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Study Design: An established rabbit intervertebral disc (IVD)/endplate explant fracture model was extended with physiologic post-traumatic dynamic loading (PTDL) and coculturing of peripheral blood mononuclear cells (PBMCs).Objective: The aim of this study was to quantify the effects of PTDL and of cocultured PBMCs on post-traumatic disc degeneration (DD) and to determine whether PTDL facilitates homing of PBMC to fractured IVD/endplates.Summary Of Background Data: DD is associated with endplate fracture. In vivo studies suggest a key role of immune cells in the pathogenesis of DD. However, the complexity of in vivo systems impedes the investigation of single factors governing the pathogenesis.Methods: Seventy-two IVD/endplate specimens were divided into 4 groups. In group A, endplate fractures were induced with a high-velocity axial load and exposed to PTDL in coculture with PBMCs for 14 days. Group A was compared with 3 control groups, with single-factor removal, in order to assess the relative contribution of PTDL (group B), PBMCs (group C), and endplate fracture (group D) to the biological response of the IVD. Disc gene transcription and serum nitric oxide (NO) serum concentration were measured to investigate differences in anabolism, catabolism, and inflammatory response between the groups. Changes in matrix composition and disc structure were assessed histologically.Results: PBMCs did not home to fractured IVDs, with or without PTDL. Group A compared with group D showed an enhanced transcription of anabolic, catabolic, and pro-inflammatory genes during the entire experiment, and an increased NO concentration for the first 3 days. Changes typical for DD were also found in histological sections. Group A compared with group C showed significant increases in catabolic and pro-inflammatory gene transcription after at least 7 days. No differences were found between groups A and B.Conclusion: Trauma induces degenerative changes; PTDL neither aggravates nor ameliorates this response. Although PBMCs do not infiltrate the disc, they aggravate the degenerative changes.Level Of Evidence: N/A. [ABSTRACT FROM AUTHOR]

Background Measuring of oxidative stress in peripheral blood mononuclear cells is a suitable model of dietary induced systemic oxidative stress. Thus, we aimed to evaluate whether a chronic high fructose intake could induce oxidative damage in peripheral blood and bone marrow mononuclear cells of rats. Methods Animals were randomly assigned to the following groups: Control group (standard rat chow and tap water n = 8), and Fructose group (standard rat chow and a 10% fructose solution in the drinking water n = 8). Reactive oxygen species and cytokines were measure using flow cytometry in peripheral blood and bone-marrow mononuclear cells. Apoptotic cell death and the advanced oxidation protein products (AOPP) were also determined. Results We observed a significant increase in ROS production in peripheral blood mononuclear cells of fructose group as compared to control rats. Apoptosis and the AOPP were higher in those animals underwent high fructose intake. Serum levels of IL-6 and IL-12 were also increased after 12 weeks of high fructose intake. Conclusion We concluded that fructose intake leads to systemic oxidative stress and pro-inflammatory condition which affect peripheral blood mononuclear cells and bone-marrow mononuclear cells viability. [ABSTRACT FROM AUTHOR]

Background: The ability for engulfment of pathogens and inert particles is the key hallmark of the phagocytic cells. Phagocytes play a significant role in the modulation of local or extended inflammation. Since fever activates a number of factors linked with the immune response it was the goal of this study to examine the in vitro effect of hyperthermia on the phagocytic capacity, the number of phagocytic cells and the viability of human peripheral blood mononuclear cells (PBMC) at 37 and 40°C. Methods: PBMC were incubated with 0.8 μm polysterene latex beads, for 2 hours at 37 and 40°C. The number of phagocytic cells, and that of latex particles internalized by each individual cell was counted with a light microscope. In addition, the percentage of viable cells and the number of active metabolic cells was evaluated. Results: A temperature of 40°C significantly increased the number of phagocytic cells and the phagocytic index by 41 and 37% respectively, as compared to cells incubated at 37°C. While the number of vital cells (trypan blue test) did not differ statistically at both temperatures, the number of active metabolic cells (XTT test) after 2 h of incubation at 40°C was 17% higher as compared with that at 37°C. However, the number of active metabolic cells after 24 h of incubation at 40°C was 51% lower compared with cells incubated at 37°C. Conclusions: The increased phagocytic capacity of human peripheral blood monocytes at high temperature further enlightens the immunomodulatory effect of fever in the immune responses during inflammation. [ABSTRACT FROM AUTHOR]

Background: Recent studies provide evidence that peripheral blood monocytes have the ability to differentiate into mesenchymal- like cells. The ability of cultured monocytes to differentiate and produce insulin in vitro is analysed in the present study. Methods: Peripheral blood monocytes were isolated from healthy donors and cultivated for fourteen days. Growth factors and liraglutide were used to induce pancreatic differentiation in most of the cultures. The growth factors were: monocyte colonystimulating factor, interleukin-3, hepatocyte growth factor and epidermal growth factor. The rest of the cultures were cultivated only with nutrient medium and human serum. Insulin levels were measured by an enzyme-linked immunosorbent assay. Cellular morphology was observed using optical and electron microscopy. Cell membrane receptors were detected by flow cytometry. Results: Monocytes were able to synthesize and excrete high levels of insulin after seven days in culture. A further increase in the excretion of insulin was observed after fourteen days. Cells were also able to differentiate and synthesize insulin, even if no growth factors were added to the culture medium. Some of the cultures were able to excrete insulin in a glucose-dependent manner. Differentiated monocytes were connected to neighbouring cells with axons and resembled the morphology of mesenchymal, dendritic and myeloid-progenitor cells. Cells retained their mature receptors and simultaneously developed immature receptors on their membrane. Conclusions: Monocytes can acquire morphological properties of multipotent cells when they are cultivated under specific conditions in vitro. Differentiated monocytes are able to synthesize and excrete insulin. [ABSTRACT FROM AUTHOR]

Morbidity and mortality caused by schistosomiasis usually results from immunopathology. But the underlying mechanisms are not yet clearly understood. Th2-type immune response is thought to be dominant during chronic schistosomiasis, and upregulation of arginase-I is one component of this milieu. A cohort study was conducted to assess arginase activity in peripheral blood of humans with intestinal schistosomiasis in Wonji-Shoa Sugar Estate, Central Ethiopia. Laboratory-confirmed 30 Schistosoma mansoni-infected patients and 18 apparently healthy controls were recruited. Faecal egg count was carried out by Kato-Katz technique. Plasma and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood. Activity of arginase in plasma and PBMC lysates was measured, and results were compared with that of controls. Twenty-one of 30 patients had light infection, whereas moderate and heavy intensity infections were observed in eight and only one patient(s), respectively. A significant increase in both PBMC (patients: 59·96 + 82·99, controls: 25·44 + 24·6 mU/mg protein, P < 0·0001) and plasma (patients: 1·61 + 2·19, controls: 0·31 + 0·73 mU/mL plasma, P < 0·0001) arginase activity was observed during human S. mansoni infection. Arginase activity increases in peripheral blood of patients with intestinal schistosomiasis. [ABSTRACT FROM AUTHOR]

Cerebral palsy (CP) is a non progressive, demyelinating disorder that affects a child's development and posture and may be associated with sensation, cognition, communication and perception abnormalities. In CP, cerebral white matter is injured resulting in the loss of oligodendrocytes. This causes damage to the myelin and disruption of nerve conduction. Cell therapy is being explored as an alternate therapeutic strategy as there is no treatment currently available for CP. To study the benefits of this treatment we have administered autologous bone marrow mononuclear cells (BMMNCs) to a 12-year-old CP case. He was clinically re-evaluated after six months and found to demonstrate positive clinical and functional outcomes. His trunk strength, upper limb control, hand functions, walking stability, balance, posture and coordination improved. His ability to perform activities of daily living improved. On repeating the Functional Independence Measure (FIM), the score increased from 90 to 113. A repeat positron emission tomography- computed tomography (PET-CT) scan of the brain six months after intervention showed progression of the mean standard deviation values towards normalization which correlated to the functional changes. At one year, all clinical improvements have remained. This indicated that cell transplantation may improve quality of life and have a potential for treatment of CP. [ABSTRACT FROM AUTHOR]

Background: Most of the blood tests aiming for breast cancer screening rely on quantification of a single or few biomarkers. The aim of this study was to evaluate the feasibility of detecting breast cancer by analyzing the total biochemical composition of plasma as well as peripheral blood mononuclear cells (PBMCs) using infrared spectroscopy. Methods: Blood was collected from 29 patients with confirmed breast cancer and 30 controls with benign or no breast tumors, undergoing screening for breast cancer. PBMCs and plasma were isolated and dried on a zinc selenide slide and measured under a Fourier transform infrared (FTIR) microscope to obtain their infrared absorption spectra. Differences in the spectra of PBMCs and plasma between the groups were analyzed as well as the specific influence of the relevant pathological characteristics of the cancer patients. Results: Several bands in the FTIR spectra of both blood components significantly distinguished patients with and without cancer. Employing feature extraction with quadratic discriminant analysis, a sensitivity of ~90 % and a specificity of ~80 % for breast cancer detection was achieved. These results were confirmed by Monte Carlo cross-validation. Further analysis of the cancer group revealed an influence of several clinical parameters, such as the involvement of lymph nodes, on the infrared spectra, with each blood component affected by different parameters. Conclusion: The present preliminary study suggests that FTIR spectroscopy of PBMCs and plasma is a potentially feasible and efficient tool for the early detection of breast neoplasms. An important application of our study is the distinction between benign lesions (considered as part of the non-cancer group) and malignant tumors thus reducing false positive results at screening. Furthermore, the correlation of specific spectral changes with clinical parameters of cancer patients indicates for possible contribution to diagnosis and prognosis. [ABSTRACT FROM AUTHOR]

Relationships between n-3 long chain polyunsaturated fatty acids (LC-PUFA) in plasma and synovial fluid (SF) were examined in 36 patients with knee effusion within the context of a variety of rheumatic diagnoses and various stated fish oil (FO) intakes (from 0 to 30 mL of standard FO daily) of variable duration. In a sub-group of patients, correlations between PUFA in SF mononuclear cells (MNC) and cell-free supernatants of SF and between SF MNC and peripheral blood (PB) MNC were examined. Correlations were also sought between clinical data (stated FO intake, pain score) and n-3 LC-PUFA. Correlations between plasma n-3 LC-PUFA and SF n-3 LC-PUFA were very strong ( r 2 >0.9, p <0.001). The LC-PUFA profiles of SF supernatants differed from those of MNC. PUFA profiles in PB MNC and SF MNC were similar, except for a higher proportion of DHA in the latter. Positive correlations were observed between stated intakes of FO and EPA in plasma and SF (for both r =0.37, p =0.02) and DHA in plasma ( r =0.37, p =0.02) and SF ( r =0.36, p =0.03). n-3 LC-PUFA in plasma and SF correlated inversely with pain score (plasma r 2 =0.16, p <0.02; SF r 2 0.32, p =0.001). In conclusion, plasma n-3 LC-PUFA is a strong indicator of SF n-3 LC-PUFA status across a broad range of rheumatic diagnoses and FO intakes. Higher n-3 LC-PUFA in plasma and SF were associated with lesser pain experience. [ABSTRACT FROM AUTHOR]

In polycystic ovary syndrome (PCOS), oxidative stress is implicated in the development of β-cell dysfunction. However, the role of mononuclear cell (MNC)-derived inflammation in this process is unclear. We determined the relationship between β-cell function and MNC-derived nuclear factor-ΰB (NF-ΰB) activation and tumor necrosis factor-α (TNF-α) secretion in response to a 2-h 75-g oral glucose tolerance test (OGTT) in normoglycemic women with PCOS (15 lean, 15 obese) and controls (16 lean, 14 obese). First- and second-phase β-cell function was calculated as glucose-stimulated insulin secretion (insulin/glucose area under the curve for 0â€"30 and 60â€"120 min, respectively) Ã-- insulin sensitivity (Matsuda Index derived from the OGTT). Glucose-stimulated NF-ΰB activation and TNF-α secretion from MNC, and fasting plasma thiobarbituric acid-reactive substances (TBARS) and high-sensitivity C-reactive protein (hs-CRP) were also assessed. In obese women with PCOS, first- and second-phase β-cell function was lower compared with lean and obese controls. Compared with lean controls, women with PCOS had greater change from baseline in NF-ΰB activation and TNF-α secretion, and higher plasma TBARS. β-Cell function was inversely related to NF-ΰB activation (1st and 2nd) and TNF-α secretion (1st), and plasma TBARS and hs-CRP (1st and 2nd). First- and second-phase β-cell function also remained independently linked to NF-ΰB activation after adjustment for body fat percentage and TBARS. In conclusion, β-cell dysfunction in PCOS is linked to hyperglycemia-induced NF-ΰB activation from MNC and systemic inflammation. These data suggest that in PCOS, inflammation may play a role in impairing insulin secretion before the development of overt hyperglycemia. [ABSTRACT FROM AUTHOR]

Background aims. The diverse phenotypic changes and clinical and economic disadvantages associated with the monolayer expansion of bone marrow-derived mesenchymal stromal cells (MSCs) have focused attention on the development of onestep intraoperative cells therapies and homing strategies. The mononuclear cell fraction of bone marrow, inclusive of discrete stem cell populations, is not well characterized, and we currently lack suitable cell culture systems in which to culture and investigate the behavior of these cells. Methods. Human bone marrow-derived mononuclear cells were cultured within fibrin for 2 weeks with or without fibroblast growth factor-2 supplementation. DNA content and cell viability of enzymatically retrieved cells were determined at days 7 and 14. Cell surface marker profiling and cell cycle analysis were performed by means of multi-color flow cytometry and a 5-ethynyl-2'-deoxyuridine incorporation assay, respectively. Results. Total mononuclear cell fractions, isolated from whole human bone marrow, was successfully cultured in fibrin gels for up to 14 days under static conditions. Discrete niche cell populations including MSCs, pericytes and hematopoietic stem cells were maintained in relative quiescence for 7 days in proportions similar to that in freshly isolated cells. Colony-forming unit efficiency of enzymatically retrieved MSCs was significantly higher at day 14 compared to day 0; and in accordance with previously published works, it was fibroblast growth factor-2-dependant. Conclusions. Fibrin gels provide a simple, novel system in which to culture and study the complete fraction of bone marrow-derived mononuclear cells and may support the development of improved bone marrow cell-based therapies. [ABSTRACT FROM AUTHOR]

Background aims. Traditional bone marrow (BM) collection is inadequate for separation of abundant mononuclear cells (MNCs). We aimed to investigate the effects of preoperative exercise on BM collection in patients with type 2 diabetes mellitus (T2DM). Methods. Sixty patients with T2DM were randomly assigned to either a control group or an exercise group (n = 30 each). The patients in the exercise group exercised before the collection. All patients underwent routine surgical care. The collected BM volume, operation duration, collecting speed, puncture times and pain scores were recorded. BM samples were tested before and after MNCs separation for CD34+ flow cytometry and whole blood cell count. Results. The collected BM volumes were significantly larger and collection speed was faster in the exercise group (379.77 ± 4.93 mL and 1.40 ± 0.14 mlVs) than those in the control group (356.67 ± 15.36 mL and 0.89 ±0.16 rnL/s, P = 0.00 for both). Puncture times were significantly less and pain scores were lower in the exercise group (2.07 ± 0.25 and 2.67 ± 1.56) than those in the control group (2.50 ± 0.63 and 3.43 ± 1.76, P = 0.00 and 0.02, respectively). CD34+ cells and whole blood cell count variables were comparable in the 2 groups. Conclusions. Preoperative exercise facilitates BM collection by increasing collected volume, improving collecting speed, relieving patients' pains and ensuring MNC quality [ABSTRACT FROM AUTHOR]

Aims Supernatants of serum-free cultured mononuclear cells (MNC) contain a mix of immunomodulating factors (secretome), which have been shown to attenuate detrimental inflammatory responses following myocardial ischaemia. Inflammatory dilated cardiomyopathy (iDCM) is a common cause of heart failure in young patients. Experimental autoimmune myocarditis (EAM) is a CD4+ T cell-dependent model, which mirrors important pathogenic aspects of iDCM. The aim of this study was to determine the influence of MNC secretome on myocardial inflammation in the EAM model. Methods and results BALB/c mice were immunized twice with an alpha myosin heavy chain peptide together with Complete Freund adjuvant. Supernatants from mouse mononuclear cells were collected, dialysed, and injected i.p. at Day 0, Day 7, or Day 14, respectively. Myocarditis severity, T cell responses, and autoantibody formation were assessed at Day 21. The impact of MNC secretome on CD4+ T cell function and viability was evaluated using in vitro proliferation and cell viability assays. A single high-dose application of MNC secretome, injected at Day 14 after the first immunization, effectively attenuated myocardial inflammation. Mechanistically, MNC secretome induced caspase-8-dependent apoptosis in autoreactive CD4+ T cells. Conclusion MNC secretome abrogated myocardial inflammation in a CD4+ T cell-dependent animal model of autoimmune myocarditis. This anti-inflammatory effect of MNC secretome suggests a novel and simple potential treatment concept for inflammatory heart diseases. [ABSTRACT FROM AUTHOR]

Tissue engineering provides a promising alternative therapy to the complex surgical reconstruction of auricular cartilage by using ear-shaped autologous costal cartilage. Bacterial nanocellulose (BNC) is proposed as a promising scaffold material for auricular cartilage reconstruction, as it exhibits excellent biocompatibility and secures tissue integration. Thus, this study evaluates a novel bilayer BNC scaffold for auricular cartilage tissue engineering. Bilayer BNC scaffolds, composed of a dense nanocellulose layer joined with a macroporous composite layer of nanocellulose and alginate, were seeded with human nasoseptal chondrocytes (NC) and cultured in vitro for up to 6 weeks. To scale up for clinical translation, bilayer BNC scaffolds were seeded with a low number of freshly isolated (uncultured) human NCs combined with freshly isolated human mononuclear cells (MNC) from bone marrow in alginate and subcutaneously implanted in nude mice for 8 weeks. 3D morphometric analysis showed that bilayer BNC scaffolds have a porosity of 75% and mean pore size of 50 ± 25 μm. Furthermore, endotoxin analysis and in vitro cytotoxicity testing revealed that the produced bilayer BNC scaffolds were non-pyrogenic (0.15 ± 0.09 EU/ml) and non-cytotoxic (cell viability: 97.8 ± 4.7%). This study demonstrates that bilayer BNC scaffolds offer a good mechanical stability and maintain a structural integrity while providing a porous architecture that supports cell ingrowth. Moreover, bilayer BNC scaffolds provide a suitable environment for culture-expanded NCs as well as a combination of freshly isolated NCs and MNCs to form cartilage in vitro and in vivo as demonstrated by immunohistochemistry, biochemical and biomechanical analyses. [ABSTRACT FROM AUTHOR]

Hereditary hemorrhagic telangiectasia (HHT) or Rendu-Osler-Weber disease is a rare genetic vascular disorder known for its endothelial dysplasia causing arteriovenous malformations and severe bleedings. HHT-1 and HHT-2 are the most prevalent variants and are caused by heterozygous mutations in endoglin and activin receptor-like kinase 1, respectively. An undervalued aspect of the disease is that HHT patients experience persistent inflammation. Although endothelial and mural cells have been the main research focus trying to unravel the mechanism behind the disease, wound healing is a process with a delicate balance between inflammatory and vascular cells. Inflammatory cells are part of the mononuclear cells (MNCs) fraction, and can, next to eliciting an immune response, also have angiogenic potential. This biphasic effect of MNC can hold a promising mechanism to further elucidate treatment strategies for HHT patients. Before MNC are able to contribute to repair, they need to home to and retain in ischemic and damaged tissue. Directed migration (homing) of MNCs following tissue damage is regulated by the stromal cell derived factor 1 (SDF1). MNCs that express the C-X-C chemokine receptor 4 (CXCR4) migrate toward the tightly regulated gradient of SDF1. This directed migration of monocytes and lymphocytes can be inhibited by dipeptidyl peptidase 4 (DPP4). Interestingly, MNC of HHT patients express elevated levels of DPP4 and show impaired homing toward damaged tissue. Impaired homing capacity of the MNCs might therefore contribute to the impaired angiogenesis and tissue repair observed in HHT patients. This review summarizes recent studies regarding the role of MNCs in the etiology of HHT and vascular repair, and evaluates the efficacy of DPP4 inhibition in tissue integrity and repair. [ABSTRACT FROM AUTHOR]

• This study focuses on the effects of Tmsb4x on human septicemia. • Tmsb4x is poorly expressed in PB of patients with septicemia. • Overexpressed Tmsb4x promotes differentiation of mononuclear cells into DCs. • Generation of DCs enhances immune response during septicemia. • Tmsb4x deficiency reduces the proportion of DCs in the PB of septicemia patients. Thymosin beta 4 × (Tmsb4x) has been highlighted as an important regulator in immune and inflammation responses. Promoted differentiation of mononuclear cells into dendritic cells (DCs) exert a beneficial effect on septicemia. Herein, we investigated the effects of Tmsb4x on the mononuclear cells to affect immune responses during septicemia. Initially, we isolated peripheral blood samples from healthy individuals and patients with septicemia for extraction of mononuclear cells, followed by Tmsb4x expression quantification. A cell model was constructed with mononuclear cells through lipopolysaccharide stimulation. The viability and apoptosis were evaluated in response to Tmsb4x silencing or re-expression. Additionally, the proportion of DCs was assessed by determining levels of inflammatory factors as well as by flow cytometric analysis. A mouse septicemia model was developed for in vivo validation. Cell and animal models demonstrated decreased Tmsb4x expression in the setting of septicemia, which led to increased inflammatory response and reduced proportion of DCs, along with inhibited mononuclear cell viability and promoted apoptosis. However, restoration of Tmsb4x facilitated the differentiation of mononuclear cells into DCs. To conclude, upregulated Tmsb4x promoted the generation of DCs from mononuclear cells, which contributed to deep understanding of underpinning mechanisms in the development of septicemia. [ABSTRACT FROM AUTHOR]

Background: This study aimed to identify the density of mononuclear cells (MNCs) and CD34+ cells in the bone marrow of patients with three neurologic conditions., Methods: The study included 88 patients with three neurologic conditions: 40 with cerebral palsy (CP) due to oxygen deprivation (OD), 23 with CP related to neonatal icterus (NI), and 25 with neurological sequelae after traumatic brain injury. Bone marrow aspiration was conducted from the patients' bilateral anterior iliac crest under general anesthesia in an operating theater. MNCs were isolated by Ficoll gradient centrifugation and then infused intrathecally., Results: There was a significant difference in the average MNC per ml and percentage of CD34+ cells by the type of disease, age group, and infusion time (p value < 0.05). The multivariable regression model showed the percentage of CD34+ association with the outcome (gross motor function 88 items- GMFM-88) in patients with CP., Conclusions: The density of MNCs was 5.22 million cells per mL and 5.03% CD34+ cells in patients with three neurologic conditions. The highest density of MNCs in each ml of bone marrow was found in patients with CP due to OD, whereas the percentage of CD34+ cells was the highest among patients with CP related to NI., (© 2023. The Author(s).)

Background: We aimed to determine whether NLRP3 inflammasomes in peripheral blood mononuclear cells (PBMC) were associated with carotid plaque instability in carotid atherosclerosis patients., Methods: Consecutive 38 carotid atherosclerosis with vulnerable plaques, 22 carotid atherosclerosis with stable plaques, and 40 healthy subjects with no carotid or coronary artery stenosis were enrolled. They were referred to the Second Hospital of Dalian Medical University from 2013-2019. Carotid plaques were evaluated by modified plaque vulnerability risk score (MPVRS) and pathological assessment. The mRNA and protein expression of NLRP3 inflammasome components in PBMC were determined by quantitative real time PCR and Western blot analysis or ELISA., Results: When consecutive study subjects undergoing carotid endarterectomy were divided into stable (≤4) and unstable (>4) plaque groups according to the MPVRS, the unstable plaque group had significantly raised mRNA and protein expression of NLRP3 inflammasome components in PBMC as compared with the stable plaque group and healthy subject group. Furthermore, subjects with higher NLRP3 protein expression in PBMC had greater incidence of cerebrovascular events., Conclusion: Increased NLRP3 inflammasome components in PBMC is associated with instability of human carotid atherosclerotic plaques, suggesting NLRP3 inflammasome as a potential biomarker for monitoring carotid plaque instability., (Copyright ©2023 Xie et al. Published by Tehran University of Medical Sciences.)

Skeletal muscle possesses a remarkable regenerative capacity, mainly relying on a population of undifferentiated and unipotent muscle progenitors, called muscle stem cells (MuSCs) or satellite cells, and their interplay with various cell types within the niche. Investigating the cellular composition of skeletal muscle tissues and the heterogeneity among various cell populations is crucial to the unbiased understanding of how cellular networks work in harmony at the population level in the context of skeletal muscle homeostasis, regeneration, aging, and diseases. As opposed to probing the average profile in a cell population, single-cell RNA-seq has unlocked access to the transcriptomic landscape characterization of individual cells in a highly parallel manner. This chapter describes the workflow for single-cell transcriptomic analysis of mononuclear cells in skeletal muscle by taking advantage of the droplet-based single-cell RNA-seq platform, Chromium Single Cell 3' solution from 10x Genomics ® . Using this protocol, we can reveal insights into muscle-resident cell-type identities, which can be exploited to study the muscle stem cell niche further., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Pyroptosis is an immunological response to infection and cellular stresses initiated by inflammasome oligomerization resulting in the release of pro-inflammatory factors including cytokines and other immune stimuli into the extracellular matrix. In order to understand the role of inflammasome activation and subsequent pyroptosis in human infection and disease pathogenesis and to explore markers of these signaling events as potential disease or response biomarkers, we must utilize quantitative, reliable, and reproducible assays to readily investigate these pathways in primary specimens. Here, we describe two methods using imaging flow cytometry for evaluation of inflammasome ASC specks in homogeneous peripheral blood monocytes and in bulk, heterogeneous peripheral blood mononuclear cells. Both methods can be applied to assess speck formation as a biomarker for inflammasome activation in primary specimens. Additionally, we describe the methods for quantification of extracellular oxidized mitochondrial DNA from primary plasma samples, serving as a proxy for pyroptosis. Collectively, these assays may be utilized to determine pyroptotic influences on viral infection and disease development or as diagnostic aids and response biomarkers., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Heme oxygenase (HO)-1 is the inducible isoform of the heme-degrading enzyme HO, which is upregulated by multiple stress stimuli. HO-1 has major immunomodulatory and anti-inflammatory effects via its cell-type-specific functions in mononuclear cells. Contradictory findings have been reported on HO-1 regulation by the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) in these cells. Therefore, we reinvestigated the effects of LPS on HO-1 gene expression in human and murine mononuclear cells in vitro and in vivo. Remarkably, LPS downregulated HO-1 in primary human peripheral blood mononuclear cells (PBMCs), CD14 + monocytes, macrophages, dendritic cells, and granulocytes, but upregulated this enzyme in primary murine macrophages and human monocytic leukemia cell lines. Furthermore, experiments with human CD14 + monocytes revealed that activation of other TLRs including TLR1, -2, -5, -6, -8, and -9 decreased HO-1 mRNA expression. LPS-dependent downregulation of HO-1 was specific, because expression of cyclooxygenase-2, NADP(H)-quinone oxidoreductase-1, and peroxiredoxin-1 was increased under the same experimental conditions. Notably, LPS upregulated expression of Bach1, a critical transcriptional repressor of HO-1. Moreover, knockdown of this nuclear factor enhanced basal and LPS-dependent HO-1 expression in mononuclear cells. Finally, downregulation of HO-1 in response to LPS was confirmed in PBMCs from human individuals subjected to experimental endotoxemia. In conclusion, LPS downregulates HO-1 expression in primary human mononuclear cells via a Bach1-mediated pathway. As LPS-dependent HO-1 regulation is cell-type- and species-specific, experimental findings in cell lines and animal models need careful interpretation. [ABSTRACT FROM AUTHOR]

Background: This study aims to examine the immunological parameters, focusing IL-10 productivity, in prophylactic sublingual immunotherapy (SLIT) in asymptomatic subjects sensitized to Japanese cedar pollen (JCP). Methods: This study was conducted as part of a randomized, double-blind, placebo-controlled, multiple center trial, and was performed for two consecutive pollen seasons in 2012 and 2013. The present results were based only on our institution. We recruited 29 participants with specific IgE against JCP of at class 2 and higher levels without history of the pollinosis symptoms at the time of JCP scattering. The SLIT group received standardized JCP extract for five months over the pollen season. We observed and judged development of the symptoms in the pollen season. The percentage of IL-10 producing CD4+T (Trl) cells, B cells and monocytes were analyzed by flow cytometry. JCP specific IgE and total IgE were also measured. Results: The ratio of development of cedar pollinosis was significantly lower in the SLIT group compared to the placebo group in 2013. In 2012, the percentage of circulating Trl cells and IL-10 producing monocytes significantly increased in the SLIT group. In 2013, the percentage of circulating Trl cells and IL-10 producing B cells increased significantly in the SLIT group. The percentage of circulating IL-10 producing monocytes significantly decreased in the placebo group. Conclusions: Prophylactic SLIT is effective for prevention of the development of pollinosis. Induction of IL-10 producing T cells, B cells and monocytes is an important mechanism of SLIT for prevention of pollinosis in asymptomatic but sensitized subjects. [ABSTRACT FROM AUTHOR]

Recent reports have highlighted the roles of free fatty acid receptor 2 (FFAR2) in the ° regulation of metabolic and inflammatory processes. However, the potential function of FFAR2 in type 1 diabetes (T1D) remains unexplored. Our results indicated that the mRNA "5 level of FFAR2 was upregulated in peripheral blood mononuclear cells of T1D patients. I The human FFAR2 promoter regions were cloned, and luciferase reporter assays revealed that NFkB activation induced FFAR2 expression. Furthermore, we showed that FFAR2 activation by overexpression induced cell apoptosis through ERK signaling. Finally, treatment with the FFAR2 agonists acetate or phenylacetamide 1 attenuated the inflammatory response in multiple-low-dose streptozocin-induced diabetic mice, and improved the impaired glucose tolerance. These results indicate that FFAR2 may play a protective role by inducing apoptosis of infiltrated macrophage in the pancreas through its feedback upregulation and activation, thus, in turn, improving glucose homeostasis in diabetic mice. These findings highlight FFAR2 as a potential therapeutic target of T1D, representing a link between immune response and glucose homeostasis. [ABSTRACT FROM AUTHOR]

TNF-like ligand 1A (TL1A), a newly identified member of the TNF superfamily, has been proved as an important mediator of inflammation and critically involved in the pathogenesis of rheumatoid arthritis and several other autoimmune diseases. The aim of our study was to determine the possible role of TL1A in the pathogenesis of psoriasis. In this study, serum levels of TL1A in patients with psoriasis vulgaris (PV) and atopic dermatitis (AD) were detected by enzyme-linked immunosorbent assay (ELISA). Then, peripheral blood mononuclear cells (PBMCs) from patients with PV were isolated, the mRNA expression of TL1A was measured by real-time quantitative PCR. The effects of TL1A on the production of T cell cytokines, such as IL-17, IFN-γ, IL-4 and IL-10 in PBMCs were determined. We demonstrated that serum TL1A levels were significantly elevated in patients with PV but not in patients with AD. Besides, the high serum TL1A levels in patients with PV decreased after treatment. PBMCs derived from psoriatic patients showed significantly increased TL1A mRNA levels. Soluble TL1A synergized with IL-23 to stimulate PBMCs from patients with PV to produce IL-17. Taken together, these findings strongly suggest that TL1A may play a role in the pathogenesis of PV. [ABSTRACT FROM AUTHOR]

Objective : To assess the effect of bone marrow mononuclear cells (BMMNCs) transplantation in the expression of nuclear factor-κB (NF-κB) in spinal cord injury (SCI) in rats. Methods : BMMNCs were isolated from tibia and femur by a density gradient centrifugation. After establishment of acute transection SCI, rats were divided into experiment (BMMNCs), experiment control (0.1 M PBS infused) and sham surgery groups (laminectomy without any SCI). Locomotor function was assessed weekly for 5 weeks post-injury using BBB locomotor score and urinary bladder function daily for 4 weeks post-injury. Activity of NF- κB in spinal cord was assessed by immunohistochemistry and reverse transcriptase polymerase chain reaction. Results : At each time point post-injury, sham surgery group had significantly higher Basso, Beattie, Bresnahan locomotor and urinary bladder function scores than experiment and experiment control group (p<0.05). At subsequent time interval there were gradual improvement in both experiment and experiment control group, but experiment group had higher score in comparison to experiment control group (p<0.05). Comparisons were also made for expression of activated NF-κB positive cells and level of NF-κB messenger RNA in spinal cord at various time points between the groups. Activated NF-κB immunoreactivity and level of NF-κB mRNA expression were significantly higher in control group in comparison to experiment and sham surgery group (p<0.05). Conclusion : BMMNCs transplantation attenuates the expression of NF-κB in injured spinal cord tissue and thus helps in recovery of neurological function in rat models with SCI. [ABSTRACT FROM AUTHOR]

We recently proposed a new surgical approach to treat ventral root avulsion, resulting in motoneuron protection. The present work combined such a surgical approach with bone marrow mononuclear cells (MC) therapy. Therefore, MC were added to the site of reimplantation. Female Lewis rats (seven weeks old) were subjected to unilateral ventral root avulsion (VRA) at L4, L5 and L6 levels and divided into the following groups (n = 5 for each group): Avulsion, sealant reimplanted roots and sealant reimplanted roots plus MC. After four weeks and 12 weeks post-surgery, the lumbar intumescences were processed by transmission electron microscopy, to analyze synaptic inputs to the repaired a motoneurons. Also, the ipsi and contralateral sciatic nerves were processed for axon counting and morphometry. The ultrastructural results indicated a significant preservation of inhibitory pre-synaptic boutons in the groups repaired with sealant alone and associated with MC therapy. Moreover, the average number of axons was higher in treated groups when compared to avulsion only. Complementary to the fiber counting, the morphometric analysis of axonal diameter and "g" ratio demonstrated that root reimplantation improved the motor component recovery. In conclusion, the data herein demonstrate that root reimplantation at the lesion site may be considered a therapeutic approach, following proximal lesions in the interface of central nervous system (CNS) and peripheral nervous system (PNS), and that MC therapy does not further improve the regenerative recovery, up to 12 weeks post lesion. [ABSTRACT FROM AUTHOR]

Our aim was to assess the use of peripheral blood mononuclear cells (PBMC) as an in vivo cellular model to evaluate diet-induced changes in the oxidative stress status by analyzing the gene expression pattern of NADPH-oxidase subunits and antioxidant genes. A randomized, controlled trial assigned metabolic syndrome patients to 4 diets for 12 weeks each: (i) high-saturated fatty acid (HSFA), (ii) high-monounsaturated fatty acid, and (iii), (iv) two low-fat, high-complex carbohydrate diets supplemented with n-3 polyunsaturated fatty acids or placebo. A fat challenge reflecting the fatty acid composition as the original diets was conducted post-intervention. The mRNA levels of gp91 phox ( P < 0.001), p22 phox ( P = 0.005), p47 phox ( P = 0.001) and p40 phox ( P < 0.001) increased at 2 h after the intake of the HSFA meal. The expression of SOD1 , SOD2 , GSR , GPx1 , GPX4 , TXN , TXNRD1 and Nrf2 increased after the HSFA meal ( p < 0.05). In contrast, the expression of these genes remained unaltered in response to the other dietary interventions. Our results suggest that the increased expression of antioxidant genes in PBMC seems to be due to the response to the postprandial oxidative stress generated mainly in adipose tissue after the consumption of an HSFA diet. [ABSTRACT FROM AUTHOR]

This study was designed to investigate the effects of physical conditioning on the expression of the insulin sensitive glucose transporter-4 protein (GLUT4) on mononuclear cells and HOMA-IR levels in dogs and compared to results reported in human skeletal muscle and the skeletal muscle of rodent models. Blood was sampled from conditioned dogs ( n = 8) and sedentary dogs ( n = 8). The conditioned dogs were exercised four months prior the experiment and were following a uniform training protocol, whereas the sedentary dogs were not. GLUT4 expression in mononuclear cells and plasma insulin levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA). Blood glucose levels were determined using blood plasma. HOMA-IR was calculated using plasma insulin and blood glucose levels using the linear approximation formula. Our results indicate that the state of conditioning had a significant effect on the GLUT4 expression at the surface of mononuclear cells. HOMA-IR was also affected by conditioning in dogs. GLUT4 levels in mononuclear cells of sled dogs were inversely correlated with the homeostasis model assessment of insulin sensitivity. This study demonstrates that conditioning increases GLUT4 levels in mononuclear cells of sled dogs as it has been previously reported in skeletal muscle. Our results support the potential of white blood cells as a proxy tissue for studying insulin signaling and may lead to development of a minimally invasive and direct marker of insulin resistance. This may be the first report of GLUT4 in mononuclear cells in response to exercise and measured with ELISA. [ABSTRACT FROM AUTHOR]

Purpose: Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol effl ux by sustained elevated blood glucose concentrations. 2 models were used to assess this potential relationship: human monocytes/leukocytes and murine bone marrow-derived macrophages (BMDM). Methods: 10 subjects (4 F/6 M, 50–85 years, BMI 25–35 kg/m2) underwent an oral glucose challenge. Baseline and 1- and 2-h post-challenge ABC-transporter mRNA expression was determined in monocytes, leukocytes and peripheral blood mononuclear cells (PBMC). In a separate study, murine-BMDM were exposed to 5 mmol/L D-glucose (control) or additional 20 mmol/L Dor L-glucose and 25 ug/mL oxidized low density lipoprotein (oxLDL). High density lipoprotein (HDL)-mediated cholesterol effl ux and ABCtransporter (ABCA1 and ABCG1) expression were determined. Results: Baseline ABCA1and ABCG1 expression was lower ( > 50 %) in human monocytes and PBMC than leukocytes ( p < 0.05). 1 h post-challenge leukocyte ABCA1 and ABCG1 expression increased by 37 % and 30 %, respectively ( p < 0.05), and began to return to baseline thereafter. There was no signifi cant change in monocyte ABCtransporter expression. In murine BMDM, higher glucose concentrations suppressed HDL-mediated cholesterol effl ux (10 %; p < 0.01) without signifi cantly aff ecting ABCA1 and ABCG1 expression. Data demonstrate that leukocytes are not a reliable indicator of monocyte ABC-transporter expression. Conclusions: Human monocyte ABC-transporter gene expression was unresponsive to a glucose challenge. Correspondingly, in BMDM, hyperglycemia attenuated macrophage cholesterol effl ux in the absence of altered ABC-transporter expression, suggesting that hyperglycemia, per se , suppresses cholesterol transporter activity. This glucose-related impairment in cholesterol effl ux may potentially contribute to diabetes-associated atherosclerosis. [ABSTRACT FROM AUTHOR]

Well into the second decade since its conception, cell transplantation continues to undergo intensive evaluation for the treatment of myocardial infarction. At a mechanistic level, its objectives remain to replace lost cardiac cell mass with new functioning cardiomyocytes and vascular cells, thereby minimizing infarct size and scar formation, and improving clinical outcomes by preventing adverse left ventricular remodeling and recurrent ischemic events. Many different cell types, including pluripotent stem cells and various adult-derived progenitor cells, have been shown to have therapeutic potential in preclinical studies, while early phase human trial experience has provided divergent outcomes and fundamental lessons, emphasizing that there remain key issues to address and challenges to overcome before cell therapy can be applied to wider clinical practice. The purpose of this review is to provide a balanced update on recent seminal developments in this exciting field and look to the next important steps to ensure its forward progression. [ABSTRACT FROM AUTHOR]

Root lesions may affect both dorsal and ventral roots. However, due to the possibility of generating further inflammation and neuropathic pain, surgical procedures do not prioritize the repair of the afferent omponent. The loss of such sensorial input directly disturbs the spinal circuits thus affecting the functionality of the injuried limb. The present study evaluated the motor and sensory improvement following dorsal root reimplantation with fibrin sealant (FS) plus bone marrow ononuclear cells (MC) after dorsal rhizotomy. MC were used to enhance the repair process. We also analyzed changes in the glial response and synaptic circuits within the spinal cord. Female Lewis rats (6-8 weeks old)were divided in three groups: rhizotomy (RZ group), rhizotomy repaired with FS (RZ+FS group) and rhizotomy repaired with FS and MC (RZ+FS+MC group). The behavioral tests electronic von-Frey and Walking track test were carried out. For immunohistochemistry we used markers to detect different synapse profiles as well as glial reaction. The behavioral results showed a significant decrease in sensory and motor function after lesion. The reimplantation decreased glial reaction and improved synaptic plasticity of afferent inputs. Cell therapy further enhanced the rewiring process. In addition, both reimplanted groups presented twice as much motor control compared to the non-treated group. In conclusion, the reimplantation with FS and MC is efficient and may be considered an approach to improve sensory-motor recovery following dorsal rhizotomy. [ABSTRACT FROM AUTHOR]

Background and Objective: Pomalidomide--a combination of Lenalidomide and Thalidomide drugs- is one of the newest anticancer drugs. Pomalidomide induces apoptosis in cancer cells. Furthermore, few studies indicating its relatively low cytotoxic effects on normal peripheral blood cells have been carried out. However, there is yet no information about the effects of Pomalidomide on bone marrow cells that contain important immune cells. In this study, the effect of Pomalidomide on normal bone marrow mononuclear cells was studied. Materials and Methods: The samples obtained from individuals who clinically needed bone marrow examination, but finally were diagnosed with no serious pathologic conditions in their leukocytes. Remained bone marrow aspirates were obtained and mononuclear cells were isolated. Half of the mononuclear cells were cultured with Pomalidomide (final concentration 10µM) and the remaining half (control group) were cultured with media alone. After 48h incubation, vital activity (MTT) and cell apoptosis induction rates were assessed. Results: The results showed that the vital activity of bone marrow mononuclear cells cultured in the presence of Pomalidomide increased significantly compared to the control group (P<0.05). However, Pomalidomide had no apoptosis induction and the rate of apoptotic cells had no significant difference with control group. Conclusion: Pomalidomide moderately stimulates the viability (or activity) of normal bone marrow mononuclear cells to improve and maintain them. Also, unlike most anticancer drugs it has no observable toxic effect or apoptosis induction on these cells. So, it may be concluded that Pomalidomide may be more favorable in preventing the side effects of anticancer drugs on normal cells. [ABSTRACT FROM AUTHOR]

Functional decline of the hematopoietic system occurs during aging and contributes to clinical consequences, including reduced competence of adaptive immunity and increased incidence of myeloid diseases. This has been linked to aging of the hematopoietic stem cell ( HSC) compartment and has implications for clinical hematopoietic cell transplantation as prolonged periods of T-cell deficiency follow transplantation of adult mobilized peripheral blood (PB), the primary transplant source. Here, we examined the gene expression profiles of young and aged HSCs from human cord blood and adult mobilized PB, respectively, and found that Wnt signaling genes are differentially expressed between young and aged human HSCs, with less activation of Wnt signaling in aged HSCs. Utilizing the OP9- DL1 in vitro co-culture system to promote T-cell development under stable Notch signaling conditions, we found that Wnt signaling activity is important for T-lineage differentiation. Examination of Wnt signaling components and target gene activation in young and aged human HSCs during T-lineage differentiation revealed an association between reduced Wnt signal transduction, increasing age, and impaired or delayed T-cell differentiation. This defect in Wnt signal activation of aged HSCs appeared to occur in the early T-progenitor cell subset derived during in vitro T-lineage differentiation. Our results reveal that reduced Wnt signaling activity may play a role in the age-related intrinsic defects of aged HSCs and early hematopoietic progenitors and suggest that manipulation of this pathway could contribute to the end goal of improving T-cell generation and immune reconstitution following clinical transplantation. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type 1 (HIV-1) genetic diversity is one of the most important features of HIV-1 infections and the result of error accumulation during reverse transcription and of high viral turnover. HIV-1 reverse transcription is influenced by factors such as the level of nucleotides and/or the cellular activation state. HIV-1 diversity was investigated after 48 h of viral propagation in peripheral blood mononuclear cells (PBMCs) obtained from healthy donors in three different cell culture conditions: (1) resting PBMCs, (2) simultaneous infection and PBMC activation, and (3) PBMC activation 72 h before infection. Cellular DNA was extracted and proviruses of each culture condition were amplified. Single-genome PCR clones were obtained and the protease and reverse transcriptase of the pol gene were sequenced. An elevated number of nucleotide substitutions in all three culture conditions were observed. In condition 1, the mutational rate observed ranged from 1.0 × 10-3 to 2.1 × 10-2, the genetic diversity was 0.6%, and hypermutation was observed in 7.1% of sequenced clones. In condition 2, the mutational rate ranged from 1.0 × 10-3 to 1.0 × 10-2, the genetic diversity was 0.8%, and hypermutation affected 6.7% of clones. In condition 3, the mutational rate ranged from 2.8 × 10-3 to 1.1 × 10-2, the genetic diversity was 1%, and 5.9% of clones were hypermutated. Substitutions occurred more frequently in some specific nucleotide stretches, and a common pattern for substitutions in all the different conditions was identified. There was a significant accumulation of mutations during the initial periods of in vitro HIV-1 propagation irrespective of culture conditions. The rapid accumulation of virus diversity might represent a viral strategy when colonizing new hosts. Complementary studies are necessary to allow for a better understanding of the initial periods of infection, which represent a crucial event related to disease progression. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Cell-based therapies have been employed to promote neovascularization mainly through the release of paracrine factors inhibiting apoptosis and supporting migration and proliferation of resident differentiated cells. We tested in vitro pro-angiogenic effects of apoptotic cord blood-derived mononuclear cells (CB-MNCs) and their conditioned medium (CM) on mature endothelial cells (HUVECs) and peripheral blood-derived endothelial progenitor cells (ECFCs). CB-MNCs were γ03b3;-irradiated to induce apoptosis and cultured for 72h to obtain the release of CM. MNCs viability, evaluated by flow cytometry, decreased progressively after γ03b3;-irradiation reaching 41% at 72h. γ03b3;-Irradiated MNCs (γ03b3;MNCs) released increasing amounts of EGF, PDGF-AB and VEGF in their CM over time, as assessed by ELISA. γ03b3;-MNCs and their CM enhanced capillary-like network formation (in a dose-dependent and time-persistent manner), proliferation and migration of HUVECs in vitro, while they primed capillary-like network formation (dose-independent and not time-persistent) and induced migration but did not support proliferation of ECFCs. Our data support the hypothesis of paracrine mechanism as prevalent in regenerative medicine and demonstrate the efficacy of MNCs secretome in inducing neovascularization. To our knowledge, this is the first paper highlighting differential pro-angiogenic effects of CM on mature and progenitor endothelial cells, adding a tile in the understanding of mechanisms involved in neovascularization. [ABSTRACT FROM AUTHOR]

Neutrophils (PMN) play diverse regulatory and effector functions in the immune system through the release of reactive nitrogen species, including nitric oxide (NO). The enzyme responsible for NO synthesis in PMN is inducible nitric oxide synthase (iNOS) that is regulated by various signaling pathways, e.g. PI3K-Akt/PKB, and transcription factors. N-Nitrosodimethylamine (NDMA), a xenobiotic widespread in the human environment, affects immune cells. The study objective here was to examine the role of the PI3K-Akt/PKB pathway in induction of NO synthesis (with involvement of iNOS) in human PMN, as well as in autologous mononuclear cells (PBMC), exposed to NDMA. Isolated cells were incubated for 2 h with a sub-lethal dose of NDMA and then the expression of several select proteins in the cell cytoplasmic and nuclear fractions were determined by Western blot analyses. The results indicated that NDMA enhanced expression of iNOS, phospho-PI3K, and phospho-IκBα in the cytoplasmic fraction of the PMN and PBMC. The nuclear fraction of these cells also had a higher NF-κB expression. Moreover, in PMN, NDMA caused an increased expression of phospho-Akt (T308), phospho-Akt (S473), and phospho-IKKαβ in the cytoplasm, and c-Jun and FosB in the nuclear fraction. Blocking of PI3K caused a decrease in expression of all these proteins in NDMA-exposed PMN. However, inhibition of PI3K led to a drop in expression of iNOS, phospho-PI3K, and phospho-IκBα in the cytoplasm, and in NF-κB in the nuclear fraction, of PBMC. The results of these studies indicated to us that NDMA activates the PI3K-Akt/PKB pathway in human PMN and that this, in turn, contributes to the activation of transcription factors NF-κB, c-Jun, and FosB involved in NO production (through modulation of iNOS expression). [ABSTRACT FROM AUTHOR]

High-fat, high-carbohydrate (HFHC) meals induce an inflammatory response in mononuclear cells (MNC). Here, we studied the interaction between metabolic and inflammatory signalling pathways by the measurement of postprandial effects of three different test meals on intracellular Akt, S6 kinase (S6K)/mammalian target of rapamycin and NF-κB signalling in human MNC. We recruited six healthy, lean individuals. Each individual ingested three different meals in the morning separated by at least 3 d: a HFHC meal; an oral lipid-tolerance test meal; a healthy breakfast. Blood samples were obtained before and 1, 2, 4, 6 and 8 h after ingestion. Plasma insulin and IL-6 levels were measured. Intracellular metabolic and inflammatory signalling pathways were assessed by measuring the phosphorylation of Akt kinase and S6K, the degradation of inhibitory κB-α (IκB-α) protein and the DNA binding activity of NF-κB in MNC. mRNA expression levels of the Akt and NF-κB target genes Mn superoxide dismutase (MnSOD), CC-chemokine-receptor 5 (CCR5), intercellular adhesion molecule 1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1) were measured by quantitative RT-PCR. We found a positive correlation of Akt phosphorylation with NF-κB activation (NF-κB binding activity: r 0·4500, P= 0·0003; IκB-α protein levels: r − 0·5435, P< 0·0001), a negative correlation of plasma insulin levels with NF-κB binding activity (r − 0·3993, P= 0·0016) and a positive correlation of plasma insulin levels with S6K activation (r 0·4786, P< 0·0001). The activation of Akt and pro-inflammatory NF-κB signalling was supported by the up-regulation of the respective target genes MnSOD and CCR5. In conclusion, the present data suggest a postprandial interaction between the metabolic and inflammatory signalling pathways Akt and NF-κB in MNC. [ABSTRACT FROM PUBLISHER]

Objective: The systematic pro-inflammatory responses after percutaneous coronary intervention with drug-eluting stents (DES) remain poorly defined. Therefore, we compared the systematic pro-inflammatory state of circulating mononuclear cells (MNCs) between DES and bare metal stent (BMS) implantation. Methods: Patients with indications for treatment with stents were randomized in a 1:1 ratio to placement of DES or BMS. The primary endpoint was a change of pro-inflammatory state at 12 weeks post-procedure. Results: Thirty-six consecutive patients received DES or BMS. At 12 weeks after stent implantation, the lipid profile and high-sensitivity C-reactive protein (hs-CRP) improved significantly in both groups. The mRNA levels and plasma concentrations of interleukin-6, tumor necrosis factor-α and matrix metalloproteinase-9 were significantly elevated in the DES group, which was not observed in the BMS group. An increase in NF-κB binding activity and a decrease in PPAR-γ expression in MNCs were observed in the DES group, along with increases in IκB phosphorylation and p50 expression. However, similar changes were not observed in the BMS group. Conclusions: Systematic inflammatory responses were accentuated after the patients were treated percutaneously with DES, despite their improved lipid profile and hs-CRP. These data may provide fundamental information for optimizing therapeutic strategy in the era of DES. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Current haematopoietic stem cell transplantation protocols rely heavily upon CD34+ cells to estimate haematopoietic stem and progenitor cell yield. However, several studies nowadays emerged with evidence that CD133+ cells represent a more primitive cell population than their CD34+ counterparts. The objective of the present study was to isolate CD133+ stem cells by a rapid negative isolation method which depletes human peripheral blood mononuclear cells (MNCs) from cells expressing haematopoietic lineage markers CD2, CD3, CD11b, CD14,CD15, CD16, CD19, CD56, CD123, and CD235a and isolates a discrete lineage negative (Lin-) cell population (11±2% of MNC, n=6). Human peripheral blood MNCs were fractionated by density gradient centrifugation. After labelling with antibody against haematopoietic lineage markers, MNCs were subjected to magnetic assisted cell sorting (MACS). The expression of stem cells markers was examined by reverse transcriptase PCR (RT-PCR). We showed that Lin- stem-progenitor cell population encompassed the common markers used to characterize adult stem cells including SLAMF1, CD90 and CD133 and was also enriched for CD117, a surface receptor for stem cell factor. This cell population is more homogenous and primitive as compared to suspension MNCs which comprised of SLAMF1 and CD117 but the cells with haematopoietic lineage marker, CD14 and CD2 were also present. In conclusion, our approach was able to isolate a homogenous Lin- stem-progenitor cell population from human peripheral blood hence offers a promising alternative to current haematopoietic stem and progenitor selection methods. [ABSTRACT FROM AUTHOR]

Wear particles and ion release from medical CoCr contribute to the risk for aseptic loosening. Nitrogen plasma immersion ion implantation (PIII) has been shown to reduce wear of CoCr but is associated with increased Co ion release. This work investigated the cytocompatibility of CoCr modified by nitrogen PIII at different temperatures (mCoCr). The osteosarcoma cell line Saos-2, mesenchymal stem cells (MSCs), and mononuclear cells (MNCs) were grown directly on CoCr/mCoCr discs or treated with CoCl2. Proliferation and metabolic activity of Saos-2 and MSC were decreased on mCoCr in correlation with increasing implantation temperature. The elevated Co ion release seemed to play a decisive role since analog effects were observed by treatment of cells with CoCl2. Proliferation of phytohemagglutinin-stimulated MNC was reduced in the presence of CoCr discs or CoCl2. For MNC-alloy cultures we observed an increase in interleukin 2 (IL-2) and a decrease in interferon γ (IFN-γ) and IL-10 secretion compared to cultures without alloys. The results of this study demonstrate that PIII process temperature and corresponding Co ion release correlate with material cytocompatibility. Therefore, the two competing parameters, reduction of wear and increase in Co ions, have to be taken into consideration for the evaluation of the clinical applicability of nitrogen-implanted CoCr. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 1744-1754, 2014. [ABSTRACT FROM AUTHOR]

The article offers information on the isolation of diploid baker's yeast. It discusses how yeast is capable of activating immune cells and also provides information on analyses of the cell wall. It mentions about cell wall of baker's yeast, Saccharomyces cerevisiae, which is composed of 85 percent polysaccharides and 15 percent proteins. Photographs depicting analyses of polysaccharides from the cell wall is also offered.

Objective: To evaluate the peripheral blood mononuclear cell ( PBMC) expression levels of hemeoxygenase 1 ( HMOX-1), superoxide dismutase 1 ( SOD-1), vascular endothelial growth factor A ( VEGF-A), transforming growth factor beta 1 ( TGF-β1), interleukin ( IL)-6, IL-15 and AdipoQ genes to study their association with preeclampsia (PE). Methods: A total of 177 pregnant women were recruited: 108 cases and 69 controls. Quantification of gene expression was measured by quantitative real-time polymerase chain reaction (PCR) using TaqMan probes. Results: Underexpression of VEGF-A and TGF-β1 was a constant in most of the cases (80.91% and 76.36%, respectively) and their expression was associated with onset and/or severity of disease ( p values < 0.05). IL-6, IL-15 and AdipoQ, showed low or no expression in PBMC samples evaluated. Conclusion: PBMC underexpression of VEGF-A and TGF-β1 is a hallmark of PE in the study population. [ABSTRACT FROM AUTHOR]

Background Several mechanisms have been proposed to explain why some platelets have a reduced response to aspirin ( ASA). Among them, it was reported an increased circulating level of vitamin-D-binding protein ( DBP). In addition, nitric oxide ( NO) released from mononuclear cells was involved in the antiplatelet effects of ASA. The aim was to analyse the relationship between platelet response to ASA and both NO generation and vitamin-D-binding protein content in mononuclear cells. Materials and methods Mononuclear cells were obtained from patients with stable coronary artery disease that were divided by a platelet functionality test ( PFA-100) as ASA-sensitive ( n = 23) and ASA resistant ( n = 27). Results Both the release of NO (determined by nitrite + nitrate concentration) and the expression of endothelial-type NO synthase ( eNOS) were higher in mononuclear cells from ASA sensitive as compared with those from ASA-resistant patients. There was a positive correlation between either the release of NO and the expression of eNOS protein in mononuclear cells with the ability of ASA to inhibit platelet activity. DBP content in mononuclear cells was higher in ASA resistant than in ASA sensitive. The level of DBP content in mononuclear cells was negatively associated with the ability of ASA to inhibit platelets. However, in vitro experiments suggested that there was no association between DBP and NO production by mononuclear cells. Conclusions Mononuclear cells from patients with platelets with lower responsiveness to ASA showed a reduced ability to produce NO. [ABSTRACT FROM AUTHOR]

Highlights: [•] Human blood mononuclear cells were stimulated with calcitriol for up to 120h. [•] We observed no substantial changes to DNA methylation on a genome scale. [•] Other mechanisms appear of more importance to the regulation of genes by vitamin D. [Copyright &y& Elsevier]