased at 48 h (d3h0). Plasma levels of all these molecules were similar between HVHF- and SVHF-treated patients at all time points considered. In addition, the levels of endothelial microparticles remained always elevated, suggesting the presence of a persistent microvascular injury. Plasma from septic patients induced leukocyte adhesion on EC and TEC through up-regulation of adhesion receptors. Moreover, on EC, septic plasma induced a cytotoxic and anti-angiogenic effect. On TEC, septic plasma exerted a direct pro-apoptotic effect via Fas up-regulation and caspase activation, loss of polarity, altered expression of megalin and tight junction molecules with an impaired ability to internalize albumin. The inhibition of plasma-induced cell injury was concomitant to the decrease of TNF-α, Fas-Ligand and CD40-Ligand levels. The protective effect of both HVHF and SVHF was time-limited, since a further increase of circulating mediators and plasma-induced cell injury was observed after 48 h (d3h0). No significant difference of EC/TEC damage were observed using HVHF- or SVHF-treated plasma. The in vitro hemofiltration model confirmed the absence of a significant modulation of cytokine adsorption between HVHF and SVHF. In comparison to SVHF, HVHF did not increase inflammatory cytokine clearance and did not reverse the detrimental effects of septic plasma-induced EC and TEC injury. Further studies using adsorptive membranes are needed to evaluate the potential role of high dose convective therapies in the limitation of the harmful activity of plasma soluble factors involved in SA-AKI.Trial registration IVOIRE randomized clinical trial; ClinicalTrials.gov (NCT00241228) (18/10/2005).

In vitro characterization of 3D culture-based differentiation of human liver stem cells.

Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro. Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system, guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study, we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation. Methods: We evaluated the expression of hepatic markers using qRT-PCR, immunofluorescence, and Western blot analysis. Additionally, we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality. Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes, including CYP450, urea cycle enzymes, and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes, evident as early as 1 day post-differentiation. Interestingly, HLSCs transiently upregulated stem cell markers during differentiation, followed by downregulation after 7 days. Furthermore, differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24 h, with accumulation over time. Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system, its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications.

Extracellular Vesicles Derived from Human Liver Stem Cells Counteract Chronic Kidney Disease Development and Cardiac Dysfunction in Remnant Kidney Murine Model: The Possible Involvement of Proteases.

Fibrosis is a marker of chronic kidney disease (CKD) and consists of the accumulation of the extracellular matrix (ECM) components, causing the progressive deterioration of kidney function. Human liver stem cells (HLSCs) have anti-fibrotic activity, and HLSC-derived extracellular vesicles (EVs) mediate this effect. Herein, we evaluated the ability of HLSC-EVs to reverse renal and cardiac alterations in a murine model of partial nephrectomy (PNx) that mimics human CKD development. Furthermore, we investigated the contribution of extracellular matrix remodeling-related proteases to the anti-fibrotic effect of HLSC-EVs. PNx was performed by ligation of both poles of the left kidney, followed one week later by the removal of the right kidney. EV treatment started 4 weeks after the nephrectomy, when renal and cardiac alternations were already established, and mice were sacrificed at week eight. HLSC-EV treatment improved renal function and morphology, significantly decreasing interstitial fibrosis, glomerular sclerosis, and capillary rarefaction. This improvement was confirmed by the decreased expression of pro-fibrotic genes. Moreover, EV treatment improved cardiac function and reduced cardiac fibrosis. HLSC-EVs shuttled different proteases with ECM remodeling activity, and matrix metalloproteinase 1 (MMP-1) was involved in their anti-fibrotic effect on renal tissue. HLSC-EV treatment interferes with CKD development and ameliorates cardiomyopathy in PNx mice.