Clinical translation of mesenchymal stromal cell extracellular vesicles: Considerations on scientific rationale and production requisites

The present paper is a commentary to ‘Identification and characterization of hADSC‐derived exosome proteins from different isolation methods’ (Huang et al. 2021; 10.1111/jcmm.16775). Given the enthusiasm for the potential of mesenchymal stromal cell‐derived extracellular vesicles (MSC‐EVs), some considerations deserve attention as they move through successive stages of research and application into humans. We herein remark the prerequisite of generating that evidence ensuring a high consistency in safety, composition and biological activity of the intended MSC‐EV preparations, and the suitability of disparate isolation techniques to produce efficacious EV preparations and fulfil requirements for standardized clinical‐grade biomanufacturing.     

The effect of A23187 on glucose production from methylglyoxal and pyruvate in isolated murine hepatocytes.

1. A23187 increased the glucose production from methylglyoxal in isolated hepatocytes, and maximal stimulation was obtained at 10(-6) M. The effect of A23187 was dependent on the presence of Ca2+. 2. Glucose production from pyruvate (less than 1 mM) in isolated hepatocytes was stimulated by A23187 in the presence of 2.5 mM Ca2+ and was depressed at pyruvate concentrations above 1 mM. Both the virtual Km and the virtual Vmax of glucose production from pyruvate were decreased by A23187.     

Expression of CD11c Is Associated with Unconventional Activated T Cell Subsets with High Migratory Potential

CD11c is an α integrin classically employed to define myeloid dendritic cells. Although there is little information about CD11c expression on human T cells, mouse models have shown an association of CD11c expression with functionally relevant T cell subsets. In the context of genital tract infection, we have previously observed increased expression of CD11c in circulating T cells from mice and women. Microarray analyses of activated effector T cells expressing CD11c derived from naïve mice demonstrated enrichment for natural killer (NK) associated genes. Here we find that murine CD11c⁺ T cells analyzed by flow cytometry display markers associated with non-conventional T cell subsets, including γδ T cells and invariant natural killer T (iNKT) cells. However, in women, only γδ T cells and CD8⁺ T cells were enriched within the CD11c fraction of blood and cervical tissue. These CD11c⁺ cells were highly activated and had greater interferon (IFN)-γ secretory capacity than CD11c^- T cells. Furthermore, circulating CD11c⁺ T cells were associated with the expression of multiple adhesion molecules in women, suggesting that these cells have high tissue homing potential. These data suggest that CD11c expression distinguishes a population of circulating T cells during bacterial infection with innate capacity and mucosal homing potential.     

Inhibition of apoptosis in human immunodeficiency virus-infected cells enhances virus production and facilitates persistent infection.

Apoptosis is one of several mechanisms by which human immunodeficiency virus type 1 (HIV-1) exerts its cytopathic effects. CD4+ Jurkat T-cell lines overexpressing the adenovirus E1B 19K protein, a potent inhibitor of apoptosis, were used to examine the consequences of inhibition of apoptosis during acute and chronic HIV-1 infections. E1B 19K protein expression inhibited HIV-induced apoptosis, enhanced virus production, and established high levels of persistent viral infection. One E1B 19K-expressing line appeared to undergo HIV-induced death via a nonapoptotic mechanism, illustrating that HIV infection results in lymphocyte depletion through multiple pathways. Increased virus production associated with sustained cell viability suggests that therapeutic approaches involving inhibition of HIV-induced programmed cell death may be problematic.     

Corticosterone-releasing activity of immune mediators

Products derived from the activated immune system have been reported to modulate neuroendocrine function. In addition, a direct connection between neuroendocrine and immune responses to stress has recently been proposed. We now provide evidence that heterogeneous lymphokine-containing supernatants from mitogen-stimulated rat spleen cells can stimulate both basal and corticotropin-induced corticosterone secretion from rat adrenal cells in an in vitro perifusion system. Moreover, thymosin alpha 1, a 28-amino acid residue peptide found both in thymus and lymphocyte-derived supernatants was also able to synergistically stimulate corticotropin-stimulated corticosterone release, without affecting basal corticosterone output in this same in vitro adrenal cell perifusion system. These results reinforce the suggestion about the existence of bidirectional interactions between the immune and neuroendocrine systems. They also indicate that this communication may occur directly at the adrenal gland level, a major effector site of the body's response to stress.     

Cytostatic peptide isolation from culture media of mouse peritoneal exudate cells

It was found that the supernatant of mouse PEC culture medium (MCM) (both resident and casein-elicited cells) has an inhibitory effect in vitro on the incorporation of [3H]TdR into DNA of mouse spleen cells. The inhibitory effect in the MCM appears in the first 24 hr and also reaches its maximum value within this time. The inhibitory effect of this factor could not be demonstrated in the extract of freshly harvested M phi cells. The factors responsible for inhibition proved to be heat stable at 80 degrees C for longer than 30 min. Following heat treatment, the crude extract was separated into four fractions absorbing uv light at 280 nm using Sephadex G-25 column chromatography, and the most potent biologically active inhibitory factor was eluted in the last fraction. This fraction could also be obtained with a more effective permeation volume using Trysacryl GF 05 gel chromatography, and the active B fraction from this chromatography could be separated into four subfractions by isotachophoresis (ITP). The active fraction, which was obtained by Trysacryl GF 05 gel chromatography and further separated by ITP, was found to be highly inhibitory. It contained a peptide-like substance with a molecular mass of approximately 2.0 kDa and had an anionic character at pH 4.0. The inhibitory effect of MCM cannot be influenced either by inhibitory compounds of protein synthesis or by proteolysis blocking agents. Furthermore, the inhibitory effect is shown to be reversible and is more pronounced on B cells than on T lymphocytes.     

The solvent-free thermal dehydration of hexitols on zeolites

Dehydration of galactitol, D-glucitol and D-mannitol at high temperature in the presence of molecular sieves without solvent under an argon atmosphere is described. Cyclodehydration products with retention or inversion of the configuration at asymmetric carbon atoms, were observed. Reaction of galactitol yielded racemic 1,4-anhydrogalactitol in a first step and then racemic 1,4:3,6-dianhydroiditol. Complete analytical separations of exhaustively O-acetylated reaction products were achieved by GC and structures were assigned using co-injection with standards.     

Conformational changes in diphtheria toxoids. Analysis with monoclonal antibodies

Monoclonal antibodies (Mab) were raised against CRM197, a non-toxic mutant of diphtheria toxin (DT). The ability of four Mabs to bind DT and the six functional mutants CRM197, CRM176, CRM228, CRM1001, CRM45 and CRM30 was assessed by immunoblotting and by a radioimmunoassay in which the protein antigen in solution competes with labeled CRM197 for the Mab binding site. The results show that the peptides recognized by Mab11.3, Mab53 and Mab23 are accessible in the mutant molecules in solution but not when they are part of the native DT structure, which could therefore be described for this purpose as 'closed' in contrast with an 'open' conformation of CRM197, CRM176 and CRM228. In particular, the behaviour of Mab53 indicates that the single amino acid substitutions in the A fragments of CRM197 and CRM176 also affect the conformation of their B fragments.     

Identification and functional expression of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals

Seed oil from castor bean (Ricinus communis) contains high amounts of hydroxy fatty acid rich triacylglycerols (TAGs) that can serve as raw material for production of bio-based products such as nylon, cosmetics, lubricants, foams, and surfactants. Diacylglycerol acyltransferase (DGAT) catalyses the terminal reaction in the acyl-CoA dependent Kennedy pathway of triglyceride biosynthesis. There is still some debate whether there are three or four enzymes in yeast that have DGAT activity and catalyse the synthesis of TAG but of these the DGAT2 homologue Dga1 contributes in a major way to TAG biosynthesis. Here we report on the cloning of a cDNA for DGAT2 from castor bean and prove its biological activity following expression in yeast and enzymatic assays using diricinolein as the acceptor and ricinoleoyl-CoA as the donor. Previous reports of DGAT in castor have focussed on DGAT1 which has little amino acid sequence homology to DGAT2. Expressional studies demonstrate that DGAT2 is 18-fold more highly expressed in seeds than in leaves and shows temporal specific expression during seed development. In contrast, DGAT1 shows little difference in expression in seeds versus leaves. We conclude that in castor bean DGAT2 is more likely to play a major role in seed TAG biosynthesis than DGAT1.