Immunomodulatory Properties of Umbilical Cord Blood-Derived Small Extracellular Vesicles and Their Therapeutic Potential for Inflammatory Skin Disorders.

Umbilical cord blood (UCB) has long been seen as a rich source of naïve cells with strong regenerative potential, likely mediated by paracrine signals. More recently, small extracellular vesicles (sEV), such as exosomes, have been shown to play essential roles in cell-to-cell communication, via the transport of numerous molecules, including small RNAs. Often explored for their potential as biomarkers, sEV are now known to have regenerative and immunomodulating characteristics, particularly if isolated from stem cell-rich tissues. In this study, we aim to characterize the immunomodulating properties of umbilical cord blood mononuclear cell-derived sEV (UCB-MNC-sEV) and explore their therapeutic potential for inflammatory skin diseases. UCB-MNC-sEV were shown to shift macrophages toward an anti-inflammatory phenotype, which in turn exert paracrine effects on fibroblasts, despite previous inflammatory stimuli. Additionally, the incubation of PBMC with UCB-MNC-sEV resulted in a reduction of total CD4+ and CD8+ T-cell proliferation and cytokine release, while specifically supporting the development of regulatory T-cells (Treg), by influencing FOXP3 expression. In a 3D model of psoriatic skin, UCB-MNC-sEV reduced the expression of inflammatory and psoriatic markers IL6, IL8, CXCL10, COX2, S100A7, and DEFB4. In vivo, UCB-MNC-sEV significantly prevented or reversed acanthosis in imiquimod-induced psoriasis, and tendentially increased the number of Treg in skin, without having an overall impact on disease burden. This work provides evidence for the anti-inflammatory and tolerogenic effect of UCB-MNC-sEV, which may be harnessed for the treatment of Th17-driven inflammatory skin diseases, such as psoriasis.

The Role of Outer Membrane Proteins in UPEC Antimicrobial Resistance: A Systematic Review.

Uropathogenic Escherichia coli (UPEC) are one of the most common agents of urinary tract infection. In the last decade, several UPEC strains have acquired antibiotic resistance mechanisms and some have become resistant to all classes of antibiotics. UPEC outer membrane proteins (OMPs) seem to have a decisive role not only in the processes of invasion and colonization of the bladder mucosa, but also in mechanisms of drug resistance, by which bacteria avoid killing by antimicrobial molecules. This systematic review was performed according to the PRISMA guidelines, aiming to characterize UPEC OMPs and identify their potential role in antimicrobial resistance. The search was limited to studies in English published during the last decade. Twenty-nine studies were included for revision and, among the 76 proteins identified, seven were associated with antibiotic resistance. Indeed, OmpC was associated with ß-lactams resistance and OmpF with ß-lactams and fluoroquinolone resistance. In turn, TolC, OmpX, YddB, TosA and murein lipoprotein (Lpp) were associated with fluoroquinolones, enrofloxacin, novobiocin, ß-lactams and globomycin resistances, respectively. The clinical implications of UPEC resistance to antimicrobial agents in both veterinary and human medicine must propel the implementation of new strategies of administration of antimicrobial agents, while also promoting the development of improved antimicrobials, protective vaccines and specific inhibitors of virulence and resistance factors.

Toxicological Profile of Umbilical Cord Blood-Derived Small Extracellular Vesicles.

The development and adoption of cell therapies has been largely limited by difficulties associated with their safety, handling, and storage. Extracellular vesicles (EV) have recently emerged as a likely mediator for the therapeutic effect of cells, offering several advantages over cell therapies. Due to their small size and inability to expand and metastasize, EV are generally considered safer than cell transplantation. Nevertheless, few studies have scrutinized the toxicity profile of EV, particularly after repeated high-dose administration. The present study aimed to evaluate a preparation of small EV obtained from umbilical cord blood mononuclear cells (UCB-MNC-sEV) for its cytotoxicity in different cell lines, as well as its differential accumulation, distribution, and toxicity following repeated intravenous (IV) administrations in a rodent model. In vitro, repeated sEV exposure in concentrations up to 1 × 1011 particles/mL had no deleterious impact on the viability or metabolic activity of peripheral blood mononuclear cells, THP-1 monocytes, THP-1-derived macrophages, normal dermal human fibroblasts, or human umbilical vein endothelial cells. DiR-labelled sEV, injected intravenously for four weeks in healthy rats, were detected in clearance organs, particularly the kidneys, spleen, and liver, similarly to control dye. Moreover, repeated administrations for six and twelve weeks of up to 1 × 1010 total particles of sEV dye were well-tolerated, with no changes in general haematological cell counts, or kidney and liver toxicity markers. More importantly, unlabelled sEV likewise did not induce significant alterations in cellular and biochemical blood parameters, nor any morphological changes in the heart, kidney, lung, spleen, or liver tissue. In sum, our data show that UCB-MNC-sEV have no significant toxicity in vitro or in vivo, even when administered repeatedly at high concentrations, therefore confirming their safety profile and potential suitability for future clinical use.

Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use.

Extracellular vesicles (EV) are a promising therapeutic tool in regenerative medicine. These particles were shown to accelerate wound healing, through delivery of regenerative mediators, such as microRNAs. Herein we describe an optimized and upscalable process for the isolation of EV smaller than 200 nm (sEV), secreted by umbilical cord blood mononuclear cells (UCB-MNC) under ischemic conditions and propose quality control thresholds for the isolated vesicles, based on the thorough characterization of their protein, lipid and RNA content. Ultrafiltration and size exclusion chromatography (UF/SEC) optimized methodology proved superior to traditional ultracentrifugation (UC), regarding production time, standardization, scalability, and vesicle yield. Using UF/SEC, we were able to recover approximately 400 times more sEV per mL of media than with UC, and upscaling this process further increases EV yield by about 3-fold. UF/SEC-isolated sEV display many of the sEV/exosomes classical markers and are enriched in molecules with anti-inflammatory and regenerative capacity, such as hemopexin and miR-150. Accordingly, treatment with sEV promotes angiogenesis and extracellular matrix remodeling, in vitro. In vivo, UCB-MNC-sEV significantly accelerate skin regeneration in a mouse model of delayed wound healing. The proposed isolation protocol constitutes a significant improvement compared to UC, the gold-standard in the field. Isolated sEV maintain their regenerative properties, whereas downstream contaminants are minimized. The use of UF/SEC allows for the standardization and upscalability required for mass production of sEV to be used in a clinical setting.

Mitochondrial microRNAs: A Putative Role in Tissue Regeneration.

The most famous role of mitochondria is to generate ATP through oxidative phosphorylation, a metabolic pathway that involves a chain of four protein complexes (the electron transport chain, ETC) that generates a proton-motive force that in turn drives the ATP synthesis by the Complex V (ATP synthase). An impressive number of more than 1000 mitochondrial proteins have been discovered. Since mitochondrial proteins have a dual genetic origin, it is predicted that ~99% of these proteins are nuclear-encoded and are synthesized in the cytoplasmatic compartment, being further imported through mitochondrial membrane transporters. The lasting 1% of mitochondrial proteins are encoded by the mitochondrial genome and synthesized by the mitochondrial ribosome (mitoribosome). As a result, an appropriate regulation of mitochondrial protein synthesis is absolutely required to achieve and maintain normal mitochondrial function. Regarding miRNAs in mitochondria, it is well-recognized nowadays that several cellular mechanisms involving mitochondria are regulated by many genetic players that originate from either nuclear- or mitochondrial-encoded small noncoding RNAs (sncRNAs). Growing evidence collected from whole genome and transcriptome sequencing highlight the role of distinct members of this class, from short interfering RNAs (siRNAs) to miRNAs and long noncoding RNAs (lncRNAs). Some of the mechanisms that have been shown to be modulated are the expression of mitochondrial proteins itself, as well as the more complex coordination of mitochondrial structure and dynamics with its function. We devote particular attention to the role of mitochondrial miRNAs and to their role in the modulation of several molecular processes that could ultimately contribute to tissue regeneration accomplishment.

Neonatal Apex Resection Triggers Cardiomyocyte Proliferation, Neovascularization and Functional Recovery Despite Local Fibrosis.

So far, opposing outcomes have been reported following neonatal apex resection in mice, questioning the validity of this injury model to investigate regenerative mechanisms. We performed a systematic evaluation, up to 180 days after surgery, of the pathophysiological events activated upon apex resection. In response to cardiac injury, we observed increased cardiomyocyte proliferation in remote and apex regions, neovascularization, and local fibrosis. In adulthood, resected hearts remain consistently shorter and display permanent fibrotic tissue deposition in the center of the resection plane, indicating limited apex regrowth. However, thickening of the left ventricle wall, explained by an upsurge in cardiomyocyte proliferation during the initial response to injury, compensated cardiomyocyte loss and supported normal systolic function. Thus, apex resection triggers both regenerative and reparative mechanisms, endorsing this injury model for studies aimed at promoting cardiomyocyte proliferation and/or downplaying fibrosis.

Subacute and chronic hypersensitivity pneumonitis: histopathological patterns and survival.

BACKGROUND: In hypersensitivity pneumonitis (HP), survival can be predicted on the basis of the severity of fibrosis in surgical lung biopsy, but few data are available on the influence of clinical, functional, tomographic and histologic findings on prognosis. OBJECTIVES: To describe the impact on survival of clinical data, histological patterns, and HRCT findings in subacute/chronic HP. METHODS: A retrospective analysis of 103 patients diagnosed with HP submitted to surgical lung biopsy. Chronic HP was characterized by HRCT findings indicative of fibrosis (n=76). RESULTS: The most relevant exposures were to molds and birds. Lung biopsies revealed typical HP with granulomas in 46 patients, bronchiolocentric interstitial pneumonia in 27, and non-specific interstitial pneumonia (NSIP) in 16. By univariate analysis, several findings were predictors of mortality: older age, male sex, velcro crackles, higher FEV(1)/FVC ratio, lower oxygen saturation during exercise, and absence of mosaic pattern/air trapping and presence of fibrosis on HRCT. By multivariate analysis, remained significant: age (p=0.007), oxygen saturation during exercise (p=0.003), and mosaic pattern/air trapping on HRCT (p=0.004). Patients with NSIP had a greater survival than did those with typical histology and those with bronchiolocentric pneumonia (p=0.033). CONCLUSIONS: A wide range of histological features are found in HP. Typical findings are seen in 45% of cases. Other common patterns are NSIP and centriacinar lesions. Survival is better in patients with NSIP and worse in those with older age, desaturation during exercise, and absence of mosaic pattern/air trapping on HRCT.