Comparison of viral inactivation methods on the characteristics of extracellular vesicles from SARS-CoV-2 infected human lung epithelial cells.

The interaction of SARS-CoV-2 infection with extracellular vesicles (EVs) is of particular interest at the moment. Studying SARS-CoV-2 contaminated-EV isolates in instruments located outside of the biosafety level-3 (BSL-3) environment requires knowing how viral inactivation methods affect the structure and function of extracellular vesicles (EVs). Therefore, three common viral inactivation methods, ultraviolet-C (UVC; 1350 mJ/cm2 ), ß-propiolactone (BPL; 0.005%), heat (56°C, 45 min) were performed on defined EV particles and their proteins, RNAs, and function. Small EVs were isolated from the supernatant of SARS-CoV-2-infected human lung epithelial Calu-3 cells by stepwise centrifugation, ultrafiltration and qEV size-exclusion chromatography. The EV isolates contained SARS-CoV-2. UVC, BPL and heat completely abolished SARS-CoV-2 infectivity of the contaminated EVs. Particle detection by electron microscopy and nanoparticle tracking was less affected by UVC and BPL than heat treatment. Western blot analysis of EV markers was not affected by any of these three methods. UVC reduced SARS-CoV-2 spike detectability by quantitative RT-PCR and slightly altered EV-derived ß-actin detection. Fibroblast migration-wound healing activity of the SARS-CoV-2 contaminated-EV isolate was only retained after UVC treatment. In conclusion, specific viral inactivation methods are compatible with specific measures in SARS-CoV-2 contaminated-EV isolates. UVC treatment seems preferable for studying functions of EVs released from SARS-CoV-2 infected cells.

How Does Human Milk Protect Against Necrotizing Enterocolitis (NEC)? Targeted Validation and Time-Course Analysis of 35 Gene Responses as NEC-Signature in Fetal Intestinal Epithelial Cells.

Breastfeeding reduces the risk of necrotizing enterocolitis (NEC), one of the most common causes of morbidity and mortality in preterm infants. However, the molecular substrates by which human milk (HM) offers protection against NEC are not well known. Using fetal intestinal epithelial cells treated with known NEC aggravators, namely lipopolysaccharide (LPS) and platelet-activating factor (PAF), we mapped the time-course of changes in targeted expression analysis of 35 NEC-associated genes, so-called the NEC signature. We found, first, that HM treatment fully rescued LPS/PAF-induced fetal intestinal cell death at 12 and 24 h (n = 5). Differential gene expression and bioinformatics revealed that HM did not mitigate inflammatory and cell death signals, but instead promoted cell proliferation and stress response pathways to mitigate LPS/PAF-induced inflammatory cell death. From this, epidermal growth factor (EGF) synthesis emerged as the central player in rescue of the fetal intestinal cell death. Functional validation was supported by reversal of the cellular rescue by HM following EGF knockdown by small interfering RNA. In conclusion, this study suggests that HM might offer protection against NEC through enhancing intestinal EGF production to rescue the inflammatory cell death. Future studies are warranted to verify these HM molecular protective effects in NEC models in vivo. The findings reported herein also support future research avenues to discover new therapeutics to boost intrinsic EGF production in the injured intestinal tissues in neonates with NEC, for example, by bioactive components in human milk, natural compounds, or small molecules.

Extracellular Vesicle-Based Method for Detecting MYCN Amplification Status of Pediatric Neuroblastoma.

MYCN amplification is the strongest predictor of high-risk neuroblastoma (NB). The standard procedure to detect MYCN status requires invasive procedures. Extracellular vesicles (EVs) contain molecular signatures of originated cells, present in biofluids, and serve as an invaluable source for cancer liquid biopsies. This study aimed to establish an EV-based method to detect the MYCN status of NB. Two EV subtypes, i.e., microvesicles (MVs) and exosomes, were sequentially isolated from the culture supernatant by step-wise centrifugation, ultrafiltration, and size-exclusion chromatography. Quantitative RT-PCR was performed to detect MYCN mRNA. As a result, MYCN mRNA was detectable in the MVs, but not exosomes, of MYCN-amplified NB cells. MYCN mRNA-containing MVs (MYCN-MV) were successfully detected in three distinct MYCN-amplified NB cell lines but absent in three MYCN non-amplification cells. The simulated samples were prepared by pulsing MVs into human serum. MYCN-MV detection in the simulated samples showed a less interfering effect from the human blood matrix. Validation using clinical specimens (2 mL bone marrow plasma) obtained from patients at various disease stages showed a promising result. Five out of six specimens of MYCN-amplified patients showed positive results, while there were no false positives in four plasma samples of the MYCN non-amplification group. This study communicated a novel EV-based method for detecting the MYCN status of pediatric NB based on MYCN mRNA contents in MVs. Future studies should be pursued in a prospective cohort to determine its true diagnostic performance.

Cell-Main Spectra Profile Screening Technique in Simulation of Circulating Tumour Cells Using MALDI-TOF Mass Spectrometry.

Circulating atypical cells (CAC) are released from a primary tumour site into peripheral blood and are indicators of cancer metastasis. CAC occur at very low frequency in circulating blood, and their detection remains challenging. Moreover, white blood cells (WBC) are the major contaminant in enriched CAC samples. Here, we developed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a novel CAC characterization platform. Main spectra profiles (MSP) of normal and cancer cells were generated by MALDI-TOF MS, and a cell-main spectra database was then compiled and analysed using the MALDI Biotyper software. Logarithmic scores accurately predicted distinct cell types. The feasibility of this workflow was then validated using simulated samples, which were prepared by 5000 WBC of three healthy individuals spiked with varying numbers (3, 6, 12, 25, 50, and 100) of lung, colon, or prostate cancer cells. MALDI-TOF MS was able to detect cancer cells down to six cells over the background noise of 5000 WBC with significantly higher predictive scores as compared to WBC alone. Further development of cell-MSP database to cover all cancer types sourced from cell lines and patient tumours may enable the use of MALDI-TOF MS as a cancer-screening platform in clinical settings in the future.

HMP-S7 Is a Novel Anti-Leukemic Peptide Discovered from Human Milk.

Chemotherapy in childhood leukemia is associated with late morbidity in leukemic survivors, while certain patient subsets are relatively resistant to standard chemotherapy. It is therefore important to identify new agents with sensitivity and selectivity towards leukemic cells, while having less systemic toxicity. Peptide-based therapeutics has gained a great deal of attention during the last few years. Here, we used an integrative workflow combining mass spectrometric peptide library construction, in silico anticancer peptide screening, and in vitro leukemic cell studies to discover a novel anti-leukemic peptide having 3+ charges and an alpha helical structure, namely HMP-S7, from human breast milk. HMP-S7 showed cytotoxic activity against four distinct leukemic cell lines in a dose-dependent manner but had no effect on solid malignancies or representative normal cells. HMP-S7 induced leukemic cell death by penetrating the plasma membrane to enter the cytoplasm and cause the leakage of lactate dehydrogenase, thus acting in a membranolytic manner. Importantly, HMP-S7 exhibited anti-leukemic effects against patient-derived leukemic cells ex vivo. In conclusion, HMP-S7 is a selective anti-leukemic peptide with promise, which requires further validation in preclinical and clinical studies.

Effects of Desflurane and Sevoflurane anesthesia on regulatory T cells in patients undergoing living donor kidney transplantation: a randomized intervention trial.

BACKGROUND: Volatile anesthetic agents used during surgery have immunomodulatory effects which could affect postoperative outcomes. Recognizing that regulatory T cells (Tregs) plays crucial roles in transplant tolerance and high peripheral blood Tregs associated with stable kidney graft function, knowing which volatile anesthetic agents can induce peripheral blood Tregs increment would have clinical implications. This study aimed to compare effects of desflurane and sevoflurane anesthesia on peripheral blood Tregs induction in patients undergoing living donor kidney transplantation. METHODS: A prospective, randomized, double-blind trial in living donor kidney transplant recipients was conducted at a single center, tertiary-care, academic university hospital in Thailand during August 2015 - June 2017. Sixty-six patients were assessed for eligibility and 40 patients who fulfilled the study requirement were equally randomized and allocated to desflurane versus sevoflurane anesthesia during transplant surgery. The primary outcome included absolute changes of peripheral blood CD4+CD25+FoxP3+Tregs which measured by flow cytometry and expressed as the percentage of the total population of CD4+ T lymphocytes at pre-exposure (0-h) and post-exposure (2-h and 24-h) to anesthetic gas. P-value < 0.05 denoted statistical significance. RESULTS: Demographic data were comparable between groups. No statistical difference of peripheral blood Tregs between desflurane and sevoflurane groups observed at the baseline pre-exposure (3.6 ± 0.4% vs. 3.1 ± 0.4%; p = 0.371) and 2-h post-exposure (3.0 ± 0.3% vs. 3.5 ± 0.4%; p = 0.319). At 24-h post-exposure, peripheral blood Tregs was significantly higher in desflurane group (5.8 ± 0.5% vs. 4.1 ± 0.3%; p = 0.008). Within group analysis showed patients receiving desflurane, but not sevoflurane, had 2.7% increase in peripheral blood Treg over 24-h period (p < 0.001). CONCLUSION: This study provides the clinical trial-based evidence that desflurane induced peripheral blood Tregs increment after 24-h exposure, which could be beneficial in the context of kidney transplantation. Mechanisms of action and clinical advantages of desflurane anesthesia based on Treg immunomodulation should be investigated in the future. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02559297 . Registered 22 September 2015 - retrospectively registered.

Capillary blood as an alternative specimen for enumeration of percentages of lymphocyte subsets.

OBJECTIVE: Capillary blood has been increasingly used in point-of-care setting for clinical monitoring in immunology and infectious diseases. We explored whether percentages of lymphocyte subsets (T-cells; CD3+, helper T-cells; CD4+, cytotoxic T-cells; CD8+, B-cells; CD19+, NK cells; CD56+, gamma delta T-cells, and regulatory T-cells) with regard to total lymphocyte count from capillary and venous blood of healthy volunteers were in good agreement. RESULTS: All percentages of lymphocyte subsets with regard to total lymphocyte count from capillary blood were significantly correlated with those from venous blood (r ≥ 0.9 for every cell type). However, Bland-Altman plots showed high agreement between capillary and venous samples only in those of CD3+, CD4+, and CD8+ cells (limit of agreement percentages from mean venous blood < 20%). However, the agreement of percentages of other lymphocyte subsets from venous and capillary blood was mediocre. We concluded that capillary blood could be used as an alternative for venous blood to determine percentages of CD3+, CD4+, and CD8+ cells with regard to total lymphocyte count.

Suppressive Characteristics of Umbilical Cord Blood-derived Regulatory T Cells After Ex Vivo Expansion on Autologous and Allogeneic T Effectors and Various Lymphoblastic Cells.

The third-party umbilical cord blood (UCB)-derived regulatory T cells (Treg) are an alternative to donor-derived Treg as cellular therapy of graft-versus-host disease following hematopoietic stem cell transplantation. However, their suppressive characteristics against autologous and allogeneic T effector cells (Teff) have rarely been documented. The exact role of UCB-Treg in hematologic malignancies is also uncertain. Here, we investigated the direct effects of UCB-Treg on the proliferation of autologous Teff, as compared with allogeneic Teff, and also determined cellular fates of lymphoblasts after UCB-Treg co-culture. UCB-Treg were isolated from 8 UCB samples using 2-step immunomagnetic bead sorting. After 10-day ex vivo expansion, up to 60-fold increase in cell number with 76.7%±4.9% of CD4CD25CD127FoxP UCB-Treg was obtained. Further characterization showed that ex vivo-expanded UCB-Treg contained a higher proportion of CD95CD45RACCR4Treg-B subpopulation compared with the CD95CD45RACCR4Treg-A subpopulation (13.0%±4.8% vs. 0.8%±0.7%; P<0.05), along with the detecting of substantial amounts of secretory IL-10 (57.7±17.8 pg/mL) and TGF-ß1 (196.5±29.7 pg/mL) in culture supernatants. After 4 days co-culture with UCB-Treg (at the ratio of 1:1), the proliferation of autologous and allogeneic Teff was decreased comparably (43.6%±17.5% vs. 37.6±17.7%; P=0.437). Suppression was independent of HLA-A, B, and DRB1 compatibility between UCB-Treg and Teff. UCB-Treg co-culture with various lymphoblasts showed proliferative suppression of Jurkat T lymphoblasts (45.4%±20.5% at the ratio of 1:1), but not Namalwa and Raji B lymphoblasts. All lymphoblasts had no significant cell apoptosis or death after co-culture. In conclusion, the ex vivo-expanded UCB-Treg had no difference in autologous and allogeneic Teff suppression. UCB-Treg therapy in patients with graft-versus-host disease who have a primary disease of T-cell leukemia may have additional benefits in the prevention of relapsed disease.

Fetal Intestinal Cell Growth as a Measure of the Comparative Biofunctionality of Human Milk and Infant Formulas: An In Vitro Study.

BACKGROUND: Infant formulas are produced to resemble human milk (HM) and to provide adequate energy and appropriate nutritional components for suitability of infant growth and development, some of which are customized for specific medical conditions. However, it has remained unclear whether formulas contain any biofunctionality equivalent to HM, particularly fetal intestinal cell growth promotion. OBJECTIVE: To evaluate the biofunctionality in HM and various formulas by using an in vitro fetal intestinal cell growth assay. MATERIALS AND METHODS: Nine specimens of HM collected from 9 milk donors and 16 formulas consisting of 5 regular formulas (RFs), 2 preterm formulas (PFs), 2 partial hydrolysate formulas (PHFs), 3 extensive hydrolysate formulas (EHFs), 2 amino acid formulas (AAFs), and 2 soy protein formulas (SPFs) were included. Fetal intestinal cell growth assay was performed in six replicates per milk specimen. Biofunctionality of HM digest (HMD) derived from in vitro tryptic digestion of HM was also examined. Statistical analysis was performed by ANOVA with post-hoc Tukey's Honestly Significant Difference test. RESULTS: The fetal intestinal cell growth-promoting activity of HM and formula groups were sorted from the highest as follows: HM, 192.8% ± 16.7%; AAF, 153.5% ± 17.8%; EHF, 149.4% ± 12.5%; RF, 123.5% ± 14.2%; PHF, 111.2% ± 17.9%; PF, 110.3% ± 8.2%; and SPF, 109.3% ± 17.3%. Statistical analysis showed that growth promotion of HM was significantly higher than that of all examined formulas (p < 0.0001). Among formulas, EHF and AAF showed greater growth-promoting activity than the others (p < 0.0001). HM and HMD had a comparable growth-promoting effect on fetal intestinal cells (198.5% ± 27.9% versus 191.2% ± 17.9%, p = 0.724), supporting the potential impact of HM biofunctionality under physiologic gastrointestinal digestion. CONCLUSIONS: Our data suggested that formulas are not equivalent to HM in respect of fetal intestinal cell growth biofunctionality. Despite having less activity than HM, EHF and AAF exhibited considerable levels of growth-promoting effect that may have clinical implications, especially when HM is unavailable.

Unravelling Pathophysiology of Crystalline Nephropathy in Ceftriaxone-Associated Acute Kidney Injury: A Cellular Proteomic Approach.

BACKGROUND: Previous studies showed that ceftriaxone can cause acute kidney injury (AKI) in the pediatric population. This study proposed a cellular model of crystalline nephropathy in ceftriaxone-associated AKI and explored the related pathophysiology by using a proteomic approach. METHODS: Ceftriaxone was crystallized with calcium in artificial urine. Madin-Darby Canine Kidney (MDCK) cells, a model of distal renal tubular cell, were cultured in the absence (untreated control) or presence of ceftriaxone crystals for 48-h (n = 5 each). MDCK cells were harvested and subsequently analyzed by proteomic analysis. Protein bioinformatics (i.e., STRING and Reactome) was used to predict functional alterations, and subsequently validated by Western blotting and cellular studies. p < 0.05 was considered statistically significant. RESULTS: Phase-contrast microscopy showed increased intracellular vesiculation and cell enlargement as a result of ceftriaxone crystal exposure. Proteome analysis revealed a total of 20 altered proteins (14 increased, 5 decreased and 1 absent) in ceftriaxone crystal-treated MDCK cells as compared to untreated cells (p < 0.05). Protein bioinformatics and validation studies supported heat stress response mediated by heat shock protein 70 (Hsp70) and downregulation of annexin A1 as the proposed pathophysiology of crystalline nephropathy in ceftriaxone-associated AKI, in which impaired proliferation and wound healing of crystal-induced distal tubular cells were outcomes. CONCLUSIONS: This study, for the first time, used the in vitro model of crystalline nephropathy to investigate the underlying pathophysiology of ceftriaxone-associated AKI, which should be investigated in vivo for potential clinical benefits in the future.

Evaluation of Fetal Intestinal Cell Growth and Antimicrobial Biofunctionalities of Donor Human Milk After Preparative Processes.

BACKGROUND: Donor human milk is considered the next best nutrition following mother's own milk to prevent neonatal infection and necrotizing enterocolitis in preterm infants who are admitted at neonatal intensive care unit. However, donor milk biofunctionalities after preparative processes have rarely been documented. OBJECTIVE: To evaluate biofunctionalities preserved in donor milk after preparative processes by cell-based assays. MATERIALS AND METHODS: Ten pools of donor milk were produced from 40 independent specimens. After preparative processes, including bacterial elimination methods (holder pasteurization and cold-sterilization microfiltration) and storage conditions (-20°C freezing storage and lyophilization) with varied duration of storage (0, 3, and 6, months), donor milk biofunctionalities were examined by fetal intestinal cell growth and antimicrobial assays. RESULTS: At baseline, raw donor milk exhibited 193.1% ± 12.3% of fetal intestinal cell growth and 42.4% ± 11.8% of antimicrobial activities against Escherichia coli. After bacteria eliminating processes, growth promoting activity was better preserved in pasteurized donor milk than microfiltrated donor milk (169.5% ± 14.3% versus 146.0% ± 11.8%, respectively; p < 0.005), whereas antimicrobial activity showed no difference between groups (38.3% ± 14.1% versus 53.7% ± 17.3%, respectively; p = 0.499). The pasteurized donor milk was further examined for the effects of storage conditions at 3 and 6 months. Freezing storage, but not lyophilization, could preserve higher growth-promoting activity during 6 months of storage (163.0% ± 9.4% versus 72.8% ± 6.2%, respectively; p < 0.005). Nonetheless, antimicrobial activity was lost at 6 months, regardless of the storage methods. CONCLUSIONS: This study revealed that fetal intestinal cell growth and antimicrobial assays could be applied to measure donor milk biofunctionalities and support the utilization of donor milk within 3 months after preparative processes.

Lamin A/C in renal tubular cells is important for tissue repair, cell proliferation, and calcium oxalate crystal adhesion, and is associated with potential crystal receptors.

A previous study reported that lamin A/C (LMNA) expression was increased in renal tubular cells adhered with calcium oxalate monohydrate (COM) crystals; however, its functional significance in kidney stone disease remained unknown. In the present study, increased levels of LMNA and its partner, nesprin-1 (SYNE1), in Madin-Darby canine kidney cells upon COM crystal adhesion were confirmed by Western blotting and immunofluorescence staining. LMNA was then knocked down by small interfering RNA. Immunofluorescence staining confirmed the efficiency of small interfering RNA of LMNA (si-LMNA), which also reduced expression of its partner, SYNE1. Scratch assay and total cell count revealed defects in tissue repair and cell proliferation, respectively, whereas cell death quantitation showed no cytotoxicity in si-LMNA-transfected cells. Crystal-binding assay highlighted the role of LMNA in crystal adhesion, whereas protein network analysis revealed interactions between LMNA and potential COM crystal receptors. Their associations were confirmed by reduced levels of these proteins, including vimentin, tubulin, enolase, S100, and annexin A2, in si-LMNA-transfected cells. These data have demonstrated for the first time, to our knowledge, that LMNA in renal tubular cells is important for tissue repair, cell proliferation, and COM crystal adhesion and is associated with potential COM crystal receptors. Therefore, LMNA may serve as a potential target for prevention of kidney stone disease and its recurrence.-Pongsakul, N., Vinaiphat, A., Chanchaem, P., Fong-ngern, K., Thongboonkerd, V. Lamin A/C in renal tubular cells is important for tissue repair, cell proliferation, and calcium oxalate crystal adhesion, and is associated with potential crystal receptors.

Isolation and characterizations of oxalate-binding proteins in the kidney.

Oxalate-binding proteins are thought to serve as potential modulators of kidney stone formation. However, only few oxalate-binding proteins have been identified from previous studies. Our present study, therefore, aimed for large-scale identification of oxalate-binding proteins in porcine kidney using an oxalate-affinity column containing oxalate-conjugated EAH Sepharose 4B beads for purification followed by two-dimensional gel electrophoresis (2-DE) to resolve the recovered proteins. Comparing with those obtained from the controlled column containing uncoupled EAH-Sepharose 4B (to subtract the background of non-specific bindings), a total of 38 protein spots were defined as oxalate-binding proteins. These protein spots were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS) as 26 unique proteins, including several nuclear proteins, mitochondrial proteins, oxidative stress regulatory proteins, metabolic enzymes and others. Identification of oxalate-binding domain using the PRATT tool revealed "L-x(3,5)-R-x(2)-[AGILPV]" as a functional domain responsible for oxalate-binding in 25 of 26 (96%) unique identified proteins. We report herein, for the first time, large-scale identification and characterizations of oxalate-binding proteins in the kidney. The presence of positively charged arginine residue in the middle of this functional domain suggested its significance for binding to the negatively charged oxalate. These data will enhance future stone research, particularly on stone modulators.

Characterizations and proteome analysis of platelet-free plasma-derived microparticles in ß-thalassemia/hemoglobin E patients.

Aggregatability and oxidative damage of red blood cells (RBCs), platelet activation and increased amount of blood cells-derived microparticles (MPs) are thought to be the etiologies for the thrombotic risk in thalassemia, but with unclear mechanisms. Here we report cellular origins and increases in number, oxidative stress status, and procoagulant activity, as well as altered proteome of MPs isolated from ß-thal/HbE patients. Flow cytometric analysis revealed that ß-thal/HbE patients had significantly higher levels of phosphatidylserine (PS)-bearing MPs in platelet-free plasma (PFP) as compared to normal subjects. The high levels of MPs correlated with not only the increased procoagulant activity but also the increased platelet counts. Additionally, these PS-bearing MPs were originated mostly from platelets and RBCs, both of which had increased levels of reactive oxygen species. Proteome analysis of MPs by 2-DE followed by Q-TOF MS and MS/MS analyses identified 29 proteins with significantly altered levels in MPs derived from ß-thal/HbE patients (e.g. the increased levels of peroxiredoxin 6, apolipoprotein E, cyclophilin A and heat shock protein 90). These findings suggest that the oxidative damage in platelets and RBCs potentially induces production of MPs with altered proteome that may, in turn, facilitate thromboembolic complications, which are commonly found in thalassemic patients. This article is part of a Special Issue entitled: Integrated omics.