Human Blood Mononuclear Cells Research Articles

Therapeutic mechanism of cord blood mononuclear cells via the IL-8-mediated angiogenic pathway in neonatal hypoxic-ischaemic brain injury

In a clinical trial of cerebral palsy, the level of plasma interleukin-8 (IL-8) was increased, correlated with motor improvement, after human umbilical cord blood mononuclear cell (hUCBC) infusion. This study aimed to elucidate the role of IL-8 in the therapeutic effects of hUCBCs in a mouse model of hypoxic-ischaemic brain injury (HI). In P7 HI mouse brains, hUCBC administration at day 7 after HI upregulated the gene expression of Cxcl2, the mouse IL-8 homologue and increased the expression of its receptor, CXCR2. hUCBC administration restored the sequential downstream signalling axis of p-p38/p-MAPKAPK2, NFκB, and angiogenic factors, which were downregulated by HI. An in vitro assay revealed the downregulation of the angiogenic pathway by CXCR2 knockdown and p38 inhibition. In vivo p38 inhibition prior to hUCBC administration in HI mouse brains produced identical results. Behavioural outcomes revealed a therapeutic effect (ps < 0.01) of hUCBC or IL-8 administration, which was correlated with decreases in infarct size and angiogenic findings in the striatum. In conclusion, the response of the host to hUCBC administration in mice upregulated Cxcl2, which led to the activation of the IL-8-mediated p-p38 signalling pathway. The upregulation of the downstream pathway and angiogenic growth factors via NFκB can be inferred to be the potential therapeutic mechanism of hUCBCs.

MARK activation in human blood mononuclear cells in types 1 and 2 diabetes

MARK activation in human blood mononuclear cells in types 1 and 2 diabetes

Hesperozygis ringens (Benth.) Epling: a study involving extraction, chemical profiling, antioxidant and biological activity

Hesperozygis ringens is a plant of the Lamiaceae family which is restricted to the Southern region of Brazil. It is popularly used as an insecticide, but knowledge on it is very scarce. This study aimed to determine the chemical markers of H. ringens extracts obtained via ultrasound-assisted (UAE-EtOH) and supercritical fluid (SFE-CO2) extractions. UAE-EtOH and SFE-CO2 extracts were characterised by UPLC-MS and GC-MS, respectively. Both products had their antioxidant activity, cytotoxicity and genotoxicity evaluated. Twelve compounds were found in the UAE-EtOH extract, including phenolic acids and flavonoids; the SFE-CO2 extract contained terpenes and phytosterols. The UAE-EtOH extract showed a greater antioxidant activity. Neither extract presented cytotoxicity or genotoxicity against human mononuclear blood cells.

Immunotropic effects in cultured human blood mononuclear cells exposed to a 900MHz pulse-modulated microwave field.

ABSTRACTThe specific biological effect of electromagnetic field (EMF) remains unknown even though devices present in our daily lives, such as smartphones and Wi-Fi antennae increase the environmental level of electromagnetic radiation. It is said that the human immune system is able to react to discrete environmental stimuli like EMF. To investigate the effect of 900 MHz microwave stimulation on the immune system our research aimed to analyze lymphocyte proliferation and observe and assess the basic immunoregulatory activities using a newly developed and improved anechoic chamber. Samples of mononuclear cells (PBMC) isolated from the blood of healthy donors were exposed to 900 MHz pulse-modulated radiofrequency radiation (20 V/m, SAR 0.024 W/kg) twice (15 min each) or left without irradiation (control group). Subsequently, the control and exposed cells were set up to determine several parameters characterizing T cell immunocompetence and monocyte immunogenic activity. Although the microcultures of PBMC exposed to radiofrequency radiation demonstrated higher immunogenic activity of monocytes (LM index) and T-cell response to concanavalin A than control cultures after first exposure, this parameter decreased after a second stimulation. Saturation of the interleukin-2 (IL-2) receptor rose significantly after the second day of exposure. On the other hand, response to mitogen dropped after EMF stimulation. The results suggest that PBMC are able to overcome stress caused by mitogens after stimulation with 900 MHz radiation.

Human umbilical cord blood mononuclear cells protect against renal tubulointerstitial fibrosis in cisplatin-treated rats

Currently, there is no effective method to prevent renal interstitial fibrosis after acute kidney injury (AKI). In this study, we established and screened a new renal interstitial fibrosis rat model after cisplatin-induced AKI. Our results indicated that rats injected with 4 mg/kg cisplatin once a week for two weeks after firstly administrated with 6.5 mg/kg loading dose of cisplatin could set up a more accurate model reflecting AKI progression to renal interstitial fibrosis. Then, we investigated the effects and possible mechanisms of human umbilical cord blood mononuclear cells (hUCBMNCs) on renal tubular interstitial fibrosis after cisplatin-induced AKI. In rats injected with hUCBMNCs for four times, level of matrix metalloproteinase 7 (MMP-7) in serum and urine, urinary albumin/creatinine ratio, tubular pathological scores, the relative collagen area of the tubulointerstitial region, endoplasmic reticulum dilation and the mitochondrial ultrastructural damage were significantly improved. The level of reactive oxygen species, α-smooth muscle actin (α-SMA), [NOD]-like pyrin domain containing protein 3 and cleaved-Caspase 3 in renal tissue decreased significantly. However, in rats injected with hUCBMNCs for two times, no significant difference was discovered in MMP-7 levels and urinary albumin/creatinine ratio. Although expression of α-SMA and the percentage areas of collagen staining in tubulointerstitial tissues were ameliorated in rats injected with hUCBMNCs for two times, the effects were significantly weaker than those in rats injected with hUCBMNCs for four times. Taken together, our study constructed a highly efficient, duplicable novel rat model of renal fibrosis after cisplatin-induced AKI. Multiple injections of hUCBMNCs may prevent renal interstitial fibrosis after cisplatin-induced AKI.

Human umbilical cord blood mononuclear cells protect against renal tubulointerstitial fibrosis in cisplatin-treated rats

Currently, there is no effective method to prevent renal interstitial fibrosis after acute kidney injury (AKI). In this study, we established and screened a new renal interstitial fibrosis rat model after cisplatin-induced AKI. Our results indicated that rats injected with 4 mg/kg cisplatin once a week for two weeks after firstly administrated with 6.5 mg/kg loading dose of cisplatin could set up a more accurate model reflecting AKI progression to renal interstitial fibrosis. Then, we investigated the effects and possible mechanisms of human umbilical cord blood mononuclear cells (hUCBMNCs) on renal tubular interstitial fibrosis after cisplatin-induced AKI. In rats injected with hUCBMNCs for four times, level of matrix metalloproteinase 7(MMP-7)in serum and urine, urinary albumin/creatinine ratio, tubular pathological scores, the relative collagen area of the tubulointerstitial region, endoplasmic reticulum dilation and the mitochondrial ultrastructural damage were significantly improved. The level of reactive oxygen species, α-smooth muscle actin (α-SMA), [NOD]-like pyrin domain containing protein 3 and cleaved-Caspase 3 in renal tissue decreased significantly. However, in rats injected with hUCBMNCs for two times, no significant difference was discovered in MMP-7 levels and urinary albumin/creatinine ratio. Although expression of α-SMA and the percentage areas of collagen staining in tubulointerstitial tissues were ameliorated in rats injected with hUCBMNCs for two times, the effects were significantly weaker than those in rats injected with hUCBMNCs for four times. Taken together, our study constructed a highly efficient, duplicable novel rat model of renal fibrosis after cisplatin-induced AKI. Multiple injections of hUCBMNCs may prevent renal interstitial fibrosis after cisplatin-induced AKI.

Mesenchymal Stem Cells Treatment for Erectile Dysfunction in Diabetic Rats.

Aging men with diabetes mellitus are more easily suffering from erectile dysfunction (ED), which was poor to respond to drugs. Mesenchymal stem cell treatment (MSCT) offers us an alternative approach that might reverse diabetes mellitus erectile dysfunction (DMED). The aim of this study was to review the current studies investigating mesenchymal stem cell approach in diabetic rat models of ED for future research. A medical literature search was performed in PubMed by using the keywords including erectile dysfunction, mesenchymal stem cells, diabetes mellitus, and rat model. Representative studies on DMED rats treated by MSCT were reviewed. Streptozocin-induced type 1 diabetes mellitus rats were used in most studies because of cost and convenience. With the development of stem cell treatment for DMED research, many kinds of stem cells were used in animal experiment, such as bone marrow-derived mesenchymal stem cells, adipose-derived stem cells, human umbilical cord blood mononuclear cells, muscle-derived stem cells, urine-derived stem cells, neural crest stem cells, and endothelial progenitor cells. Although diverse stem cells were applied for DMED treatment, the mechanism behind these approaches was identical, including improving vascular injury, recovering smooth muscle, restoring neuronal cells, inhibiting the generation of inflammatory cytokines, homing mesenchymal stem cells, and decreasing apoptosis in corpus cavernosum. Meanwhile, combination therapies, including MSCT with drug, herb, and low-energy extracorporeal shockwave treatment showed satisfactory results for ED. It has been proved that MSCT is an effective and safe treatment for the DMED rats. What's more, MSCT might be a potential and promising approach for patients with DMED as a minimally invasive treatment. Combination of MSCT with various methods was proved to be a more efficient treatment and dependable option to make up for deficiencies of MSCT. Kim SW, Zhu GQ, Bae WJ. Mesenchymal Stem Cells Treatment for Erectile Dysfunction in Diabetic Rats. Sex Med Rev 2020;8:114-121.

MicroRNA signature of human blood mononuclear cells.

MicroRNAs (miRNAs) regulate a wide range of cellular processes and functions. Blood mononuclear cells (BMNCs) participate in the immune response, inflammatory reaction and angiogenesis. In 2010, a total of 157 miRNAs were quantified by RT-qPCR and a miRNA signature was determined for human peripheral BMNCs. With the advent of technologies such as RNA sequencing, many new miRNAs have been identified. This study was designed to provide an up-to-date miRNA signature for human BMNCs. Peripheral BMNCs were isolated by Ficoll density gradient centrifugation. Using the qPCR array assay, we identified 108 highly expressed miRNAs (Ct value < 30) in human BMNCs. Further validation of the array results by quantifying select miRNAs with RT-qPCR revealed a strong correlation between Ct values derived from array analysis and RT-qPCR, suggesting the array results presented in this study are accurate and reliable. Of note, the function of the majority of the highly expressed miRNAs we have identified has not yet been studied. Our findings may help direct further studies of the regulatory roles of miRNAs in BMNC function.

Abstract 021: Single Cell Multiplex Mass Cytometry Reveals Differential Abundance of Specific Memory and Regulatory T Cell Populations in Human Hypertension

Hypertension is the leading risk factor for morbidity and mortality worldwide. Emerging evidence in animal models demonstrates the importance of a variety of innate and adaptive immune cells in hypertension. We hypothesized that the abundance and phenotype of specific circulating immune cell subsets is altered in human hypertension reflecting disease pathophysiology. We performed unbiased high dimensional, single cell profiling of peripheral blood mononuclear cells in humans with a panel of 31 cell surface markers using mass cytometry. Unsupervised computational analysis from 11 control and 10 hypertensive individuals matched for age, gender, race, and body mass index revealed consistent increases in a novel memory helper T cell subset in hypertension. Manual two dimensional gating revealed that this CD4 + CD45RO + CD62L + CCR7 - CD161 lo memory cell population is nearly 2-fold increased in hypertensive subjects (1.0 vs 1.9%). As an alternative to starting with unsupervised analysis, manual gating for major known immune cell populations revealed a 40% decrease in memory cytotoxic T cells and a 29% decrease in regulatory T cells in hypertensive individuals (3.2 vs 1.9% and 1.3 vs 0.93%, respectively). Unsupervised analysis of the cellular subsets of these populations using Phenograph revealed a selective 63% decrease in PD-1 + memory cytotoxic T cells and 49% decrease in CCR10 + regulatory T cells in hypertension (0.67 vs 0.25% and 0.37 vs 0.19%, respectively). Taken together, these results demonstrate that in human hypertension there are increases in a novel memory helper T cell subset and decreases in PD-1 + memory cytotoxic and CCR10 + regulatory T cell populations. These findings may provide new insights into hypertension pathogenesis and potential therapeutic targets.

Leishmanicidal and antimicrobial activity of primin and primin-containing extracts from Miconia willdenowii

As a part of an ongoing bioprospective project, searching for potential medicinal plants from the Brazilian Atlantic Forest, Miconia willdenowii was selected for its potential leishmanicidal and antimicrobial activities. The crude ethanolic extract of M. willdenowii showed an inhibition of 99.7% of the promastigote forms of Leishmania amazonensis at the concentration of 80 μg/mL. Further investigation of its antimicrobial activity against pathogenic fungi and Gram positive and negative bacteria, revealed a significant antimicrobial activity. A bioguided study with its liquid-liquid partition fractions revealed the hexane fraction (Hex) as the most active against Leishmania, inhibiting 99.2% and 46.9% of the protozoan at concentrations of 40 and 20 μg/mL, respectively. Hex also showed significant antimicrobial activity against Staphylococcus aureus and Candida krusei with IC50 of 15.6 and 62.5 μg/mL, respectively. Purification of Hex led to the isolation of 2-methoxy-6-pentyl-benzoquinone (1, also known as primin) as the active metabolite, probably responsible for the observed antimicrobial and anti-leishmania effects. Primin (1) disclosed leishmanicidal activity (IC50 = 1.25 μM), showing higher potency than the standard drug amphotericin B (IC50 = 5.08 μM), with additional antifungal effects against all tested fungi species. Compound 1 also showed significant activity against S. aureus (IC50 = 8.94 μM), showing a comparable potency with the reference drug chloramphenicol (IC50 = 6.19 μM), but with a potential cytotoxicity towards peripheral human blood mononuclear cells (CC50 = 255.15 μM). Here in, the antimicrobial and anti-L. amazonensis effects of M. willdenowii are reported for the first time, as well as Primin (1) as its probable bioactive metabolite.

Abstract 4051: First step for development of checkpoint inhibitors immune monitoring panels in human whole blood and peripheral mononuclear cells

Abstract Cancer immunotherapies represent more than half of treatments approved for oncology, as well as those currently in development. Among the key targets of these therapies are immune-checkpoint molecules (i.e. PD-1, LAG3, PD-L1, CTLA-4). Novel immunotherapies targeting these molecules are designed to impact tumor microenvironment immune-suppression. The characterization and monitoring of circulating and tumor infiltrating immune cells by flow cytometry is a critical aspect of pre-clinical and clinical drug development. In order to generated robust data to support drug development, flow cytometric methods must be fully optimized. In this presentation will discuss the critical aspects of assay optimization for flow cytometric methods with an emphasis on screening monoclonal antibody clones. The importance of the data is illustrated in the differences in assay performance when the correct clones are identified. Methods: Four different anti-PD-1 obtained from different providers were screened on activated human PBMC. Additionally, four PD-L1 and five LAG-3 monoclonal antibody clones were screened on activated or rested PBMC spiked in whole blood. All antibodies were all titrated to obtain saturating concentration and optimal titers of different clones were compared. Results: On CD4+ T cells from thawed cryopreserved peripheral blood mononuclear cells (PBMC), the best performance was observed with EH12.2H7 or EH12.1 clones which identified 30-33% of CD4+ T cells as PD-1 positive and up to 55-60% of CD8+ T cells as PD-1 positive. Clone eBioJ105 staining of the same samples on the same day identified 14% PD-1 positive in CD4+ T cell and around 40% PD-1 positive in CD8+ T cell, while clone MIH4 staining revealed low level of PD-1 expression on CD4+ T cells and CD8+ T cells. After ex vivo stimulation with staphylococcus enterotoxin B (SEB), EH12.2H7, EH12.1 and eBioJ105 clones all yielded similar results with 76-85% PD-1 positive on both CD4+ T cells and CD8+ T cells but fewer positive cells were identified with MIH4 clone. Out of the four PD-L1 antibodies tested, only two were able to distinguish a clear PD-L1 population on PBMC rested for 4 days and spiked in whole blood used as positive control for PD-L1 induction. For LAG-3 antibodies, all five were able to reveal the induction of this checkpoint molecule on cells activated by SEB and spiked in whole blood with % of LAG3+ cells in lymphocytes ranging from 13% to 25%. Conclusions: These results illustrate the importance of thorough reagent evaluation in order to achieve a sensitivity coherent with the information-rich promises of flow cytometry. Citation Format: Laila-Aicha Hanfi, Scott Sugden. First step for development of checkpoint inhibitors immune monitoring panels in human whole blood and peripheral mononuclear cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4051.

Administration of human umbilical cord blood mononuclear cells restores bladder dysfunction in streptozotocin-induced diabetic rats.

This study evaluated the effect of human umbilical cord blood mononuclear cells (HUCB-MNCs) on bladder dysfunction in streptozotocin (STZ; 35 mg/kg, i.v.)-induced diabetic rats. Adult male Sprague-Dawley rats (n = 30) were equally divided into three groups: control group, STZ-diabetic group, and HUCB-MNC-treated group (1 × 106 cells). HUCB-MNCs were isolated by density gradient centrifugation from eight healthy donors and injected into the corpus cavenosum in STZ-diabetic rats 4 weeks after the induction of diabetes. Studies were performed 4 weeks after HUCB-MNC or vehicle injection. In vitro organ bath studies were performed on bladder strips, whereas protein expression of hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), and α-smooth muscle actin (SMA) in the bladder and the ratio of smooth muscle cells (SMCs) to collagen were determined using western blotting and Masson trichrome staining. Neurogenic contractions of detrusor smooth muscle strips were 55% smaller in the diabetic group than control group (P < 0.05); these contractions were normalized by HUCB-MNC treatment. In addition, HUCB-MNC treatment restored the impaired maximal carbachol-induced contractile response in detrusor strips in the diabetic group (29%; P < 0.05). HUCB-MNC treatment improved the KCl-induced contractile response in the diabetic bladder (68%; P < 0.05), but had no effect on ATP-induced contractile responses. Increased expression of HIF-1α and VEGF protein and decreased expression of α-SMA protein and the SMC/collagen ratio in diabetic rats were reversed by HUCB-MNC. Administration of HUCB-MNCs facilitates bladder function recovery, which is likely related to downregulation of HIF-1α expression and attenuation of fibrosis in STZ-diabetic rats.

Phospho Tensin Homolog in Human and Lipid Peroxides in Peripheral Blood Mononuclear Cells Following Exposure to Flavonoids

Objectives: Studies have shown that human and peripheral blood mononuclear cells (PBMCs) are mostly used for research purposes to study several biochemical endpoints. The effects of the flavonoids, genistein, kaempferol, and quercetin on phospho tensin homolog (PTEN) levels in cancer cells (i.e., breast [BT549], lung [A549]), human embryonic kidney cells (HEK293), and the levels of lipid peroxides (LP) in PBMCs were respectively investigated.Materials and methods: Cancer, kidney, and PBMCs from several donors were each exposed to each of the flavonoids at concentrations of 0, 5, 10, 15, 20, and 25 µM. Our hypotheses were that exposure of cancer and kidney cells to genistein, kaempferol, and quercetin can increase PTEN and decrease lipid peroxides in PBMCs levels respectively to better cope with oxidative stress.Results: The results indicate that the flavonoids increased total PTEN levels in a dose-dependent manner. The effect of quercetin was more pronounced followed by genistein and kaempferol. Furthermore, decreases in lipid peroxides were observed in the PBMCs for the flavonoid-treated samples compared to those exposed to flavonoids and with oxidative stress as described by Fenton’s chemistry. Levels of LP in quercetin-treated samples were lower compared to kaempferol and genistein.Conclusions: The findings suggest that the flavonoids play an important role in controlling oxidative stress in several human cells.

Saturation of acyl chains converts cardiolipin from an antagonist to an activator of Toll-like receptor-4

Cardiolipins (CLs) are tetra-acylated diphosphatidylglycerols found in bacteria, yeast, plants, and animals. In healthy mammals, CLs are unsaturated, whereas saturated CLs are found in blood cells from Barth syndrome patients and in some Gram-positive bacteria. Here, we show that unsaturated but not saturated CLs block LPS-induced NF-κB activation, TNF-α and IP-10 secretion in human and murine macrophages, as well as LPS-induced TNF-α and IL-1β release in human blood mononuclear cells. Using HEK293 cells transfected with Toll-like receptor 4 (TLR4) and its co-receptor Myeloid Differentiation 2 (MD2), we demonstrate that unsaturated CLs compete with LPS for binding TLR4/MD2 preventing its activation, whereas saturated CLs are TLR4/MD2 agonists. As a consequence, saturated CLs induce a pro-inflammatory response in macrophages characterized by TNF-α and IP-10 secretion, and activate the alternative NLRP3 inflammasome pathway in human blood-derived monocytes. Thus, we identify that double bonds discriminate between anti- and pro-inflammatory properties of tetra-acylated molecules, providing a rationale for the development of TLR4 activators and inhibitors for use as vaccine adjuvants or in the treatment of TLR4-related diseases.Graphical abstract

Augmented Survival, Angiogenic Properties and Mobilization of Human Endothelial Progenitor Cells Following Exposure to Sevoflurane

Anesthetic preconditioning (APC) with volatile agents has cardiac anti‐ischemic virtues that may be linked to stimulating effects of volatile anesthetics on stem cells biology. We investigated the influence of sevoflurane (Sevo) on apoptosis, proliferation, incorporation in tube‐like structure and mobilization of human endothelial progenitor cells (EPCs). 7‐days old cultured EPCs derived from human cord blood mononuclear cells were exposed in vitro to sevoflurane 2% or 4% in air/5% CO2, or only to air/5% CO2 (sham control) for 2 hrs in a modular chamber. Proliferation, apoptosis and incorporation of EPCs into tube‐like structures of HUVEC was evaluated 24 hrs later. EPCs plasma levels were evaluated by flow‐cytometry and in vitro culture assay before exposure to sevoflurane (1 MAC in 50% O2) or only to 50% O2 (sham control) and 24 hrs afterwards, in blood samples collected from coronary artery disease patients. Values are expressed as means ± SEM, compared by ANOVA. In the in vitro preconditioning setting, sevoflurane stimulated EPCs proliferation estimated by the MTS assay (n = 12, p &lt; 0.01 for 2% Sevo, p &lt; 0.001 for 4% Sevo) and by counting adherent DilAcLDL+/ FITC‐UEAI+ cells (n = 4, p &lt; 0.01 for 2% Sevo, p &lt; 0.001 for 4% Sevo), diminished apoptosis measured by annexin V/ propidium iodide assay (n = 5, p &lt; 0.05 for both 2% and 4% Sevo), and raised the number of incorporated DiIAcLDL‐EPCs into HUVEC tube‐like structures (n = 4, p &lt; 0.01 for 2% Sevo, p &lt; 0.001 for 4% Sevo) when compared to control. Plasma CD45dim/CD34+/CD309+ and CD45dim/CD34+/CD309+/CD133+ mononuclear cell populations were increased in Sevo treated patients (n = 15) vs. the control group (n = 10) at 24 h post‐exposure vs. baseline (p &lt; 0.01), and the number of adherent DilAcLDL+/FITC‐UEAI+ cells in 7 days old EPCs cultures was significantly higher in the study group vs. control between the same time points (p &lt; 0.05). Our data prove that sevoflurane positively influences the biology of EPCs both in vivo and vitro, and may have an important cardioprotective impact in various interventional and surgical heart procedures by improving angiogenesis and ischemic tissue repair.Support or Funding InformationThe financial support was provided by UEFISCDI Romania, research grant PN‐II‐RUTE‐2012‐3‐0463.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing.

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5'-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird's-eye view of the antibody repertoire, and our computational methods.

Effects of Light on In Vitro Production of Melatonin by Human Peripheral Blood Mononuclear, Polymorphonuclear, and Whole Blood Cells

Recent studies have shown that melatonin can be secreted, in addition to other sources, by peripheral blood mononuclear cells (PBMCs). We investigated whether melatonin could also be secreted by cultured polymorphonuclear (PMNs) and whole blood (WB) cells under resting and stimulated conditions. In addition, influences of the intensity and color of light on the production of melatonin by these cells were estimated. Blood samples were collected from a healthy volunteer, and PBMCs and PMNs were isolated. The PBMC, PMN, and WB cultures were exposed for 8 h to light intensities of 0, 20, 50, 100, 300, and 500 lux and to white, red, blue, and green lights in the presence/absence of mitogens. Levels of melatonin in supernatants were detected by enzyme-linked immunosorbent assay (ELISA). In addition to PBMCs, PMNs and WB cells were found to also produce melatonin, and this production was increased due to mitogen activations. The most effective intensity of light, which suppresses melatonin secretion, was 100 lux. There were no significant differences between the influences of different monochromatic lights on the melatonin production by the examined blood cell cultures. Thus, PBMCs, PMNs, and WB cultures can produce melatonin, and mitogen activation causes a higher production of this hormone. Moreover, although white light suppressed melatonin secretion at 100 lux, there was no difference among various types of lights. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.

Immobilization of Denosumab on Titanium Affects Osteoclastogenesis of Human Peripheral Blood Monocytes.

Immobilization of proteins has been examined to improve implant surfaces. In this study, titanium surfaces were modified with nanofunctionalized denosumab (cDMAB), a human monoclonal anti-RANKL IgG. Noncoding DNA oligonucleotides (ODN) served as linker molecules between titanium and DMAB. Binding and release experiments demonstrated a high binding capacity of cDMAB and continuous release. Human peripheral mononuclear blood cells (PBMCs) were cultured in the presence of RANKL/MCSF for 28 days and differentiated into osteoclasts. Adding soluble DMAB to the medium inhibited osteoclast differentiation. On nanofunctionalized titanium specimens, the osteoclast-specific TRAP5b protein was monitored and showed a significantly decreased amount on cDMAB-titanium in PBMCs + RANKL/MCSF. PBMCs on cDMAB-titanium also changed SEM cell morphology. In conclusion, the results indicate that cDMAB reduces osteoclast formation and has the potential to reduce osteoclastogenesis on titanium surfaces.

Stem Cell from Human Exfoliated Deciduous Teeth (SHED) versus Human Umbilical Cord Blood Mononuclear Cells (cbMNC) Transplantation in Neural Damage Reduction in Rat Model of Cerebral Ischemia

Ischemic stroke is one of major cause of mortality and disability in Indonesia. Stem Cells are considered as a promising therapy for ischemic stroke. In this study, we compared therapeutic potency of Stem cell from human exfoliated deciduous teeth (SHED) and Human umbilical cord blood mononuclear cell (cbMNC) using rat models of ischemic stroke. Following middle cerebral artery occlusion (MCAO), twenty male wistar rats were divided into four groups : normal rats (n=5), rats undergone permanent MCAO (n=5) as the control (stroke) group, rats undergone permanent MCAO and SHED transplantation (n=5) and rats undergone permanent MCAO and cbMNC transplantation (n=5) as the treatment group. SHED transplantation was performed at the acute phase after MCAO by intravenous injection. Histopathological evaluation of the neuron death ratio with hematoxylin and eosin staining confirmed that there was no significant differences at comparative study of neuron death ratio in rats transplanted with SHED and rats transplanted with cbMNC (p=0,81). SHED and cbMNC transplantation at acute stroke showed reduction in the neuron death ratio in the brain of rat models with ischemic stroke, and may provide an opportunity for neuroprotection and neural regeneration after ischemic stroke.

Umbilical cord mesenchymal stem cells and umbilical cord blood mononuclear cells improve neonatal rat memory after hypoxia-ischemia

Current treatment options for hypoxic-ischemic encephalopathy (HIE) are limited. Human umbilical cord mesenchymal stem cells (UC-MSCs) and cord blood mononuclear cells (CB-MNCs) offer great potential for the treatment of many neurological diseases. The aim of the present study was to identify which cell type is more effective for the treatment of HIE. PKH26-labeled UC-MSCs and CB-MNCs were transplanted into rats with hypoxia-ischemia (HI)-induced brain damage. Apoptotic cell numbers in the brain, as labeled by TUNEL, were assessed. Myelination and gliosis were investigated using myelin basic protein and glial fibrillary acidic protein immunohistochemistry, respectively. The Morris water maze was used to assess animal learning abilities. Our data show that transplantation of UC-MSCs or CB-MNCs after HI reduced astrogliosis, prevented neuronal loss in the striatum, and markedly improved functional brain outcomes after a 28-day recovery period. Moreover, treatment with CB-MNCs increased the proportion of mature oligodendrocytes and improved myelination in cortical areas. Both UC-MSCs and CB-MNCs may result in the recovery of neurological function in HI rats. Based on our data, UC-MSCs and UCB-MNCs may be particularly effective stem cell subsets for treatment of neonatal HIE.