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Background: Ligation of intersphincteric fistula tract (LIFT) is a sphincter-saving procedure used for treatment of complex anal fistula. The current study aimed to assess the outcome of local injection of bone marrow mononuclear cells (BM-MNCs) in conjunction with LIFT as compared to LIFT alone in regards to healing rate, time to healing, and ultimate success rate.Methods: This was a prospective randomized trial on patients with trans-sphincteric anal fistula. Patients were randomly allocated to one of two equal groups: LIFT and LIFT with BM-MNC injection. The main outcome measures were healing at 10 weeks of follow-up, recurrence after healing, and complications.Results: Seventy patients (48 male and 22 female) of a mean age of 37.9 ± 10.4 years were included. The mean time to complete healing after LIFT + BM-MNCs was significantly shorter than after LIFT alone (20.5 ± 5.2 vs 28.04 ± 5.8 days; P < 0.0001). The ultimate success rates of both groups were similar (LIFT = 60% vs LIFT with BM-MNCs = 68.6%, P = 0.62). There was no significant difference in the mean operation time or complication rate between the two groups. Secondary extension and previous anal surgery were significant independent predictors of failure of healing.Conclusion: LIFT combined with BM-MNC injection was associated with a shorter time to complete healing than LIFT alone. However, BM-MNC injection did not have a significant impact on the overall healing and ultimate success rate. [ABSTRACT FROM AUTHOR]

The mechanism of action of Platelet Rich Plasma (PRP) is thought to be related to the biomolecules present in α-granules. However, for the healing process to occur, an inflammatory phase is also deemed necessary. Leukocytes present in the inflammatory phase release both pro- and anti-inflammatory molecules. The latter may play an important role in the process of "inflammatory regeneration". Thus, we propose that in the context of healing, both platelets and leukocytes play an important role, specifically due to the macrophage's plasticity to switch from the M1 to M2 fraction. Therefore, we propose that PRP products derived from the buffy coat may be more beneficial than detrimental from a standpoint of the regenerative potential of PRP. [ABSTRACT FROM AUTHOR]

Calcium ion-crosslinked nanofibrillated cellulose (NFC) hydrogels were investigated as potential materials for wound healing dressings. The physicochemical properties of the hydrogels were examined by rheology and water retention tests. Skin cells and monocytes were selected for application-oriented biocompatibility studies. The NFC hydrogels presented entangled fibrous networks and solid-like behavior. Water retention tests showed the material´s potential to maintain a suitable moist environment for different type of wounds. The hydrogels did not affect dermal fibroblasts monolayer cultures upon direct contact, as cell monolayers remained intact after application, incubation and removal of the materials. Inflammatory response studies with blood-derived mononuclear cells revealed the inert nature of the hydrogels in terms of cytokine secretion and reactive oxygen species production. Results highlight the great potential of ion-crosslinked NFC hydrogels for the development of advanced wound dressings, where further functionalization of the material could lead to improved properties towards the healing of specific wound types. [ABSTRACT FROM AUTHOR]

Background. Glioblastoma (GBM) represents the most aggressive malignant brain tumor in adults, with a risible median life expectancy despite gold standard treatment. Novel drug-delivery methods have been explored. Here we evaluated the possibility to use mononuclear cells (MCs) belonging to the monocytic-dendritic lineage as drug-carrier. Methods. MCs were obtained from 10 patients harboring a GBM, and from healthy volunteers, considered as controls. GBM tissue was also obtained from patients. MCs were cultured and the adherent population on fibronectin (FN-MCs), after immunocytochemistry and flow cytometry characterization, was loaded with Paclitaxel (FN-MCs-PTX). Antiproliferative and migration activity of FN-MCs-PTX was evaluated in two-dimensional (2D) and three-dimensional (3D) co-culture assays with red fluorescent U87 Malignant Glioma cells and primary GBM cells. Antiangiogenic properties of FN-MCs-PTX were tested on cultures with endothelial cells. Results. Phenotypical characterization showed a high expression of monocytic-dendritic markers in GBM cells and FN-MCs. FN-MCs demonstrated to effectively uptake PTX and to strongly inhibit GBM growth in vitro (P < 0.01). Moreover, tumor-induced migration of MCs, although partially affected by the PTX cargo, remained statistically significant when compared with unprimed cells and this was confirmed in a 3D Matrigel model (P < 0.01) and in aTrans-well assay (P < 0.01). FN-MCs-PTX also disclosed considerable antiangiogenic properties. Discussion. Our results suggest that the fibronectin-adherent population of MCs isolated from peripheral blood can be an effective tool to inhibit GBM growth. Given the relative facility to obtain such cells and the short time needed for their culture and drug loading this approach may have potential as an adjuvant therapy for GBM. [ABSTRACT FROM AUTHOR]

Biomedical implants have been widely used in bone repair applications. However, nanosized degradation products from these implants could elicit an inflammatory reaction, which may lead to implant failure. It is well known that the size, chemistry, and charge of these nanoparticles can modulate this response, but little is known regarding the role that the particle's morphology plays in inducing inflammation. The present study aims to investigate the effect of hydroxyapatite nanoparticle (HANPs) morphology on inflammation, in-vitro and in-vivo . Four distinct HANP morphologies were fabricated and characterized: long rods, dots, sheets, and fibers. Primary human polymorphonuclear cells (PMNCs), mononuclear cells (MNCs), and human dermal fibroblasts (hDFs) were exposed to HANPs and alterations in cell viability, morphology, apoptotic activity, and reactive oxygen species (ROS) production were evaluated, in vitro . PMNCs and hDFs experienced a 2-fold decrease in viability following exposure to fibers, while MNC viability decreased 5-fold after treatment with the dots. Additionally, the fibers stimulated an elevated ROS response in both PMNCs and MNCs, and the largest apoptotic behavior for all cell types. Furthermore, exposure to fibers and dots resulted in greater capsule thickness when implanted subcutaneously in mice. Collectively, these results suggest that nanoparticle morphology can significantly impact the inflammatory response. [ABSTRACT FROM AUTHOR]

Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells. Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution. Particulate matter induces a dose- and time- dependent, rapid (1 h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50–1000 nm). Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity. [ABSTRACT FROM AUTHOR]

Objectives This study is one in series measuring RAGE axis (receptor for advanced glycation end products, its isoforms, and ligands) as a biomarker in multiple sclerosis (MS). We identified and quantified membrane-bound RAGE (mRAGE) expression levels on freshly isolated PBMCs and its subpopulation (monocytes and T cells), and determined the relationship between mRAGE expression levels and MS disease severity. Materials and methods mRAGE expression was determined for 28 MS patients and 16HCs, by flow cytometry, using fluorochrome unconjugated primary RAGE monoclonal antibody and a polyclonal secondary antibody conjugated to R-Phycoerythrin (PE). Results After adjusting for multiple comparisons and correcting for group differences in age and gender, MS patients showed higher percentages of mRAGE-positive on PBMCs (12.4 ± 2.1 vs. 4.08 ± 0.8, P = 0.02), monocytes (37.4 ± 5.8 vs. 20.1 ± 5.0, P = 0.08) and T cells (4.1 ± 1.2 vs. 2.1 ± 0.3, P = 0.05). SPMS patients’ showed lower percentages of RAGE-positive monocytes (13.7 ± 5.5 vs. 49.5 ± 6.6, P = 0.0006) and RAGE-positive T cells (4.1 ± 1.8 vs. 6.6 ± 1.5, P = 0.04) than RRMS patients. We observed a negative relationship between the percentages of mRAGE-positive PBMCs and MS severity scale (MSSS) ( r = − 0.39, P = 0.04), monocytes and EDSS ( r = −0.48, P = 0.01), monocytes and MSSS ( r = − 0.58, P = 0.001), and T cells and MSSS ( r = −0.40, P = 0.04). Monocytes expression of mRAGE showed 0.811 area under the curve (95% CI: 0.64–0.98) sensitivity/specificity for MSSS. Conclusion The reduced mRAGE expression on PBMCs in general, and on monocytes in particular, can be used as biomarker of MS disease severity and progression. [ABSTRACT FROM AUTHOR]

• A systematic review suggests five miRNAs consistently dysregulated in MG: miR-106b-3p and miR-21-5p were upregulated, and miR-20b, miR-15b, and miR-16 were downregulated. • Five other miRNAs were mostly upregulated or downregulated across studies: miR-146a, miR-30e-5p, and miR-338-3p were mostly upregulated and miR-324-3p was mostly downregulated. • The major targets of these dysregulated miRNAs are less understood in MG. • Prior to these consistently and mostly dysregulated miRNAs can be used for diagnostic and therapeutic purposes, it is, thus, required to pursue molecular mechanisms these miRNAs specifically use in the context of MG. • The small number of studies was the important limitation of our review; further studies are warranted to quantitatively measure the effect size of the difference in miRNA levels between patients with MG and HC subjects. Studies have described the role of microRNAs (miRNAs) in thymic function, along with directly observing the altered expression of miRNAs in thymuses of myasthenia gravis (MG) patients; so, miRNAs became a core component in the pathophysiology of MG. However, because the miRNA analysis results are contradictory, the identification of MG-related miRNAs is daunting. We did a systematic review of studies analyzing the miRNA expression profile of peripheral blood and mononuclear cells for patients with MG. We ran a database search in PubMed, Scopus, and Web of Science on August 17, 2021. Original articles that analyzed miRNA profiles in peripheral blood (serum, plasma, and whole blood) and peripheral blood mononuclear cells (PBMCs) for patients with MG in comparison with a non-MG or healthy control (HC) group were eligible. The quality of studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2). 26 studies were included. The quality of studies was fair (median score, 5). Among 226 different miRNAs that were deregulated in at least one study (range, 1–87), ten miRNAs were significantly deregulated in three or more studies. Five miRNAs (50%) showed the same deregulation: miR-106b-3p and miR-21-5p were consistently upregulated, and miR-20b, miR-15b, and miR-16 were consistently downregulated. Also, there were five miRNAs that were mostly upregulated, miR-150-5p, miR-146a, miR-30e-5p, and miR-338-3p, or downregulated, miR-324-3p, across studies. These miRNAs contribute to different pathways, importantly neural apoptosis and autophagy, inflammation, T regulatory cell development, and T helper cell balance. Prior to being used for diagnostic and therapeutic purposes, it is required to pursue molecular mechanisms these consistently and mostly dysregulated miRNAs specifically use in the context of MG. [ABSTRACT FROM AUTHOR]

• High levels of IL-4 and cell-free mitochondrial DNA were detected in CL patients infected with L. tropica or L. major. • In both groups of patients, the level of CD4+ T - cells was higher while no significant difference in levels of CD8+ T - cells was observed. • Levels of CF mt-DNA and IL-4 in CL can be used to determine the disease progression. • These biomarkers could help in designing vaccine strategies and immunotherapies. The pathophysiology of Cutaneous Leishmaniasis (CL), an infection caused by Leishmania tropica (L. tropica) and Leishmania major (L. major) is primarily determined by inflammation-mediated immune cells. The immune response mainly depends on cells and molecules related to T-cells that influence susceptibility and disease development. Understanding the immunological mechanisms that cause tissue injury or lesion healing is critical for developing appropriate treatment strategies. In the present study, T-cells profile and cell-free mitochondrial DNA (CF mt-DNA) were investigated in CL patients infected with L. tropica (n = 34) and L. major (n = 2) and compared with non-infected healthy controls (n = 20). There was a significant (p <0.0001) difference between CD4+ T - cells among L. tropica and L. major CL-infected groups as compared to control while no significant difference (p = 0.8597) was found in the percentages of CD8+ T - cells. When L. tropica and L. major CL-infected individuals were compared to controls, the levels of IL-4 and expression of CF mt-DNA were significantly higher (p <0.0001). Higher levels of CF mt-DNA were detected in CL patients, irrespective of the infective Leishmania species. We proposed that the levels of CF mt-DNA and IL-4 in CL-infected individuals can be used to determine the disease progression. A better understanding of these biomarkers and evaluation of the immune responses in CL patients might benefit the development of vaccines and immunotherapies. [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]

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]

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]

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]

• 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]

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]

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]

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]

Abstract: According to the World Health Organization statistics, dementias are the largest contributors to disease burden in advanced market economies, and the leading cause of disability and dependence among older people worldwide. So far, several techniques have been developed to identify dementias with reasonable accuracy while the patient is still alive, however, no single of them has proven to be ideal, especially if you need to have a satisfactory early diagnosis. Studies of early onset dementia are largely limited by the inaccessibility to direct examination of the living human brain: it appears therefore that for a correct biochemical and molecular characterization of dementias, potential surrogate tissues must be identified. In this context, peripheral blood mononuclear cells (PBMCs) appear particularly attractive because they can be obtained in a minimally invasive manner and can be easily analyzed. This review focuses on the most representative methodologies and strategies in detecting and quantifying fluctuation in dementia that are currently being developed. In addition it provides a comprehensive evaluation of the diagnostic sensitivity of PBMCs in patients with dementia. Finally, it discusses the data supporting the use of the determination of neutral lipids (NLs) in PBMCs by Oil Red O (ORO) staining, which is a minimally invasive, cheap, easy and fast procedure, as the promising method for early detection of dementia and to search for new effective treatments. [Copyright &y& Elsevier]

Abstract: Immune cells often express various nicotinic ACh receptor (nAChR) subtypes, including α7 nAChRs, as well as mRNA encoding secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide (SLURP)-1, an endogenous α7 nAChR allosteric ligand. We detected SLURP-1 immunoreactivity in CD205+ dendritic cells (DCs) residing in human tonsils. Phytohemagglutinin (PHA, 10μg/ml), a T cell activator, attenuated cell proliferation and increased the ACh content of MOLT-3 human leukemic T cells compared with the vehicle control. Methyllycaconitine (MLA, 100nM), a specific α7 nAChR antagonist, abolished all effects elicited by PHA. Recombinant (r)SLURP-1 (0.5μg/ml) attenuated peripheral blood mononuclear cell proliferation and increased ChAT gene expression and the ACh content in MOLT-3 cells compared with the control, all of which were abolished by MLA. This suggests SLURP-1 activates cholinergic transmission by potentiating ACh synthesis and its action at α7 nAChRs, thereby facilitating functional development of T cells. These findings support the notion that SLURP-1 acts as a key modulator of immune responses. [Copyright &y& Elsevier]

Abstract: The aim of the study was to examine the effect of androgen deprivation drugs, i.e. leuprolide and bicalutamide on the immune cross-talk between human peripheral blood mononuclear cells (PBMC) and cells from PC-3 and LNCaP human prostate cancer lines. PBMC, PC-3 and LNCaP were separately incubated without and with two androgen-deprivation drugs, i.e. leuprolide and bicalutamide, and the secretion of IL-1β, IL-6, IL-1ra and IL-10 was examined. In addition, the effect of both drugs on the production of those cytokines was carried out after 24hours incubation of PBMC with both types of cancer cells. Leuprolide or bicalutamide did not affect the production of the cytokines by PBMC or by the prostate cancer cells from the two lines. Incubation of PBMC with PC-3 or LNCaP cells caused increased production of IL-1β, IL-6 and IL-10 as compared with PBMC incubated without malignant cells. While 10−7 M and 10−8 M of leuprolide caused a decreased secretion of IL-1β by PBMC previously incubated with prostate cancer cells without the drug, bicalutamide did not affect this PBMC activity at any drug concentration. This observation suggests the existence of an additional mechanism explaining the effect of androgen deprivation therapy in prostate cancer patients. [Copyright &y& Elsevier]

Background aims. The use of bone marrow mononuclear cells (BM-MNCs) has achieved great outcomes in clinical practice. We aim to evaluate the efficacy and safety of autologous BM-MNC infusion and hyperbaric oxygen therapy (HOT) in type 2 diabetes mellitus. Methods. This single-center, randomized, open-label, controlled clinical trial with a factorial design included two phases. The patients received standard medical therapy in the run-in phase; in the treatment phase, patients with glycated hemoglobin of 7.5--9.5% were randomly assigned into four groups and underwent BM-MNC infusion along with HOT (BM-MNC+HOT group), BM-MNC infusion (BM-MNC group), HOT (HOT group) and standard medical therapy (control group), respectively. The area under the curve of C-peptide was recorded as a primary end point. Our research is registered at ClinicalTrials.gov (NCT00767260). Results. A total of 80 patients completed the follow-up. At 12 months after treatment, the area under the curve of C-peptide (ng/mL per 180 min) of the BM-MNC+HOT group and the BM-MNC group were significantly improved (34.0% and 43.8% from the baseline, respectively). The changes were both significant compared with that in the control group, but no remarkable change was observed in the HOT group. Treatment-related adverse events were mild, including transient abdominal pain (n = 5) and punctual hemorrhage (n = 3). Conclusions. BM-MNC infusion for type 2 diabetes mellitus improves islet function and metabolic control, with mild adverse effects. HOT does not synergize with BM-MNC infusion. [ABSTRACT FROM AUTHOR]

Abstract: The objective of this research was to explore whether supplementation of Se above the recommended levels or application directly to the abomasum is superior to rumen supplementation in terms of boosting the immune system and promoting the growth of suckling calves. Thirty newborn calves, 18 Jersey breed and 12 crossbred (Holstein×Jersey), during 75 days, were assigned in a randomized block design (sex and breed) with the following treatments C=control (no supplementation and selenium derived only from milk and concentrate); SeA=0.80mg of Se/animal per day to the abomasum (milk); and SeR=0.80mg of Se/animal per day to the rumen (oral). The blood concentration of Se was higher in the SeR- and SeA-treated animals than in the control animals (P=0.001) at 30 days of age. The phagocytic activity of macrophages was higher in animals receiving Se supplementation compared to the control and in SeA animals compared to SeR (P=0.007). No difference in the oxidative activity of neutrophils or hematocrit was observed among treatments regimes when the calves were 30 days old. Dry matter intake (P=0.059) and HGG (P=0.059) tended to be higher in SeR-treated animals than in SeA-treated animals at 30 days of age. Feed conversion tended to be more efficient in Se-supplemented animals compared to the control (P=0.075). There were no significant differences in WG, HG or LG among treatment regimes at 30 days of age. At 75 days of age, Se-supplemented animals tended to present higher concentrations of Se in the blood (P=0.079) and greater heart girth gain (P=0.088). Supplementation of suckling calves with 0.80mg of organic Se increased the serum levels of this trace mineral in the animals and boosted their immune systems at 30 days of age. A superior immune parameter response was observed in calves supplemented with Se to the abomasum. In addition, Se supplementation maintained the performance of animals that had a diagnosis of diarrhea. Selenium supplementation did not act as a growth promoter but did improve immune system function during this phase of compromised health. [Copyright &y& Elsevier]

Abstract: Aims: This cross-sectional study tested the hypothesis that treatment with the combination of Ezetimibe/Simvastatin (Vytorin) leads to broader changes in the expression levels of immunomodulatory genes as compared to Simvastatin monotherapy. Methods: Illumina's GenomeStudio gene expression module was used to compare gene profiles of Vytorin and Simvastatin in the peripheral blood mononuclear cells of 20 hypercholesterolemic subjects. Results: The characteristics of the immunomodulatory genes, which were altered by Vytorin, differed from those genes which were altered by Simvastatin. Vytorin mostly altered the expression levels of genes related to inflammation/oxidative stress; it downregulated the NF-KappaB and upregulated the expression of anti-inflammatory cytokine, IL-10, and anti-oxidant enzymes, GPX1 and SOD2, but also upregulated the expression levels of genes involved in cellular activation, adhesion, and coagulation cascade, including VWF, F7, PF4, PF4V1 SELP, ITGB3, ITGB5. Simvastatin mostly altered the expression levels of genes related to cellular apoptosis/proliferation. It upregulated the expression levels of apoptosis-related genes APAF1, BAX, IER3, and CSF1R, and downregulated the expression levels of genes related to cellular proliferation, including PTN and CD69. Treatment with Vytorin combination therapy modulated lipid profile and serum levels of the C-reactive protein more effectively, than treatment with Simvastatin monotherapy. Conclusion: The nature of the pleiotropic effects may play a role in Vytorin's and Simvastatin's clinical efficacies. [Copyright &y& Elsevier]

Abstract: Objectives: The relative contribution of the local vs. peripheral inflammation to the atherothrombotic processes is unknown. We compared the inflammatory status of the immune cells of the carotid plaque with similar cells in peripheral circulation of patients with advanced carotid disease (PCDs). Methods: Mononuclear cells (MNCs) were extracted from carotid endarterectomy (CEA) samples by enzymatic digestion and subsequent magnetic cell sorting. The cell surface antigenic expressions, and mRNA expression levels were compared between CEA MNCs and peripheral MNCs, using flow cytometry and RT-PCR techniques. Results: The percentages of resting MNCs were lower, and activated MNCs, particularly monocytes, were higher in the CEAMNCs, as compared to the peripheral MNCs. The percentages of activated T cells and B cells were higher in the peripheral MNCs of PCDs, than in healthy controls (HCs), but the percentages of activated monocytes did not differ between the two groups. The expression levels of both pro-inflammatory/pro-thrombotic (P38, JNKB-1, Egr-1 PAI-1, MCP-1, TF, MMP-9, HMGB-1, TNF-α, mTOR) and anti-inflammatory (PPAR-γ, TGF-β) mediators were significantly higher in the CEA MNCs as compared to the peripheral MNCs. Furthermore, MMP-9 and PPAR-γ expression levels were higher in the peripheral MNCs of PCDs than HCs. Conclusion: The inflammatory status is higher in the immune cells of the carotid plaque, as compared to those cells in the peripheral blood. The altered expression levels of both pro-inflammatory/pro-thrombotic and anti-inflammatory mediators in the milieu of the plaque suggest that the balance between these various mediators may play a key role in carotid disease progression. [Copyright &y& Elsevier]

Background aims. Long-bone pseudoarthrosis is a major orthopedic concern because of numerous factors such as difficulty of the treatment, high recurrence, high costs and the devastating effects on the patients' quality of life, which sometimes ends in amputation: Although the "gold standard" for the treatment of this pathology is autologous bone grafting, which has high ósteogenic, osteoconductive and osteoinductive properties, this treatment presents some restrictions such as the limited amount of bone that can be taken from the patient and donor site morbidity. Bone marrow mononuclear cells (BM-MNCs) comprise progenitor and stem cells with pro-angiogenic and pro-osteogenic properties. Allogenic cancellous bone graft is a natural and biodegradable osteoconductive and osteoinductive scaffold. Combination of these two components could mimic the advantages of autologous bone grafting while avoiding its main limitations. Methods. Long-bone pseudoarthrosis was treated in seven patients with autologous BM-MNCs from iliac crest combined with frozen allogenic cancellous bone graft obtained from the tissue bank. Results. All patients showed complete bone consolidation 5.3 ± 0.9 months (range, 2-9 months) after cell transplantation. Moreover, limb pain disappeared in all of them. Themean follow-up was 35.8 ± 4.6 months after transplantation (range, 24-5 1 months) without pseudoarthrosis recurrence or pain reappearing. Conclusions. Combination of autologous BM-MNCs and allogenic bone graft could constitute an easy, safe, inexpensive and efficacious attempt to treat long-bone pseudoarthrosis and non-union by reproducing the beneficial properties of autologous bone grafting while restricting its disadvantages. [ABSTRACT FROM AUTHOR]

Abstract: Resveratrol, a natural polyphenolic compound found mainly in grapes and their seeds, is gaining a widespread appreciation as a therapeutic adjuvant in a variety of diseases including cancer prevention. We examined the effect of resveratrol as a modulator of the immune dialog between peripheral blood mononuclear cells and those from two human colon carcinoma lines, expressed by a possible alteration of cytokine production. Resveratrol, incubated with non-stimulated mononuclear cells, caused a certain reduction of IL-6, IL-1ra and IL-10, and a moderate increase of TNFα release. On the other hand, resveratrol did not affect cytokine production by cancer cells from both lines. When resveratrol was added to immune and cancer cells jointly, an altered dose-dependent decreased production of the examined cytokines was obtained. These results favor the existence of a mechanism, additional to those already described, that may explain the preventive effect of resveratrol on tumorigenesis. [Copyright &y& Elsevier]

Abstract: The carotenoid lycopene has been reported to possess anti-metastatic activity which may be associated with immunomodulation. However, the anti-angiogenic effects and mechanisms of action of lycopene have not been reported. In this study, we investigated the immunomodulatory effect on in vitro and ex vivo angiogenesis of lycopene. We found that the proliferation, migration and the matrigel tube formation of human umbilical vein endothelial cells (HUVECs) was remarkably inhibited by conditioned medium (CM) of human peripheral blood mononuclear cells (MNC-CM) stimulated with various dose (1-10 μmol/L) of lycopene (LP-MNC-CM). LP-MNC-CM treatment inhibited ex vivo angiogenesis, as revealed by chicken egg chorioallantoic membrane assay. We further examined the effects of lycopene stimulation on cytokine levels in MNC and showed that, as compared to the control, lycopene (10 μmol/L) significantly (P<.001) up-regulated interleukin (IL)-12 by 163% and interferon (IFN)-γ by 531%. Furthermore, pre-treatment of HUVECs with dexamethasone, an IL-12 inhibitor, blocked the anti-angiogenic effects of LP-MNC-CM in parallel with inhibition of IL-12 and IFN-γ induction in MNC. These results demonstrate that lycopene has a potent anti-angiogenic effect and that these effect may be associated with its up-regulation of IL-12 and IFN-γ. [Copyright &y& Elsevier]

Summary: Cytokines have gained increasing attention as therapeutic targets in inflammation-related disorders and inflammatory conditions have been investigated in sheep. Monoclonal antibodies (mAbs) specific for the ovine pro-inflammatory cytokines interleukin (IL)-1β and IL-6 could be used to study the effects of blocking pro-inflammatory cytokines in sheep. Ovine-specific IL-1β and IL-6 proteins and mAbs specific for these molecules were produced and the ability of the mAbs to neutralize the proteins was tested in cultures of ovine splenic mononuclear cells. Expression of nuclear factor (NF)-κβ and signal transducer and activator of transcription (STAT)-3 was evaluated by western blotting and densitometric quantification. Treatment with purified IL-1β and IL-6 proteins increased NF-κβ (P < 0.001) and STAT-3 (P < 0.01) expression, respectively, in cell culture. Treatment with these proteins that were pre-incubated with IL-1β and IL-6 mAbs attenuated (P < 0.01) these effects. These results confirm the bioactivity of ovine IL-1β and IL-6 proteins and the neutralizing capacity of anti-ovine-IL-1β and -IL-6 mAbs in vitro. These mAbs could be used to investigate anti-inflammatory strategies for attenuation of the effects of these pro-inflammatory cytokines in sheep. [ABSTRACT FROM AUTHOR]

Abstract: Oxidative stress and apoptotic cell death in kidney have been suggested as contributing factors in the development and complication of diabetes especially in diabetic nephropathy (DN). This study investigated the effects of trigonelline (TG) on the renal functional, morphological changes and renal apoptosis in neonatal diabetic rats, a model of non-insulin-dependent diabetes mellitus. Diabetes mellitus was induced in one day old neonatal Wistar rat pups by an intraperitoneal (i.p.) injection of streptozotocin (STZ) (50mg/kg) and monitored for 16weeks thereafter. The diabetic rats were divided as follows: the nSTZ diabetic group, the TG (50mg/kg) treated diabetic group, and the TG (100mg/kg) treated diabetic group. The age matched nondiabetic group received an injection of citrate buffer (0.1M, pH4.5). At the end kidney samples were taken for light microscopic examinations. The levels of serum creatinine and BUN were significantly low in TG (100mg/kg) treated diabetic rats. Glomerular filtration rate was improved in TG treated rats. The activities of antioxidant enzyme and membrane bound enzyme were decreased and the levels of tumor necrotic factor (TNF-α) and hydroxyproline content were increased in renal tissues of the diabetic group. TG (100mg/kg/day) treatment for a period of 4weeks showed significant ameliorative effects on all the biochemical parameters studied. Biochemical findings were supported by histological studies. The degenerative changes in kidney tissue and fibrosis were alleviated in the TG treated groups. These results suggested that TG might have a significant role in alleviating kidney damage in nSTZ-diabetic rats. [Copyright &y& Elsevier]

Abstract: The human pentraxin proteins, serum amyloid P component (SAP) and C‐reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non‐specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required. We report here the production from normal human donor plasma and the characterization of the first such preparations. Importantly, we demonstrate that, contrary to reports using recombinant proteins and less well characterized preparations, neither CRP nor SAP stimulate the release by human peripheral blood mononuclear cells in vitro of any TNFα, IL‐6 or IL‐8, nor does SAP cause release of IL‐1β or IL‐10. Furthermore neither of our preparations was pro‐inflammatory in mice in vivo. [Copyright &y& Elsevier]

Abstract: A substantial number of studies provide evidence that inflammation may play a significant role in the pathogenesis of prostate cancer via increased activity of inflammatory cytokines, particularly IL-6. We have previously shown that peripheral blood mononuclear cells (PBMC) are capable of carrying out an in vitro “immunomodulatory dialog” with colon cancer cells expressed by an increased production of pro-inflammatory cytokines by PBMC. The aim of the current study was to examine the model of cell-to-cell interaction between PBMC and prostate cancer cells from two lines – androgen resistant (PC-3) and androgen-dependent (LNCaP). For that purpose, cancer cells from both lines were incubated with PBMC, and cytokine secretion by PBMC was evaluated. The results showed a cell-concentration dependent increase in secretion of the pro-inflammatory cytokine IL-6 by PBMC induced by cells from both lines, whereas generation of IL-1β and the anti-inflammatory cytokine IL-10 were found to be increased after incubation with PC-3 cells only. The secretion of IL-10 was slightly lower following incubation of PBMC with supernatants derived from PC-3 cells. The results of the study support the possibility that prostate cancer cell-induced cytokine production by PBMC, and particularly IL-6, are involved in prostate cancer development. The discrepancy between the effect of the two prostate cancer cell lines on cytokine secretion by PBMC may be due to their different androgen dependency. [Copyright &y& Elsevier]

Abstract: Background: CXCR4 receptor antagonist plerixafor is used to mobilize hematopoietic stem cells. No detailed data regarding the effects of plerixafor on other blood cell components have been published but may be of importance in regard to graft composition collected after plerixafor injection. Patients and methods: The study included thirty-nine patients with non-Hodgkin lymphoma (NHL) mobilized with chemotherapy plus G-CSF. Plerixafor was given pre-emptively in twenty patients due to poor mobilization or low collection yield. Nineteen NHL patients served as controls. We evaluated CD34+ counts and WBC counts and differential from the morning of the first plerixafor injection and 8h after the plerixafor injection. From the control patients the corresponding values were evaluated on the morning of the first apheresis and 24h before it. Results: The first plerixafor dose increased CD34+ counts and number of leukocytes, neutrophils, lymphocytes, eosinophils and monocytes. Leukocyte, neutrophil, lymphocyte, monocyte and eosinophil counts were higher after plerixafor injection compared to the control group at the time of the first apheresis. Minimal graft (⩾2×106/kg CD34+ cells) was collected in 85% of plerixafor treated patients, with a single apheresis in 45% of the patients. Discussion: Plerixafor significantly increases B-CD34+ cell counts on the next morning making effective blood stem cell collection possible in the majority of the patients mobilizing poorly. It also influences other blood cell components but impact of this observation in regard to graft content and post-transplant course needs to be assessed in further studies. [Copyright &y& Elsevier]

Abstract: Objectives: Circulating adiponectin levels in cigarette smokers are lower than those in nonsmokers. We have previously shown that adiponectin is expressed in human monocytes. The aim of this study was to further investigate the effect of smoking on adiponectin expression in peripheral blood mononuclear cells (PBMCs). Methods: A group of 77 cigarette smokers and 51 nonsmokers were consecutively enrolled in this study. The participants’ body weight, blood pressure, and metabolic parameters, including plasma glucose and plasma adiponectin levels, were recorded. The RNA from the PBMCs was assessed with real-time polymerase chain reaction (PCR) to determine the levels of adiponectin mRNA. Results: Of the 77 smokers, 67 (87.0%) were male. Their mean (standard deviation) age was 43.17 (11.47) years, and they smoked 24.56 (12.53) cigarettes/day. The duration of smoking was 23.73 (11.69) years. Both circulating adiponectin levels (p =0.0262) and adiponectin mRNA levels in PBMCs (p <0.0001) of smokers were significantly lower than those in nonsmokers. Both circulating adiponectin levels and adiponectin mRNA levels were negatively correlated with the number of cigarettes smoked per day (p <0.01). In multiple linear regression analysis, smoking was an independent factor affecting adiponectin mRNA expression in PBMCs (p <0.0001). Conclusions: Circulating adiponectin levels and adiponectin expression in PBMCs were lower in smokers; this finding suggested that attenuation of both systemic and local actions of adiponectin might contribute to the atherosclerotic process in cigarette smokers. [Copyright &y& Elsevier]

Abstract: Equine herpesvirus type 1 (EHV-1) replicates extensively in the epithelium of the upper respiratory tract, after which it can spread throughout the body via a cell-associated viremia in mononuclear leukocytes reaching the pregnant uterus and central nervous system. In a previous study, we were able to mimic the in vivo situation in an in vitro respiratory mucosal explant system. A plaquewise spread of EHV-1 was observed in the epithelial cells, whereas in the connective tissue below the basement membrane (BM), EHV-1-infected mononuclear leukocytes were noticed. Equine herpesvirus type 4 (EHV-4), a close relative of EHV-1, can also cause mild respiratory disease, but a cell-associated viremia in leukocytes is scarce and secondary symptoms are rarely observed. Based on this striking difference in pathogenicity, we aimed to evaluate how EHV-4 behaves in equine mucosal explants. Upon inoculation of equine mucosal explants with the EHV-4 strains VLS 829, EQ1 012 and V01-3-13, replication of EHV-4 in epithelial cells was evidenced by the presence of viral plaques in the epithelium. Interestingly, EHV-4-infected mononuclear leukocytes in the connective tissue below the BM were extremely rare and were only present for one of the three strains. The inefficient capacity of EHV-4 to infect mononuclear cells explains in part the rarity of EHV-4-induced viremia, and subsequently, the rarity of EHV-4-induced abortion or EHM. [Copyright &y& Elsevier]

Abstract: Objective: The inflammatory responses after percutaneous coronary intervention (PCI) with drug-eluting stent (DES) remain poorly understood; therefore, this study aims to investigate the changes of metabolic parameters and systematic inflammatory status of circulating mononuclear cells (MNC) in patients after percutaneous treatment with DES implantation. Methods and results: Twenty-seven patients with acute coronary syndrome who would undergo PCI with DES implantation were consecutively recruited and treated with standard drug therapy from the start of hospitalization. Metabolic parameters including total cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein improved significantly after 12 weeks of standard medication, whereas the plasma levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), macrophage migration inhibitory factor (MIF), and matrix metalloproteinase-9 (MMP-9) increased (P =0.012, 0.035, 0.062 and 0.112, respectively, compared to the baseline). The NF-κB DNA binding activity in MNC increased significantly compared to the baseline (P =0.015), whereas IκB-β and PPAR-γ were significantly suppressed (P =0.046 and 0.002, respectively). There were strong correlations among the changes of metabolic parameters and the changes of proinflammatory factors; however, none of them is statistically significant. Conclusions: Standard drug therapy can improve the metabolic parameters but fail to restrain the proinflammatory state after PCI with DES implantation. Longer term endpoint-based studies are still needed for further exploration of the relationship between inflammatory factors and clinical cardiovascular events in the era of DES. [Copyright &y& Elsevier]

Abstract: Scatophagus argus of the family Scatophagidae inflicts painful wounds in fishermen while handling it. The venom induces prominent local tissue damage characterized by pain, edema and necrosis. The pathogenesis of acute muscle damage in gastrocnemius muscle induced by S. argus venom was studied in mice. The inflammatory response induced by S. argus venom in the mice hind paw was studied measuring paw edema. Intramuscular injection of S. argus venom induced motoxicity. The effect of S. argus venom on the cellular components of inflammatory response was investigated. Venom from S. argus were quantitatively analyzed for enzymic and biochemical activity. The biochemical changes induced by the sublethal concentration of S. argus venom and histopathological studies of effect of venom on mice were carried out. Venom induced a rapid increment in serum creatine kinase (CK) and lactate dehydrogenase (LDH) showing the myotoxicity of venom. Concomitant with this a reduction of muscle CK and LDH activity was observed, where as no increment in muscle lactate was detected. Our findings showed that the edematic activity was dose dependent and remained significantly elevated over 48h after injection. Administration of S. argus venom caused a significant cell accumulation of neutrophils in to peritoneal cavity as well as foot pad up to 24h with maximal being at 4–6h. The venom components analyzed showed the presence of phosphodiesterase, acid phosphatases, alkaline phosphatases, proteinase, and caseinolytic activity. SDS PAGE revealed the presence of major and minor protein bands between 6.5 and 68kDa. The biochemical changes induced by the sublethal concentration of S. argus venom showed reversible changes in the hematological (blood cell count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin and platelet count) parameters which were significantly altered at 6 and 24h (GLM repeated measures p <0.05). Serum enzymes such as AST, ALT, ACP, ALP, LDH and urea were altered significantly which in turn confirmed the damage of vital organ tissue. [Copyright &y& Elsevier]

Abstract: Objective: Adult bone marrow mononuclear cells (BMMNCs) can restore cardiac function following myocardial necrosis. Protocols used to date have administered cells relatively late after ischaemia/reperfusion injury, but there is the opportunity with elective procedures to infuse cells shortly after restoration of blood flow, for example after angioplasty. Our aim was therefore to try and quantify protection from myocardial injury by early infusion of BMMNCs in a rat ischaemia reperfusion (I/R) model. Methods and results: Male Wistar rats underwent 25min of ischaemia followed by 2h reperfusion of the left anterior descending coronary artery. Ten million BMMNCs were injected i.v. at reperfusion. We found BMMNCs caused a significant reduction in infarct size at 2h when assessed by staining the area at risk with p-nitro blue tetrazolium (42% reduction, P <0.01). Apoptosis and necrosis of isolated cardiomyocytes was significantly reduced in the area at risk. Functional assessment at 7 days using echocardiography and left ventricular catheterisation showed improved systolic and diastolic function in the BMMNC treatment group (LVEF: BMMNC 71±3% vs. PBS 48±4%, P <0.0001). In functional studies BMMNC injected animals showed increased activation of Akt, inhibition of GSK-3β, amelioration of p38 MAP kinase phosphorylation and NF-κB activity compared to control myocardium. Inhibition of PI3K with LY294002 abolished all beneficial effects of BMMNC treatment. Proteomic analysis also demonstrated that BMMNC treatment induced alterations in proteins within known cardioprotective pathways, e.g., heat shock proteins, stress-70 protein as well as the chaperone protein 14-3-3 epsilon. Conclusions: Early BMMNC injection during reperfusion preserves the myocardium, with evidence of reduced apoptosis, necrosis, and activation of survival pathways. [Copyright &y& Elsevier]

Abstract: Purpose: Use of tissue-engineered vascular grafts (TEVGs) in the repair of congenital heart defects provides growth and remodeling potential. Little is known about the mechanisms involved in neovessel formation. We sought to define the role of seeded monocytes derived from bone marrow mononuclear cells (BM-MNCs) on neovessel formation. Methods: Small diameter biodegradable tubular scaffolds were constructed. Scaffolds were seeded with the entire population of BM-MNC (n = 15), BM-MNC excluding monocytes (n = 15), or only monocytes (n = 15) and implanted as infrarenal inferior vena cava (IVC) interposition grafts into severe combined immunodeficiency/bg mice. Grafts were evaluated at 1 week, 10 weeks, or 6 months via ultrasonography and microcomputed tomography, as well as by histologic and immunohistochemical techniques. Results: All grafts remained patent without stenosis or aneurysm formation. Neovessels contained a luminal endothelial lining surrounded by concentric smooth muscle cell layer and collagen similar to that seen in the native mouse IVC. Graft diameters differed significantly between those scaffolds seeded with only monocytes (1.022 ± 0.155 mm) and those seeded without monocytes (0.771 ± 0.121 mm; P = .021) at 6 months. Conclusions: Monocytes may play a role in maintaining graft patency. Incorporation of such findings into the development of second-generation TEVGs will promote graft patency and success. [Copyright &y& Elsevier]

Abstract: Toll-like receptors (TLRs) play an important role in the activation of innate and adaptive immune responses. Stimulation with multiple TLR agonists may result in synergistic, complimentary or inhibitory effects on innate immune responses. In this study, we investigated the effects of co-stimulation of sheep peripheral blood mononuclear cells (PBMC) and B cells with agonists for TLR3, 4, 7/8 and 9. Sheep PBMC stimulated with either CpG (TLR9 agonist) or RNA oligoribonucleotides ([ORNs], TLR7/8 agonist) exhibited significant IL-12 production, but only CpG induced IFNα, IgM and proliferative responses. In contrast, poly(I:C) (TLR3 agonist) and LPS (TLR4 agonist) did not induce any of these responses. Interestingly, we observed that co-stimulation of PBMC with CpG+ORN or CpG+imiquimod (another TLR7/8 agonist) resulted in significant reduction in CpG-induced IFNα production, B cell proliferation and IgM responses. Pre-incubation of cells with CpG prior to exposure of the cells to imiquimod resulted in similar inhibitory responses indicating that the down-regulatory mechanisms are not associated with competition for cellular uptake or for receptors of the two agonists. Sheep B cells constitutively expressed TLR7, TLR8 and TLR9 mRNA transcripts, suggesting a possible role of TLR cross-talk in the down-regulatory mechanisms. Down-regulation of responses by co-stimulation with closely related TLRs may be a regulatory mechanism by which the host prevents overstimulation of innate immune responses. [Copyright &y& Elsevier]

Abstract: Disturbed circadian rhythmicity is associated with human diseases such as sleep and mood disorders. However, study of human endogenous circadian rhythm is laborious and time-consuming, which hampers the elucidation of diseases. It has been reported that peripheral tissues exhibit circadian rhythmicity as the suprachiasmatic nucleus—the center of the biological clock. We tried to study human circadian rhythm using cultured peripheral blood mononuclear cells (PBMCs) obtained from a single collection of venous blood. Activated human PBMCs showed self-sustained circadian rhythm of clock gene expression, which indicates that they are useful for investigating human endogenous circadian rhythm. [Copyright &y& Elsevier]

Abstract: ICR mice were injected with ketamine for 1, 3 and 6 months and the kidneys and urinary bladders were excised and processed for histology. Starting from 1 month, all addicted mice showed invasion of mononuclear white cells, either surrounding the glomerulus or the other tubules in the kidney. The aggregation of these cells extended all the way to the pelvis and ureter. As well, in the urinary bladder, the epithelium became thin and there was submucosal infiltration of mononuclear inflammatory cells. Silver staining revealed a loss of nerve fibers amongst the muscles of the urinary bladder of the treated. Immunohistochemistry on choline acetyltransferase which is a marker for cholinergic neurons also demonstrated a decrease of those cells. We hypothesized that prolonged ketamine addiction resulted in the animals prone to urinary infection. [Copyright &y& Elsevier]

Abstract: Transient receptor potential vanilloid (TRPV) 1 channels function as sensors for a variety of noxious and inflammatory signals, including capsaicin, heat and protons, and are up-regulated under inflammatory conditions. As end-stage kidney disease (ESKD) is associated with chronic inflammation, impaired immunity and depressed lymphocyte numbers, we sought to determine whether altered TRPV1 (and related TRPV2) expression in immune cells might be a contributing factor. TRPV1 and TRPV2 mRNA expression in peripheral blood mononuclear cells (PBMC) was similar in controls and ESKD patients by quantitative real-time RT-PCR. However, using immunocytochemistry, TRPV1-immunoreactivity was significantly higher and TRPV2-immunoreactivity was significantly lower in PBMC from ESKD patients compared to controls. The plant-derived TRPV1 agonists, capsaicin and resiniferatoxin (RTX) and the putative endovanilloid/endocannabinoids, N-arachidonoyl-dopamine (NADA) and N-oleoyl-dopamine (OLDA), induced concentration-dependent death of PBMC from healthy donors with a rank order of potency of RTX>NADA>OLDA>>capsaicin. TRPV1 (5′-iodoresiniferatoxin) and cannabinoid (CB2; AM630) receptor antagonists blocked the cytotoxic effect of NADA. In subsequent experiments, PBMC from ESKD patients exhibited significantly increased susceptibility to NADA-induced death compared to PBMC from controls. The apparent up-regulation of TRPV1 may be a response to the inflammatory milieu in which PBMC exist in ESKD and may be responsible for the increased susceptibility of these cells to NADA-induced death, providing a possible explanation as to why ESKD patients have reduced lymphocyte counts and impaired immune function. Thus, TRPV1 (and possibly CB2) antagonists may have potential for the treatment of immune dysfunction in ESKD. [Copyright &y& Elsevier]

Summary: Background & aims: This study investigated the influence of four commercial lipid emulsions, Ivelip, ClinOleic, Omegaven and SMOFlipid®, on lipid body formation, fatty acid composition and eicosanoid production by cultured human peripheral blood polymorphonuclear cells (PMN) and mononuclear cells (PBMC). Methods: PMN and PBMC were exposed to emulsions at concentrations ranging from 0.01 to 0.04%. Lipid body formation was assessed by microscopy, fatty acid composition by gas chromatography and eicosanoids by ELISA. Results: Stimulation of inflammatory cells and exposure to lipid emulsions promoted the formation of lipid bodies, but there did not appear to be differential effects of the emulsions tested. In contrast, there were differential effects of lipid emulsions on eicosanoid formation, particularly with regards to LTB4 production by PMN. Omegaven dramatically increased production of eicosanoids compared with the other emulsions in a dose-dependent manner. This effect was associated with a significantly higher level of lipid peroxides in the supernatants of cells exposed to Omegaven. Conclusions: Stimulation of inflammatory cells and exposure to lipid emulsions promotes lipid body formation and eicosanoid production, although the differential effects of different emulsions appear to be largely due to lipid peroxidation of unsaturated fatty acids in some emulsions in this in vitro system. [Copyright &y& Elsevier]

Abstract: Objective: We tested the hypothesis that lycopene supplementation reduces the expression of oxidant-responsive heme oxygenase-1 (HO-1) in basal conditions and in response to an oxidant challenge and determined whether this is temporally associated with increased cell viability. Methods: We determined basal and stimulated ex vivo expression of HO-1 and cell viability in lymphocytes from volunteers after lycopene supplementation. Twenty-four healthy young men on a low lycopene diet consumed 1) 170 g of passata sauce with butter or 2) butter alone for 3 wk in a randomized crossover design. Results: Plasma lycopene concentrations at the end of the tomato and control trials were 0.54 ± 0.20 versus 0.20 ± 0.15 μmol/L, respectively (P < 0.05). There was a significant increase in the proportion of live cells (91 ± 5% versus 87 ± 9%) and a corresponding reduction in apoptosis (6 ± 4% versus 11 ± 9%) in untreated lymphocytes after supplementation (P < 0.05), with no effect on cell viability in response to hydrogen peroxide treatment. HO-1 protein expression in basal conditions and induction of HO-1 after hydrogen peroxide treatment was not different between trials. Conclusion: Lycopene supplementation did not affect basal oxidative stress or susceptibility to oxidant-induced stress as indicated by the expression of the oxidant-responsive protein HO-1 and cell viability in response to hydrogen peroxide treatment. However, lycopene supplementation significantly reduced apoptosis in freshly harvested untreated lymphocytes. We conclude that this was not through an oxidant-mediated mechanism because of the lack of an effect on oxidant-responsive HO-1. [Copyright &y& Elsevier]

Abstract: Objective: Coenzyme Q10 (CoQ10) is an essential electron carrier in the mitochondrial respiratory chain and a strong antioxidant. Signs and symptoms associated with muscular alteration and mitochondrial dysfunction, including oxidative stress, have been observed in patients with fibromyalgia (FM). The aim was to study CoQ10 levels in plasma and mononuclear cells, and oxidative stress in FM patients. Methods: We studied CoQ10 level by HPLC in plasma and peripheral mononuclear cells obtained from patients with FM and healthy control subjects. Oxidative stress markers were analyzed in both plasma and mononuclear cells from FM patients. Results: Higher level of oxidative stress markers in plasma was observed respect to control subjects. CoQ10 level in plasma samples from FM patients was doubled compared to healthy controls and in blood mononuclear cells isolated from 37 FM patients was found to be about 40% lower. Higher levels of ROS production was observed in mononuclear cells from FM patients compared to control, and a significant decrease was induced by the presence of CoQ10. Conclusion: The distribution of CoQ10 in blood components was altered in FM patients. Also, our results confirm the oxidative stress background of this disease probably due to a defect on the distribution and metabolism of CoQ10 in cells and tissues. The protection caused in mononuclear cells by CoQ10 would indicate the benefit of its supplementation in FM patients. [Copyright &y& Elsevier]

Abstract: Objectives:: This work aims to evaluate the resistance of mononuclear cells to oxidative stress using a “KRL” test, formerly utilized to evaluate the resistance of erythrocyte to free radicals. Methods:: The “KRL” test evaluates the resistance to lysis of cells treated by free radicals generated under standardized conditions. Results:: We defined new analytical parameters (level of radical production, time course, number of cells) to obtain an accurate assay determining the resistance to oxidative stress of mononuclear cells, in comparison to that of erythrocytes. This test allows the evaluation of change in the redox state of mononuclear cells (improved by an antioxidant mix or deteriorated by antimycin A-induced mitochondrial radical overproduction). Interestingly, our data show that the sensitivity of mononuclear cells to oxidative stress is not correlated with the susceptibility of erythrocytes to oxidative stress. Conclusions:: The quantification of the susceptibility of mononuclear cells to oxidative stress gives additional information (in addition to erythrocyte resistance) and could be helpful for patients with chronic inflammation. [Copyright &y& Elsevier]

Abstract: The type I interferons interferon alpha (IFNα) and IFNβ are the first line of defense potently induced upon viral infection, and at the same time are immunomodulatory cytokines bridging innate and adaptive immunity. T cells secreting interleukin-17 (IL-17) have recently been identified to regulate neutrophil-mediated inflammation, and have been implicated in the pathogenesis of experimental colitis and human inflammatory bowel disease, and are considered to regulate the inflammatory response in these models. We therefore hypothesized that type I IFNs as sentinels of viral infection might counteract the development of Th17 cells. We studied the effects of IFNα on IL-17 mRNA and protein expression in human peripheral blood mononuclear cells (PBMC) and during differentiation of human and murine naïve T cells into Th17 cells. In patients with ulcerative colitis (UC) treated systemically with IFNα, we studied colonic expression of IL-17 before and 4 weeks after therapy. IFNα potently suppressed IL-17 production in PBMC both at the mRNA and protein level. Th17 differentiation of human and murine naïve T cells was markedly suppressed in the presence of IFNα. UC patients exhibited increased IL-17 expression in colonic tissue biopsies compared to healthy controls, which was down-regulated during IFNα therapy. IL-17 expression in colonic tissue correlated with clinical remission in these patients. Our data suggest that IFNα down-regulates IL-17 expression and Th17 differentiation in vitro and in vivo. As a corollary, these effects might play a role in the mode of action of type I IFNs in the treatment of various diseases. [Copyright &y& Elsevier]

Abstract: Effects of seselin (C14H12O3; MW 228) identified from Plumbago zeylanica on phytohemagglutinin (PHA)-stimulated cell proliferation were studied in human peripheral blood mononuclear cells (PBMC). The data demonstrated that seselin inhibited PBMC proliferation-activated with PHA with an IC50 of 53.87±0.74μM. Cell viability test indicated that inhibitory effects of seselin on PBMC proliferation were not through direct cytotoxicity. The action mechanisms of seselin may involve the regulation of cell cycle progression, interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in PBMC. Since cell cycle analysis indicated that seselin arrested the cell cycle progression of activated PBMC from the G1 transition to the S phase. Seselin suppressed IL-2 and IFN-γ production in a concentration-dependent manner. Furthermore, seselin significantly decreased the IL-2 and IFN-γ gene expression in PHA-activated PBMC. Therefore, results elucidated for the first time that seselin is likely an immunomodulatory agent for PBMC. [Copyright &y& Elsevier]