Cultures Of Peripheral Blood Monocytes Research Articles

Systemic production of cytokines and growth factors in rats with metabolic syndrome

ABSTRACT. An important characteristic of the state of the immune system is the production of cytokines, which determine the development of metabolic processes and are aimed at the immune response. Changes in the immune response occur with the development of homeostasis system disorders that change the rheological characteristics of the blood and contribute to the development of the metabolic syndrome (MS). The aim of the study was to study changes in the functional activity of cytokines and angiogenic growth factors (GF) during experimental MS in rats depending on age. Мaterials. MS modeling was carried out on 21 rats, which were divided into 3 groups, 7 animals each, of the same age (1a subgroup - 6 months of age, 2a subgroup - 9 months of age, 3a subgroup - 12 months). In the pathogenesis of the development of MS The role of MS in the development of systemic production of cytokines and FR has been studied, and data on the content of pro-inflammatory cytokines in the peripheral blood depending on the age of rats have been presented. The results of the study of the content of growth factor (GF) and endothelin in peripheral blood in MS tended to decrease with increasing age of rats providing morpho- and angiogenesis (p<0.01). An increase in the concentration of endothelin in the blood serum of rats with MS may contribute to the activation of vascular endothelial cells and an increase in the level of fibrinoid in the vascular system with increasing age. The level of cytokines circulating in the peripheral blood in rats with MS significantly differed from that, which was manifested by an increase in the level of IL-4, IL-8 and IL-10. Spontaneous production of cytokines by peripheral blood lymphocytes in MS increased with the age of rats, which indicated an increase in the secretion of IL-6 and IL-10, and a decrease in the production of IFN-1 and TNF-α. The decrease in spontaneous production of IL-1β and IL-10 in 24-hour cultures of peripheral blood monocytes in rats was significantly higher compared to IL-6 (p<0.001) compared to the 2nd and 3rd - observation groups. The content of IL-8 and TNF-α did not differ significantly (p>0.05), and IL-12 was below the sensitivity limit of the test system (less than 3 pg/ml). Conclusion. The data obtained indicate that in the peripheral blood of rats with MS, the secretion of pro-inflammatory and regulatory cytokines, as well as FR, decreases with age. Hormones also have an inhibitory effect on the production of cytokines by macrophages, the level of which changes significantly during MS. The results of the study indicate various pathogenetic mechanisms of the formation of MS in rats, accompanied by a violation of the immune response of mononuclear cells and angiogenic RF at the system level.

SCD1-Fatty Acid Desaturase Inhibitor MF-438 Alleviates Latent Inflammation Induced by Preservative-Free Prostaglandin Analog Eye Drops.

IntroductionProstaglandin analogs are the first line of treatment in patients with glaucoma. Recently, many preservative-free prostaglandin analogs have been marketed to increase their tolerance in chronic use. However, potentially safer formulations have been reported to induce inflammation within ocular surface and adnexa, associated with pronounced activation of tissue macrophages.AimWe aimed to evaluate the effect of a Stearoyl-CoA desaturase-1 (SCD1) inhibitor, MF-438, on the differentiation of monocytes exposed to eye drop detergents, representing saturated fatty acid derivatives.MethodsA culture of human peripheral blood monocytes was exposed to eye drops containing fatty acid derivatives (eye drop detergents), pf-latanoprost (Monoprost®, hydroxystearate macrogolglycerol – MGHS40) or pf-tafluprost (Taflotan®, polysorbate 80 – PS80), as well as pf-latanoprost+MF-438, MGHS40, and PS80. For the negative control C(-), monocytes were cultured in basal medium, and for the positive controls, monocytes were stimulated with Lipopolysaccharide (LPS) and Interferon γ (IFNγ) (M1 macrophages) or Interleukin-4 (IL-4) (M2 macrophages). The concentration of desaturase in the cell homogenates was determined by ELISA. The number of cells was counted under a microscope at 20x magnification.ResultsThe following concentrations of SCD1 (ng/mL) were measured: 7.8±0.3 – pf-latanoprost group; 1.5±0.4 – pf-tafluprost group; 6.8±0.7 – MGHS40 group; 0.4±0.002 – PS80 group; 0.9±0.02 – pf-latanoprost+MF-438 group; 5.4±1.6 – C(-) control; 0.5±0.04 – M1 control; 2.2±0.13 – M2 control. The percentages of macrophages in culture were 33.6%, 17.6%, 33%, 0%, 13.5%, 18.6%, 36.3%, and 39.3% for the pf-latanoprost, pf-tafluprost, MGHS40, PS80, pf-latanoprost+MF-438, C(-), M1, and M2 cultures, respectively. There was a strong correlation between SCD1 concentration and macrophage count in the culture (r=0.8, p<0.05).ConclusionInhibition of SCD1 in monocytes prevents their transformation into macrophages after exposure to saturated fatty acid derivatives contained in eye drops, which may contribute to the limitation of latent inflammation within ocular adnexa and could possibly translate into better tolerability of the topical treatment.

Activation of ERBB3 Receptor Prevents TNFα Production from Human Non‐Classical Monocytes

Immune cells amplify the progression of chronic heart failure (CHF). Recombinant neuregulin‐1 (NRG1) represents a potential therapy for the treatment of various forms of heart failure. We hypothesized that NRG1, acting via ERBB receptors, inhibits pro‐inflammatory cell activation in CHF. Using flow cytometry, we found that peripheral blood monocytes (PBMs) represent a major subpopulation of mononuclear cells expressing both ERBB2 and ERBB3 receptors. A strong inverse correlation was found between expression of ERBB3 and TNFa mRNA in PBMs from patients with heart failure (rs=‐0.682, p=0.001) but not in control subjects (rs=‐0.042, p=0.886). To determine direct effects of ERBB3 signaling on activation of PBMs we used a model of LPS‐induced TNFa production in ex vivo short‐term culture of PBMs. We found that glial growth factor 2(GGF2; USAN ‐ cimaglermin alfa), NRG1 isoform type II, decreased LPS‐induced TNFa production in CD14lowCD16+ non‐classical monocytes. The inhibitory effect of GGF2 on LPS‐induced TNFa production in CD14lowCD16+ monocytes strongly correlated with the expression of ERBB3 (rs=0.769, p=0.013). In contrast to non‐classical PBMs, we found no effect of GGF2 in CD14highCD16‐ subset of classical monocytes irrespective of their expression of ERBB3. Taken together, our results suggest that ERBB3 signaling plays an important role in inhibition of TNFa production from CD14lowCD16+ cells. Given that non‐classical monocytes play a central role in initiation of vascular inflammation, the stimulation of ERBB3 signaling in these cells may potentially slow progression of CHF.

Biji kopi meningkatkan ekspresi glutathione padamonosit secara in vitro (Coffee beans increase glutathione expression to monocyes in vitro)

Some research stated that coffee may act as an antioxidant, but has not mentioned concentrations can distinguish the production of antioxidants. One of the important antioxidant roles in preventing various diseases is GSH. The researchis intended to analyze the expression of antioxidant GSH of coffee beans in vitro. Furthermore, the culture ofperipheral blood monocytes of healthy people as much as 6 cc, mixed with the anticoagulant heparin. Ficoll-Hypaquecentrifugation and suspended in RPMI 1640 medium Buffy coat separated, washed, resuspended. Cells were placedon plate 96-well microtiter of 8 x 105 cells/well, and incubated. Then added RPMI, incubated and lymphocytesdiscarded. After washing, complete medium was added and incubation. Monocytes reacted with coffee with variousconcentrations (4 groups) and incubated 18 hours. GSH then analyzed by immunocytochemistry method. GSH cellsexpressing brown and counted under a light microscope (400x). The results indicated that a concentration of 3 g coffeebrewed in 300 mL of hot water has the greatest ability to increase GSH. It was concluded that coffee can improveGSHexpression on monocytes were exposed to DMBA, and the higher the concentration of the coffee bean, the higherthe expression of GSH.

Pluripotency gene expression and growth control in cultures of peripheral blood monocytes during their conversion into programmable cells of monocytic origin (PCMO): evidence for a regulatory role of autocrine activin and TGF-β.

Previous studies have shown that peripheral blood monocytes can be converted in vitro to a stem cell-like cell termed PCMO as evidenced by the re-expression of pluripotency-associated genes, transient proliferation, and the ability to adopt the phenotype of hepatocytes and insulin-producing cells upon tissue-specific differentiation. However, the regulatory interactions between cultured cells governing pluripotency and mitotic activity have remained elusive. Here we asked whether activin(s) and TGF-β(s), are involved in PCMO generation. De novo proliferation of PCMO was higher under adherent vs. suspended culture conditions as revealed by the appearance of a subset of Ki67-positive monocytes and correlated with down-regulation of p21WAF1 beyond day 2 of culture. Realtime-PCR analysis showed that PCMO express ActRIIA, ALK4, TβRII, ALK5 as well as TGF-β1 and the βA subunit of activin. Interestingly, expression of ActRIIA and ALK4, and activin A levels in the culture supernatants increased until day 4 of culture, while levels of total and active TGF-β1 strongly declined. PCMO responded to both growth factors in an autocrine fashion with intracellular signaling as evidenced by a rise in the levels of phospho-Smad2 and a drop in those of phospho-Smad3. Stimulation of PCMO with recombinant activins (A, B, AB) and TGF-β1 induced phosphorylation of Smad2 but not Smad3. Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation. Inhibition of autocrine TGF-β signaling by either SB431542 or anti-TGF-β antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells. Furthermore, anti-TGF-β antibody moderately enhanced Oct4A/Nanog expression. Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-β/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-β/Smad3, and to a lesser extent, activin/Smad2 signaling.

Endometrial cells sense and react to tissue damage during infection of the bovine endometrium via interleukin 1.

Cells generate inflammatory responses to bacteria when pattern recognition receptors bind pathogen-associated molecules such as lipopolysaccharide. Cells may also respond to tissue damage by sensing damage-associated molecules. Postpartum bacterial infections of the bovine uterus cause endometritis but the risk of disease is increased by tissue trauma triggered by dystocia. Animals that suffered dystocia had increased concentrations of inflammatory mediators IL-8, IL-1β and IL-1α in vaginal mucus 3 weeks postpartum, but they also had more bacteria than normal animals. Ex vivo organ cultures of endometrium, endometrial cells and peripheral blood monocytes did not generate inflammatory responses to prototypical damage molecules, HMGB1 or hyaluronan, or to necrotic cells; although they secreted IL-6 and IL-8 in a concentration-dependent manner when treated with IL-1α. However, necrotic endometrial cells did not accumulate intracellular IL-1α or release IL-1α, except when pre-treated with lipopolysaccharide or bacteria. Endometrial cell inflammatory responses to IL-1α were dependent on the cognate receptor IL-1R1, and the receptor adaptor protein MyD88, and the inflammatory response to IL-1α was independent of the response to lipopolysaccharide. Rather than a typical damage-associated molecule, IL-1α acts to scale the inflammatory response in recognition that there is a combination of pathogen challenge followed by endometrial cell damage.

Llama-derived nanobodies can act as P2X7 antagonists in vitro and in vivo

used to measure BAFF RNA at 6 h. Beta-actin was used as the reference housekeeping gene. BAFF protein in the culture supernatants was measured by ELISA at 96 h. All interferons upregulated BAFF expression (RNA & protein); however, IFN-beta was the most potent (IFN-beta N IFN-alpha N IFN-gamma). We also tested various combinations of cytokines to assess synergistic or antagonistic effects on BAFF expression. TNF-alpha and IL-10 inhibited BAFF expression by IFN-beta or IFN-alpha-stimulated cells. TNF-alpha enhanced BAFF expression by IFN-gamma-stimulated cells. Similar results were obtained with primary cultures of human peripheral blood monocytes. This study was supported by the Muscular Dystrophy Association (MDA).

Cytokine regulation of BAFF expression in human myeloid cells (P3005)

Abstract B-cell activating factor (BAFF) has an essential role in B cell homeostasis. It is synthesized and released by myeloid cells (monocytes &amp; macrophages). BAFF over-expression promotes the survival and maturation of autoreactive B cells. Increased BAFF levels are found in several human autoimmune diseases, suggesting a role for BAFF in disease pathogenesis and progression. However, the regulatory signals that affect BAFF production are largely unknown. Using the human monocytic cell line THP-1, we examined the capacity of several cytokines to modulate BAFF expression; these included tumor necrosis factor-α (TNF-α), IL-4, IL-10, IL-12, IL-17, and the type I &amp; type II interferons (IFN-α, IFN-β, and IFN-γ). Replicate cultures were used to measure BAFF RNA transcripts and released BAFF protein. Quantitative real-time PCR (QRT-PCR) was used to measure BAFF RNA at 6 hrs; β-actin was used as the reference housekeeping gene. BAFF protein in the culture supernatants was measured by ELISA at 96 hrs. All interferons upregulated BAFF expression (RNA &amp; protein); however, IFN-β was the most potent (IFN-β &amp;gt; IFN-α &amp;gt; IFN-γ). We also tested various combinations of cytokines to assess synergistic or antagonistic effects on BAFF expression. TNF-α inhibited BAFF expression by IFN-β−stimulated cells. Similar results were obtained with primary cultures of human peripheral blood monocytes.

Titanium particle-challenged osteoblasts promote osteoclastogenesis and osteolysis in a murine model of periprosthestic osteolysis

The current study investigates the interactive behavior of titanium alloy particle-challenged osteoblastic bone marrow stromal cells (BMSCs) and macrophage lineage cells in a murine knee-prosthesis failure model. BMSCs were isolated from male BALB/c mice femurs and induced in osteogenic medium. At 24h after isolation, BMSCs in complete induction medium were challenged with 1, 3 or 5mgml−1 titanium particles for 7days. Culture media were collected at 2, 4 and 6days and cells were harvested at 7days for alkaline phosphatase (ALP) assay/stains. Cell proliferation in the presence of Ti particles was periodically evaluated by MTT assay. Mice implanted with titanium-pin tibial implants were given an intra-articular injection of 50μl medium containing 5×105 Ti particles-challenged bone-marrow-derived osteoblastic cells, followed by a repeat injection at 2weeks post-operation. Control mice with titanium-pin implants received a naïve osteoblastic cell transfusion. After sacrifice at 4weeks, the implanted knee joint of each group was collected for biomechanical pin-pullout testing, histological evaluation and reverse transcriptase polymerase chain reaction analysis of mRNA extracted from the joint tissues. Ti particles significantly stimulated the proliferation of BMSC-derived osteoblastic cells at both high and low particle concentrations (p<0.05), with no marked differences between the particle doses. ALP expression was diminished following Ti particle interactions, especially in the high-dose particle group (p<0.05). In addition, the culture media collected from short-term challenged (48h) osteoblasts significantly increased the numbers of TRAP+ cells when added to mouse peripheral blood monocytes cultures, in comparison with the monocytes cells receiving naïve osteoblasts media (p<0.05). Intra-articular introduction of the osteoblastic cells to the mouse pin-implant failure model resulted in reduced implant interfacial shear strength and thicker peri-implant soft-tissue formation, suggesting that titanium particles-challenged osteoblasts contributed to periprosthetic osteolysis. Comparison of the gene expression profiles among the peri-implant tissue samples following osteoblast injection did not find significant difference in RunX2 or Osterix/Sp7 between the groups. However, MMP-2, IL-1, TNF-α, RANKL, and TRAP gene expressions were elevated in the challenged-osteoblast group (p<0.05). In conclusion, titanium alloy particles were shown to interfere with the growth, maturation, and functions of the bone marrow osteoblast progenitor cells. Particle-challenged osteoblasts appear to express mediators that regulate osteoclastogenesis and peri-prosthetic osteolysis.

Increased production of hydrogen peroxide by peripheral blood monocytes associated with smoking exposure intensity in smokers.

BackgroundSmoking is known to be associated with oxidative stress; however, it has not been elucidated whether the oxidative response is influenced by the intensity of smoking exposure.ObjectivesEvaluate the effect of smoking exposure on the secretion of hydrogen peroxide (H2O2) by the peripheral blood monocytes of smokers.MethodsA total of 25 smokers (50.3±8.8 years, 48% male) underwent the following evaluations: spirometry, pulse oximetry, body composition and total peripheral blood count. Peripheral blood monocyte (PBM) cultures were isolated and maintained, and IL-6 and TNF-α were measured in the plasma and in the supernatants of spontaneous and stimulated cultures. H2O2 was evaluated in the supernatants of the PBM cultures, and a subset of the PBM culture supernatants was stimulated with phorbol myristate acetate (PMA). We also evaluated 38 healthy controls (49.1±8.2 years, 42% male).ResultsThe spontaneous and stimulated monocytes’ secretion of H2O2 were statistically higher in the smokers than in the healthy controls (p<0.001). The H2O2 secretions were statistically significant higher after stimulation with PMA in both groups (p<0.001). In the multiple regression analysis, we identified a positive, statistically significant association between pack-years of smoking and the spontaneous secretion of H2O2 by PBM culture, adjusted for potential confounding variables. The association between PBM culture secretion of H2O2 and the production of TNF-α and IL-6 was not significant.ConclusionWe identified a positive association between higher production of H2O2 in smokers and higher smoking exposure during life. The influence of pack-years smoking may be a key modifiable factor in oxidative stress associated to smoking.

In vitro cytokine stimulation assay for glycolipid biosurfactant from Rhodococcus ruber: role of monocyte adhesion.

Glycolipid biosurfactant (GLB) from Rhodococcus ruber IEGM 231 was found to stimulate tumor necrosis factor-α (TNF-α), interleukin (IL) -1β and IL-6 production when applied as an ultrasonic emulsion to the adherent human peripheral blood monocyte culture. However, a lack of cytokine-stimulating activity was registered with the GLB applied as a hydrophobic film coating in 24-well culture plates, indicating that it may have been due to its inhibitory effect on monocyte adhesion. The mode of GLB application may therefore play an important role in in vitro assay of immunostimulatory activity of this compound as well as other bacterial glycolipids. Additionally, GLB from R. ruber displayed no cytotoxicity against human lymphocytes and therefore could be proposed as a potential immunomodulating and antitumor agent.

The effect of hyperbaric oxygen therapy on osteoclast formation and bone resorption

Event Abstract Back to Event The effect of hyperbaric oxygen therapy on osteoclast formation and bone resorption HW Al-Hadi1*, SW Fox1 and GR Smerdon2 1 University of Plymouth, School of Biomedical and Biological Science, United Kingdom 2 the Diving Diseases Research Centre, United Kingdom Hyperbaric oxygen therapy (HBO) is the breathing of pure oxygen in a sealed chamber at greater than normal atmospheric pressure. HBO is employed as an adjunctive therapy in a number of skeletal disorders such radio and bisphosphonate induced osteonecrosis of the jaw and chronic osteomyelitis. These disorders are associated with aberrant remodelling and excessive levels of resorption leading to increased fracture risk. The purpose of the present study was to evaluate the effect of HBO, pressure and hyperoxia on RANKL-induced osteoclast formation in RAW 264.7 cells and human peripheral blood monocytes (PBMC). Daily, HBO (2.4 ATA, 97% O2, 90 minutes per day) or hyperoxia (1 ATM, 95% O2, 90 minutes per day) treatments significantly decreased the number of RANKL-induced TRAP positive mononuclear and multinuclear osteoclasts forming in RAW and PBMC cultures (p <0.05). Similarly, HBO and hyperoxia significantly reduced bone resorption as assessed by the bone slice assay. Furthermore, pressure alone (2.4 ATA, 8.8% O2, 90 minutes per day) also significantly suppressed osteoclast differentiation and bone resorption to a similar extent as HBO and hyperoxia. Quantitative PCR analysis of key regulators of osteoclast differentiation (RANK) and multinuclearity (DC-STAMP) indicated that HBO, hyperoxia and pressure all significantly suppressed RANK and DC-STAMP mRNA expression. Interestingly, intermittent HBO, hyperoxia and pressure (daily, 90 minutes per day) also significantly reduced RANKL-induced osteoclast formation in hypoxic conditions (2% O2, 22.5 hours per day). This data suggests that HBO, elevated O2 and pressure suppress osteoclast differentiation and bone resorption in mouse and human monocytes. Furthermore the data indicates that elevated pressure and O2 also directly inhibit osteoclast formation in hypoxic conditions a hallmark of many skeletal disorders. This provides evidence supporting the use of HBO as an adjunctive therapy to prevent bone loss in a range of skeletal disorders associated with low oxygen partial pressure. Keywords: Bones, Bone Research Conference: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society, Cambridge, United Kingdom, 27 Jun - 29 Jun, 2011. Presentation Type: Oral Poster Topic: Abstracts Citation: Al-Hadi H, Fox S and Smerdon G (2011). The effect of hyperbaric oxygen therapy on osteoclast formation and bone resorption. Front. Endocrinol. Conference Abstract: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society. doi: 10.3389/conf.fendo.2011.02.00002 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2011; Published Online: 30 Sep 2011. * Correspondence: Mrs. HW Al-Hadi, University of Plymouth, School of Biomedical and Biological Science, United Kingdom, hadil.al-hadi@plymouth.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers HW Al-Hadi SW Fox GR Smerdon Google HW Al-Hadi SW Fox GR Smerdon Google Scholar HW Al-Hadi SW Fox GR Smerdon PubMed HW Al-Hadi SW Fox GR Smerdon Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Role of CXC chemokine ligand 13 in oral squamous cell carcinoma associated osteolysis in athymic mice

Oral squamous cell carcinomas (OSCC) are malignant tumors with a potent activity of local bone invasion; however, the molecular mechanisms of tumor osteolysis are unclear. In this study, we identified high level expression of chemokine ligand, CXCL13 and RANK ligand (RANKL) in OSCC cells (SCC1, SCC12 and SCC14a). OSCC cell-conditioned media (20%) induced osteoclast differentiation which was inhibited by OPG in peripheral blood monocyte cultures indicating that OSCC cells produce soluble RANKL. Recombinant hCXCL13 (10 ng/ml) significantly enhanced RANKL-stimulated osteoclast differentiation in these cultures. Trans-well migration assay identified that CXCL13 induces chemotaxis of peripheral blood monocytes in vitro which was inhibited by addition of anti-CXCR5 receptor antibody. Zymogram analysis of conditioned media from OSCC cells revealed matrix metalloproteinase-9 (MMP-9) activity. Interestingly, CXCL13 treatment to OSCC cells induced CXCR5 and MMP-9 expression suggesting an autocrine regulatory function in OSCC cells. To examine the OSCC tumor cell bone invasion/osteolysis, we established an in vivo model for OSCC by subcutaneous injection of OSCC cells onto the surface of calvaria in NCr-nu/nu athymic mice, which developed tumors in 4-5 weeks. muCT analysis revealed numerous osteolytic lesions in calvaria from OSCC tumor-bearing mice. Histochemical staining of calvarial sections from these mice revealed a significant increase in the numbers of TRAP-positive osteoclasts at the tumor-bone interface. Immunohistochemical analysis confirmed CXCL13 and MMP-9 expression in tumor cells. Thus, our data implicate a functional role for CXCL13 in bone invasion and may be a potential therapeutic target to prevent osteolysis associated with OSCC tumors in vivo.

Citocinas e proteínas de fase aguda do soro como marcadores de regressão da resposta inflamatória ao tratamento da tuberculose pulmonar

To evaluate the pattern of pro-inflammatory cytokines, anti-inflammatory cytokines and the acute phase response (APR) as markers of the response to treatment of pulmonary tuberculosis. Twenty-eight patients with pulmonary tuberculosis were evaluated at three time points: pretreatment (T0), treatment month 3 (T3) and treatment month 6 (T6). Levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukine-10 (IL-10) and transforming growth factor-beta (TGF-beta) were determined using ELISA in the supernatant of peripheral blood mononuclear cell and monocyte culture. Levels of total protein, albumin, globulins, C-reactive protein (CRP), alpha-1-acid glycoprotein (AAG) and erythrocyte sedimentation rate (ESR) were also determined. All of these parameters were also evaluated, only once, in a group of healthy controls. In relation to controls, patients presented cytokine levels and APR that were higher at T0, lower at T3 and either lower (TNF-alpha, IL-10, TGF-beta, AAG and ESR) or normal (IFN-gamma and CRP) at T6. For individuals with negative smear sputum microscopy, CRP, AAG and ESR are potential markers of pulmonary tuberculosis and of the need for treatment; CRP (T0 > T3 > T6 = reference) can also be a marker of treatment response. In the patients, the Th0 profile (IFN-gamma, IL-10, TNF-alpha and TGF-beta), inducer of and protector against inflammation, predominated at T0, whereas the Th2 profile (IL-10, TNF-alpha and TGF-beta), protecting against the harmful pro-inflammatory effect of the remaining TNF-alpha, predominated at T6. The behavior of IFN-gamma (T0 > T3 > T6 = controls) suggests its use as a marker of treatment response.

Monocytes secrete factors regulating glycosaminoglycan synthesis in mesangial cellsin vitro

The present study was conducted to determine the manner in which monocytes increase mesangial matrices, particularly glycosaminoglycans (GAGs) which interact with various other matrix components such as collagens, laminin, fibronectin and lipoproteins. A supernatant of human peripheral blood monocyte cultures activated by lipopolysaccharide (LPS) contains stimulating factors for glycosaminoglycan synthesis in rat mesangial cells (MCs). The culture supernatant in this study was concentrated and fractionated by gel chromatography and the GAG-stimulatory factor was found to have a molecular weight of 10-17 kD. This factor was shown to be present in fractions different from that of IL-1. Gel and ion-exchange chromatography studies of GAGs synthesized by MCs indicated the elution patterns of GAGs in the presence and absence of the monocyte culture supernatant to be essentially the same. Local infiltration of monocytes into the glomerulus, often seen in various types of glomerular injury, may be an important factor in the accumulation of the mesangial matrix.

Ex vivo recovery and activation of dysfunctional, anergic, monocyte-derived dendritic cells from patients with operable breast cancer: critical role of IFN-alpha

BackgroundDendritic cells (DCs) play a crucial role in initiating effective cell-mediated immune responses, but are dysfunctional and anergic in breast cancer. Reversal of this dysfunction and establishment of optimal DC function is a key prerequisite for the induction of effective anti-cancer immune responses.ResultsPeripheral blood DCs (PBDCs) and lymph node DCs (LNDCs) generated in vitro from adherent cultures of peripheral blood monocytes (PBMs) and lymph node monocytes (LNMs), respectively, using the 4 cytokine conditioned medium (CCM) (GM-CSF+IL-4+TNF-α+IFN-α) or 3 CCM (GM-CSF+IL-4+TNF-α) demonstrated a significantly higher degree of recovery and functional capacity in a mixed lymphocyte DC reaction (MLDCR, p < 0.001), expressed significantly higher levels of HLA-DR, CD86, compared with 2 CCM (GM-CSF+IL-4) or medium alone generated DCs from PBMs and LNMs (p < 0.001). The PBDCs generated with 3 CCM or 4 CCM showed a significantly (p < 0.001) enhanced macropinocytotic capability (dextran particles) and induced increased production and secretion of interleukin-12p40 (IL-12p40) in vitro (p < 0.001), compared with PBDCs generated from monocytes using 2 CCM or medium alone. Lipopolysaccharide (LPS) stimulation of PBDCs generated with 4 CCM demonstrated enhanced secretion of IL-6 but not IL-12p70, compared with control DCs unstimulated with LPS (p < 0.001).ConclusionDysfunctional and anergic PBDCs and LNDCs from patients with operable breast cancer can be optimally reversed by ex vivo culturing of precursor adherent monocytes using a 4 CCM containing IFN-α. Maximal immunophenotypic recovery and functional reactivation of DCs is seen in the presence of IFN-α. However, 4 CCM containing IFN-α generated-PBDCs, do not produce and secrete IL-12p70 in vitro.

Identification of Molecular Factors Required for Transdifferentiation of Human Circulating Monocytes into Multipotential Cells.

Recently, we have identified a primitive cell population termed monocyte-derived multipotential cells (MOMCs), which have a fibroblast-like morphology and a unique phenotype positive for CD14, CD45, CD34, and type I collagen. MOMCs are derived from in vitro culture of circulating CD14+ monocytes, and contain progenitors capable of differentiating into a variety of mesenchymal cells, neuron, and endothelium. Thus, human MOMCs are attractive candidates for an autologous transplantable cell source for therapeutic tissue regeneration. In vitro induction of MOMCs from circulating CD14+ monocytes requires their binding to matrix proteins such as fibronectin, and exposure to soluble factors derived from peripheral blood CD14− cells, but detailed processes remain unknown. Here, we investigated molecular factors involved in MOMC induction using human peripheral blood monocyte cultures. Induction of MOMCs was defined as having all of features, including fibroblastic morphology, CD34+ phenotype, and potentials of differentiating into multiple cell lineages. First, we set up MOMC cultures on fibronectin, type I collagen, laminin, or poly-L-lysine, and found that MOMCs were efficiently obtained exclusively in the presence of fibronectin. Since fibronectin is a ligand for β1 integrins, we examined effects of blockade of these interactions on MOMC generation. Induction of MOMCs was completely inhibited by anti-α5-integrin monoclonal antibody, but not by anti-α4-integrin antibody. In addition, a synthetic peptide that competed with the RGD domain of fibronectin suppressed generation of MOMC, but a CS-1 domain peptide had no effect, indicating that binding of α5β1 integrin to the RGD domain of fibronectin is required for monocytes to acquire a multipotential property. Next, highly enriched monocytes were cultured on fibronectin with various peripheral blood cell subsets to identify cells producing soluble factors required for MOMC generation. As a result, MOMCs were obtained exclusively in the presence of platelets. MOMC induction was also observed when conditioned medium prepared by stimulating platelets with various agonists, such as thrombin and ADP, was used instead of whole platelets. When platelet-derived condition medium was fractionated based on the molecular weight (MW), MOMC induction activity was retained in the fraction of MW < 30,000. Then, we selected a list of 16 candidate factors that are released from activated platelets and < 30,000 in MW, and screened them using two different strategies: evaluation of MOMC induction efficiency in monocyte cultures by adding individual candidate factors; and FACS sorting of circulating monocytes into cells with high and low expressions of receptor for individual candidate factors, followed by evaluation of MOMC induction. As a result, SDF-1was selected as a sole factor with capacity to promote generation of MOMCs. Moreover, MOMC generation was significantly more prominent in cultures with monocytes with high expression of CXCR4, a receptor for SDF-1, than in cultures of monocytes with low CXCR4 expression. In summary, circulating monocytes are able to transdifferentiate into multipotential cells through the fibronectin-α5β1 integrin and SDF-1-CXCR4 interactions. This information is helpful in establishing an optimal MOMC culture condition used in cell transplantation for tissue regeneration.

Epigallocatechin gallate affects human dendritic cell differentiation and maturation

Epigallocatechin gallate (EGCG), a component of green tea catechin with the strongest biological activity, has been focused in recent years because of its anti-inflammatory and immunomodulatory activities. Dendritic cells (DCs) are professional antigen-presenting cells, capable of priming naive T cells, and play the key roles in the activation of T-cell-mediated immune responses. We aimed to investigate the effect of EGCG on human monocyte-derived DCs (MODCs) and, consequently, on the T-cell-mediated immune response. The induction of apoptosis, and the detailed phenotypic and functional changes of MODCs, generated by culture of peripheral blood monocytes in the presence of GM-CSF and IL-4, induced by EGCG was investigated and compared with the effects of dexamethasone. Epigallocatechin gallate induced apoptosis and affected the phenotype of the developing DCs. The expressions of CD83, CD80, CD11c, and MHC class II, which are molecules essential for antigen presentation by DCs, were downregulated by EGCG. EGCG also suppressed the endocytotic ability of immature DCs, whereas dexamethasone-treated DCs had higher endocytotic ability than control DCs. Most importantly, mature DCs treated with EGCG inhibited stimulatory activity toward allogeneic T cells while secreting high amounts of IL-10. Epigallocatechin gallate induces immunosuppressive alterations on human MODCs, both by induction of apoptosis and suppression of cell surface molecules and antigen presentation.

Increased nitric oxide production by peripheral blood monocytes and selective elevation of serum IgE in patients with systemic lupus erythematosus (SLE). (101.13)

Abstract Prospective studies of patients with SLE have reported a prevalence of clinically apparent coronary artery disease (CAD) of up to 20%, a marked increase compared with the estimated CAD prevalence rate of 3.5% among healthy females 20–35 years (AHA 1997 Heart and Stroke Statistical Update). Nitric oxide is an important mediator of inflammation associated with vascular damage. We measured nitric oxide production by cultures of peripheral blood monocytes (N= 26) from patients with SLE and determined serum immunoglobulin concentrations (N=19). Elevated immunoglobulin concentrations were observed in sera from 8 patients (42%), and 4 individuals had a selective increase in IgE (&amp;gt;100 IU/ml), with other immunoglobulin isotypes in the normal range. Nitric oxide (NO) production by adherent monocyte cultures from patients with SLE was determined on day 7 using a modified colorimetric Griess reaction to measure nitrite, the stable NO metabolite. The results showed activation in 31% of the patients, with nitrite concentrations exceeding the baseline of 2.8μM more than threefold, reaching 8.8 μM. The mechanism of this in vivo activation of blood monocytes among some patients with SLE remains to be established. Activated monocyte-derived nitric oxide may contribute to the development of atherosclerosis and cardiovascular complications seen in many patients with SLE.

Adiponectin Stimulates RANKL and Inhibits OPG Expression in Human Osteoblasts Through the MAPK Signaling Pathway

Our study indicates that recombinant adiponectin induced RANKL and inhibited OPG expression in human osteoblasts through the AdipoR1/p38 MAPK pathway, and these responses contributed to the adiponectin-induced osteoclasts formation in the co-culture of osteoblast and peripheral blood monocytes systems. These findings showed that adiponectin increased osteoclast formation indirectly through stimulating RANKL and inhibiting OPG production in osteoblasts. It also suggests the pharmacological nature of recombinant adiponectin that indirectly induces osteoclasts formation. Recently, adiponectin has emerged as an element in the regulation of bone metabolism, but the mechanism remains. This study was undertaken to investigate the action of adiponectin on osteoclastogenesis through revealing RANKL and osteoprotegerin (OPG) expression in osteoblasts and osteoclast formation. Real-time quantitative PCR and ELISA were used to detect RANKL and OPG mRNA and protein expression in cultured human osteoblasts. The involved signal pathway was studied using mitogen-activated protein kinase (MAPK) inhibitor and adiponectin receptor 1 (AdipoR1) siRNA. The effects of recombinant adiponectin on osteoclasts formation also were examined in the co-culture systems of osteoblast and peripheral blood monocytes (PBMCs) systems or purified CD14 + PBMCs cultures. Our study showed that recombinant adiponectin induced RANKL and inhibited OPG mRNA expression in human osteoblasts in a dose- and time-dependent manner. Adiponectin also increased soluble RANKL and decreased OPG secretion in osteoblasts conditioned media. Suppression of AdipoR1 with siRNA abolished the adiponectin-regulated RANKL and OPG mRNA expression in osteoblasts. Furthermore, pretreatment of osteoblasts with the MAPK inhibitor SB203580 abolished adiponectin-regulated RANKL and OPG mRNA expression. Adiponectin induced osteoclast formation in the co-culture systems of osteoblast and PBMCs systems, and OPG entirely blocked this response. However, adiponectin had no direct effect on the differentiation of osteoclast precursor purified CD14 + PBMCs. These data indicate that recombinant adiponectin induced RANKL and inhibited OPG expression in human osteoblasts through the AdipoR1/p38 MAPK pathway, and these responses contributed to the adiponectin-induced osteoclast formation in the co-culture of osteoblast and PBMCs systems. These findings showed that adiponectin increased osteoclast formation indirectly through stimulating RANKL and inhibiting OPG production in osteoblasts. It suggests the pharmacological nature of recombinant adiponectin that indirectly induces osteoclasts formation.