Metabolic Activity Of Macrophages Research Articles

The RNA binding protein IGF2BP2/IMP2 alters the cargo of cancer cell-derived extracellular vesicles supporting tumor-associated macrophages

BackgroundTumor cells release extracellular vesicles (EVs) that contribute to the polarization of macrophages towards tumor-associated macrophages (TAMs). High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. However, there is a lack of understanding of whether IMP2 affects the cargo of cancer cell-derived EVs, thereby modulating macrophage polarization.MethodsEVs were isolated from IMP2-expressing HCT116 parental cells (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells. EVs were characterized according to MISEV guidelines, microRNA cargo was assessed by microRNA-Seq, and the protein cargo was analyzed by proteomics. Primary human monocyte-derived macrophages (HMDMs) were polarized by EVs, and the expression of genes and surface markers was assessed using qPCR and flow cytometry, respectively. Morphological changes of macrophages, as well as the migratory potential of cancer cells, were assessed by the Incucyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified using a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish larvae, and macrophages were isolated by fluorescence-activated cell sorting.ResultsEVs from WT and KO cells had a similar size and concentration and were positive for 25 vesicle markers. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs than KO EVs, while the expression of TNF and IL6 was reduced. A similar pattern was observed in macrophages from zebrafish larvae treated in vivo. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. MicroRNA-Seq revealed a significant difference in the microRNA composition of WT and KO EVs, particularly a high abundance of miR-181a-5p in WT EVs, which was absent in KO EVs. Inhibitors of macropinocytosis and phagocytosis antagonized the delivery of miR-181a-5p into macrophages and the downregulation of the miR-181a-5p target DUSP6. Proteomics data showed differences in protein cargo in KO vs. WT EVs, with the differentially abundant proteins mainly involved in metabolic pathways. WT EV-treated macrophages exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate than KO EV-treated cells.ConclusionOur results show that IMP2 determines the cargo of EVs released by cancer cells, thereby modulating the EVs' actions on macrophages. Expression of IMP2 is linked to the secretion of EVs that polarize macrophages towards a tumor-promoting phenotype.

Endotoxin contamination alters macrophage-cancer cell interaction and therapeutic efficacy in pre-clinical 3D in vitro models

The rapid developments in biofabrication, in particular 3D bioprinting, in the recent years have facilitated the need for novel biomaterials that aim to replicate the target tissue in great detail. The presence of endotoxins in these biomaterials is often an overlooked problem. In pre-clinical 3D in vitro models, endotoxins can have significant influence on cell behavior and credibility of the model. In this study we demonstrate the effects of high levels of endotoxins in commercially-available gelatin on the macrophage-cancer cell crosstalk in a 3D bioprinted co-culture model. First, it is demonstrated that, while presenting the same mechanical and structural stimuli, high levels of endotoxin can have significant influence on the metabolic activity of macrophages and cancer cells. Furthermore, this study shows that high endotoxin contamination causes a strong inflammatory reaction in macrophages and significantly inhibits the effects of a paracrine macrophage-cancer cell co-culture. At last, it is demonstrated that the differences in endotoxin levels can drastically alter the efficacy of novel macrophage modulating immunotherapies, AS1517499 and 3-methyladenine. Altogether, this study shows that endotoxin contamination in biomaterials can significantly alter intra- and intercellular communication and thereby drug efficacy, which might lead to misinterpretation of the potency and safety of the tested compounds.

РОЛЬ ИММУНОЛОГИЧЕСКИХ ФАКТОРОВ В НЕБЛАГОПРИЯТНОМ ИСХОДЕ ХРОНИЧЕСКОЙ ОБСТРУКТИВНОЙ БОЛЕЗНИ ЛЕГКИХ

Aim. The aim of the research was to study the role of immunological mechanisms in the unfavorable outcome of chronic obstructive pulmonary disease (COPD). Material and methods. The research included 116 patients hospitalized in 2005-2006 in the pulmonology department of patients with the exacerbation of mild and moderate COPD. Research protocol: general clinical and special (immunological) methods of examination were performed in patients with COPD. Instrumental research methods included: spirography, ECG, EchoCG, fibrobronchoscopy, chest X-ray. After diagnostic bronchoscopy, bronchoalveolar fluid (BAF) was taken for cytological and immunological examination. On the first and second days of inpatient treatment, patients underwent immunological studies: immunophenotyping of mononuclear cells (MNC), assessment of phagocytic activity of leukocytes (latex test, nitroblue tetrazolium (NBT) test) in blood and BAF, determination of the content of immunoglobulins in blood serum and BAF, study of cytokine levels in blood serum, BAF and MNC culture supernatant under conditions of their spontaneous production and activation in the mitogen-stimulated (phytohemagglutinin "Difco" 5 mg/ml) proliferation. Statistical data processing was carried out using the Statistica 10.0 program. In 2020, the long-term survival of COPD patients was assessed. The cohort of patients was divided into two groups. Survivors were included in the first group (n=44), and patients who died by 2020 were included in the other group (n=72). The retrospective comparison of the studied indicators was determined at the time of COPD exacerbation in 2005- 2006 in these patient groups. Results and discussion. In the surviving patients, the BAF cytogram was distinguished by the higher macrophage content and the increased number of NBT-positive cells in this population with the reduced neutrophil content in both blood and BAF. The deceased had elevated levels of IgM and IgA in the blood serum and BAF. The results of the study of cytokine production in MNC cultures in vitro indicate that the ratio of IFN-γ/IL-4 in the conditions of spontaneous cytokine production was higher in the deceased compared with the indicators of the surviving patients. This fact indicates the polarization of the immune response of the deceased in the direction of the cell type mediated by Th1 cells, which is confirmed by the peculiarities of the BAF cytokine profile in the deceased – the significant increase in the value of IFN-γ/IL-4 relative to the indicators in the survivors. Conclusion. The unfavorable outcome of COPD is associated with the increase in the number of neutrophil cells in BAF, in the blood, with the predominance of Th1-type activation of adaptive immunity at the local level and activation of both cellular and humoral mechanisms of adaptive immunity at the systemic level. The increased activity of innate immunity in COPD exacerbation, manifested by the increase in the number and metabolic activity of macrophages, is associated with the long-term survival of patients.

Copper Acyl Salicylate Has Potential as an Anti-Cryptococcus Antifungal Agent.

The in vitro antifungal activity of aspirin against cryptococcal cells has been reported. However, the unwanted effects of aspirin may limit its clinical application. Conceivably, a derivative of aspirin could overcome this challenge. Toward this end, this study considered the usage of an aspirinate-metal complex, namely, copper acyl salicylate (CAS), as an anti-Cryptococcus antifungal agent. Additionally, the study examined the effects of this compound on macrophage function. The in vitro susceptibility results revealed that cryptococcal cells were vulnerable (in a dose-dependent manner) to CAS, which might have effected growth inhibition by damaging cryptococcal cell membranes. Interestingly, when CAS was used in combination with fluconazole or amphotericin B, synergism was observed. Furthermore, CAS did not negatively affect the growth or metabolic activity of macrophages; rather, it sensitized those immune cells to produce interferon gamma and interleukin 6, which, in turn, might have aided in the phagocytosis of cryptococcal cells. Compared to our aspirin data, CAS was noted to be more effective in killing cryptococcal cells (based on susceptibility results) and less toxic toward macrophages (based on growth inhibition results). Taking these findings together, it is reasonable to conclude that CAS may be a better anti-Cryptococcus drug that could deliver better therapeutic outcomes, compared to aspirin.

Abstract 428: An Unexpected Role for ACC in Lipid Droplet Formation in Macrophages in Response to Acellular Adipocyte Fat

In adipose tissues of obese mice, macrophages accumulate in crown-like structures (CLS) formed around dying adipocytes. The CLS macrophages are thought to clear the dead adipocytes by exophagy, which involves exocytosis of lysosomes and digestion of apoptotic adipocytes. After digesting the plasma membrane and other cytosolic contents of the adipocyte, CLS macrophages come in contact with the large lipid droplet, however, it is unknown how macrophages response to such acellular adipocytes. We hypothesized that the acelular adipocytes in CLSs promote inflammation and metabolically activate the macrophages to promote clearance of the lipid. To mimic the in vivo scenario, we exposed cultured murine bone marrow-derived macrophages to lipid droplets isolated from the adipocytes of high-fat diet-induced obese C57BL/6 mice. In response to adipocyte lipid, macrophages accumulated lipid droplets, which were similar in size and number to lipid droplets of CLS macrophages in vivo . Acellular lipid exposure significantly increased TNF-α gene expression, which was suppressed by the CD36 inhibitor sulfo-N-succinimidyl oleate, supporting CD36-mediated inflammatory response. Moreover, lipid exposure significantly lowered metabolic activity of macrophages and impaired clearance of apoptotic bodies from 3T3-L1 adipocytes. Using specific inhibitors, unexpectedly we found that lipid droplet accumulation in macrophages was independent of CD36 activity or processes involved in exophagy such as exocytosis of lysosomes, extracellular lipase activity, lipolysis and phagocytosis. Interestingly, lipid droplet accumulation was dependent on acetyl-CoA carboxylase (ACC) as determined by use of ACC inhibitors soraphen A and TOFA, and siRNA knock-down of ACC in macrophages. Furthermore, confocal microscopy of whole mount adipose tissue revealed expression of ACC in CLS macrophages. Altogether, using a novel in vitro model we demonstrate that acelular adipocytes suppress metabolic activity, but induce inflammation and de novo lipogenesis-mediated lipid droplet formation in macrophages.

Unique metabolic activation of adipose tissue macrophages in obesity promotes inflammatory responses

Aims/hypothesisRecent studies have identified intracellular metabolism as a fundamental determinant of macrophage function. In obesity, proinflammatory macrophages accumulate in adipose tissue and trigger chronic low-grade inflammation, that promotes the development of systemic insulin resistance, yet changes in their intracellular energy metabolism are currently unknown. We therefore set out to study metabolic signatures of adipose tissue macrophages (ATMs) in lean and obese conditions.MethodsF4/80-positive ATMs were isolated from obese vs lean mice. High-fat feeding of wild-type mice and myeloid-specific Hif1α−/− mice was used to examine the role of hypoxia-inducible factor-1α (HIF-1α) in ATMs part of obese adipose tissue. In vitro, bone marrow-derived macrophages were co-cultured with adipose tissue explants to examine adipose tissue-induced changes in macrophage phenotypes. Transcriptome analysis, real-time flux measurements, ELISA and several other approaches were used to determine the metabolic signatures and inflammatory status of macrophages. In addition, various metabolic routes were inhibited to determine their relevance for cytokine production.ResultsTranscriptome analysis and extracellular flux measurements of mouse ATMs revealed unique metabolic rewiring in obesity characterised by both increased glycolysis and oxidative phosphorylation. Similar metabolic activation of CD14+ cells in obese individuals was associated with diabetes outcome. These changes were not observed in peritoneal macrophages from obese vs lean mice and did not resemble metabolic rewiring in M1-primed macrophages. Instead, metabolic activation of macrophages was dose-dependently induced by a set of adipose tissue-derived factors that could not be reduced to leptin or lactate. Using metabolic inhibitors, we identified various metabolic routes, including fatty acid oxidation, glycolysis and glutaminolysis, that contributed to cytokine release by ATMs in lean adipose tissue. Glycolysis appeared to be the main contributor to the proinflammatory trait of macrophages in obese adipose tissue. HIF-1α, a key regulator of glycolysis, nonetheless appeared to play no critical role in proinflammatory activation of ATMs during early stages of obesity.Conclusions/interpretationOur results reveal unique metabolic activation of ATMs in obesity that promotes inflammatory cytokine release. Further understanding of metabolic programming in ATMs will most likely lead to novel therapeutic targets to curtail inflammatory responses in obesity.Data availabilityMicroarray data of ATMs isolated from obese or lean mice have been submitted to the Gene Expression Omnibus (accession no. GSE84000).

Abstract 4621: Metabolic activation of macrophages by CpG stimulates anti-tumor activity and overcomes CD47-independent inhibition by tumor cells in pancreatic ductal adenocarcinoma

Abstract Macrophages dominate the immune infiltrate present in pancreatic ductal adenocarcinoma (PDAC). Although tumor-infiltrating macrophages have the potential to mediate anti-tumor activity, they most commonly acquire an immunosuppressive role in cancer. We have found that CpG oligonucleotides - containing unmethylated cytosine-guanine motifs - potently induce macrophages to phagocytose PDAC cells and mediate anti-tumor effects. When delivered in vivo, in a fully immunocompetent murine model of PDAC, CpG increased the presence of macrophages within PDAC tumors and suppressed tumor outgrowth in a macrophage-dependent manner. Though CpG did not polarize macrophages toward classical anti-tumor (M1) or pro-tumor (M2) phenotypes, CpG treatment increased the basal rate of oxygen consumption in macrophages. Reversing this metabolic shift by inhibiting fatty acid oxidation abolished pro-phagocytic and anti-tumor effects by macrophages in vitro and in vivo. We investigated the impact of CpG in overcoming anti-phagocytic signals from CD47, a membrane protein overexpressed in multiple malignancies, including PDAC. While targeted knockout of CD47 in PDAC cells was not sufficient to activate in vitro phagocytosis by macrophages or anti-tumor effects in vivo, delivery of CpG potentiated anti-tumor and phagocytic activity that was independent of CD47 expression by tumor cells. However, the combination of CD47 loss in tumor cells and systemic CpG treatment enhanced the survival of tumor-bearing mice, indicating that disruption of CD47 can sensitize tumors to macrophage-directed immunotherapy. Together, our findings demonstrate a key role for cellular metabolism in directing the anti-tumor functions of macrophages and overcoming negative regulatory signals imposed by malignant cells. Citation Format: Mingen Liu, Gregory Beatty. Metabolic activation of macrophages by CpG stimulates anti-tumor activity and overcomes CD47-independent inhibition by tumor cells in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4621. doi:10.1158/1538-7445.AM2017-4621

Macrophage interactions with polylactic acid and chitosan scaffolds lead to improved recruitment of human mesenchymal stem/stromal cells: a comprehensive study with different immune cells.

Despite the importance of immune cell-biomaterial interactions for the regenerative outcome, few studies have investigated how distinct three-dimensional biomaterials modulate the immune cell-mediated mesenchymal stem/stromal cells (MSC) recruitment and function. Thus, this work compares the response of varied primary human immune cell populations triggered by different model scaffolds and describes its functional consequence on recruitment and motility of bone marrow MSC. It was found that polylactic acid (PLA) and chitosan scaffolds lead to an increase in the metabolic activity of macrophages but not of peripheral blood mononuclear cells (PBMC), natural killer (NK) cells or monocytes. PBMC and NK cells increase their cell number in PLA scaffolds and express a secretion profile that does not promote MSC recruitment. Importantly, chitosan increases IL-8, MIP-1, MCP-1 and RANTES secretion by macrophages while PLA stimulates IL-6, IL-8 and MCP-1 production, all chemokines that can lead to MSC recruitment. This secretion profile of macrophages in contact with biomaterials correlates with the highest MSC invasion. Furthermore, macrophages enhance stem cell motility within chitosan scaffolds by 44% but not in PLA scaffolds. Thus, macrophages are the cells that in contact with engineered biomaterials become activated to secrete bioactive molecules that stimulate MSC recruitment.

Establishment of macrophage model of iron overload in vitro and the injury induced by oxidative stress on macrophage with iron overload

To establish macrophage iron overload model in vitro by co-culture macrophages with iron, and to explore the effect of iron overload on cell reactive oxygen species (ROS) and the impact of ROS on macrophages. Iron overload group were treated with different concentrations (0, 5, 10, 20, 40, 80 μmol/L respectively) of ferric ammonium citrate (FAC). The control group was the group of macrophages without FAC treatment. We detected the number and state of cells, metabolic activity, the change of phagocytosis, the levels of ROS and reactive nitrogen, and changes of related oxidative stress signaling pathways in different groups. Changes in the above indexes were detected after application of deferasirox (DFX) to remove iron and the antioxidant N -acetylcysteine (NAC) to clear excess oxidative stress. (1)The levels of labile iron pool (LIP) in macrophages co-cultivated with iron was increased with the increase of iron concentration in a dose-dependent manner. The LIP levels was the highest in the macrophages treated with 80 μmol/L. (2)The increase of FAC concentration, the metabolic activity of macrophages in the 5 FAC-treated groups decreased to 51.58%, 40.98%, 16.23%, 3.46%, and 0.05% of the activity level of the control group (all P< 0.05). The group with the metabolic activity decreased to 16.23% (20 μmol/L) was selected as the iron overload group for the following experiments. (3)Compared with the control group, the number of macrophages in the iron overload group reduced to 32.80% (P<0.05), and the state of cells changed from adherence to partial suspension. The phagocytosis of macrophages in the iron overload group reduced to 20.40% of the control group (P<0.05). (4)Our further experiment showed that the levels of ROS and the activity nitrogen in the iron overload group increased by 7.71-and 1.45-fold compared with the control group (both P<0.05). The RT-PCR showed up-regulated mRNA expression of genes related with ROS production, i. e. nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX 4) gene related with ROS production and inducible nitric oxide synthase (iNOS) gene related with reactive nitrogen production, down-regulated mRNA expression of glutathione peroxidase 1 (GPX1) gene which participated in ROS clearance. Moreover, mRNA expression of phosphatidylinositol-3-kinase (PI3K) gene involved in oxidative stress signaling pathway in the iron overload group was up-regulated, while fork head protein O3 (FOXO3) which regulated oxidative stress through negative feedback showed a down-regulation level of mRNA expression compared with the control group. (5)After iron chelation and antioxidant treatment, the above-mentioned damage in the iron overload group were partially reversed. The damages of iron overload on macrophages may be mediated by inducing oxidative stress and activating oxidative stress signaling pathways. Our established model provides a method to explore the mechanism of iron overload on macrophage, and may shed some new light on possible therapeutic target in treating iron overload patients.

Abstract 2888: Metabolically activated macrophages in obesity associated TNBC

Abstract Triple negative breast cancer (TNBC) accounts for about 20% of all breast cancers. TNBC patients have an extremely poor prognosis due to their high metastatic potential and lack of targeted drug therapies. Emerging epidemiological data suggest that obesity is strongly linked to the incidence and severity of TNBC; obese women have a 35% higher risk of developing TNBC and 46% higher risk of developing distant metastases. Thus, understanding the biological processes that link obesity and TNBC has important clinical applications for prognosis and treatment. Mechanisms by which obesity worsens TNBC prognosis are incompletely understood. One clue to its action is that obesity causes chronic inflammation, and macrophage infiltration into adipose tissue is a key mediator of this inflammation. Recent studies demonstrated that macrophages are enriched in breast adipose tissue of obese humans and mice. Moreover, depletion of macrophages in mice diminished the effects of obesity on TNBC growth and metastasis. Although anti-inflammatory ‘M2-like’ TAMs are key effector cells that promote tumorigenesis, it is well accepted that obesity stimulates macrophages to adopt a pro-inflammatory ‘M1-like’ state. However, M1 macrophages exhibit potent anti-tumor functions. From these discordant observations an important paradox emerges: How can obesity promote breast cancer if it elicits an M1, anti-tumor macrophage phenotype? Using a combination of proteomics, immunology, and cell biology, we recently identified a novel metabolically activated (MMe) macrophage phenotype produced by exposure to high levels of insulin, glucose, and palmitate, conditions characteristic of obese and diabetic patients. Here we show that gene expression patterns in MMe (but not M1) macrophages are strongly associated with pathways involved in breast cancer. Moreover, pre-treating BM1 cells with conditioned media derived from MMe macrophages promotes TNBC cell invasion by 2.3 fold, suggesting that MMe macrophages potentiate metastasis. We further show that breast adipose tissue macrophages isolated from obese women express cell surface markers of MMe (CD36, ABCA1), but not M1 (CD38, CD319, CD274), macrophages. Furthermore, treating naïve macrophages with media conditioned by human mammary adipose tissue from an obese subject (BMI = 37), but not a lean subject (BMI = 19), induced genes diagnostic of the MMe phenotype. Thus, mammary adipose tissue from obese women supports metabolic activation of macrophages. Together, these observations suggest that obesity-induced changes to adipose tissue reprogram macrophages to an MMe phenotype that potentiates TNBC. A comprehensive understanding of signaling mechanisms involved in metabolic activation would enable development of directed therapies towards this specific pro-tumorigenic macrophage phenotype, thereby leaving the immune system of cancer patients intact. Citation Format: Payal Tiwari, Kelly Schoenfelt, Swati Kulkarni, Marsha Rosner, Lev Becker. Metabolically activated macrophages in obesity associated TNBC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2888. doi:10.1158/1538-7445.AM2015-2888

Effect of heavy metal intoxication on macrophage metabolic activity of mice infected with Ascaris suum

Abstract The effect of heavy metal intoxication on superoxide anion (O2 −) production and larval burden during experimental Ascaris suum infection was studied. Mice were chronically intoxicated with lead (Pb), cadmium (Cd) or mercury (Hg) and subsequently infected with A. suum. The metabolic activity of peritoneal macrophages in mice intoxicated with Pb was suppressed and subsequent parasitic infection did not change this inhibition. Cd intoxication increased the superoxide production and also stimulated the activity of this oxygen radical after A. suum infection. Intoxication with Hg had a stimulative effect on the macrophage metabolic activity and subsequent A. suum infection moderately reduced this activity. Parasite burden was different depending on a type of heavy metal intoxication. Pb intoxication moderately increased the parasite burden in the liver and lungs of intoxicated mice. In contrast, Cd and Hg intoxication triggered a marked reduction of A. suum larvae in the liver and lungs of intoxicated mice, respectively. Monitored heavy metals differed in their immunomodulatory effect on metabolic activity of macrophages what also altered the intensity of the parasite infection in the hosts.

A2B Adenosine Receptors Prevent Insulin Resistance by Inhibiting Adipose Tissue Inflammation via Maintaining Alternative Macrophage Activation

Obesity causes increased classical and decreased alternative macrophage activation, which in turn cause insulin resistance in target organs. Because A2B adenosine receptors (ARs) are important regulators of macrophage activation, we examined the role of A2B ARs in adipose tissue inflammation and insulin resistance. A2B AR deletion impaired glucose and lipid metabolism in mice fed chow but not a high-fat diet, which was paralleled by dysregulation of the adipokine system, and increased classical macrophage activation and inhibited alternative macrophage activation. The expression of alternative macrophage activation–specific transcriptions factors, including CCAAT/enhancer-binding protein-β, interferon regulatory factor 4, and peroxisome proliferator–activated receptor-γ, was decreased in adipose tissue of A2B AR–deficient mice. Furthermore, in in vitro studies, we found that stimulation of A2B ARs suppressed free fatty acid–induced deleterious inflammatory and metabolic activation of macrophages. Moreover, AR activation upregulated the interleukin-4–induced expression of CCAAT/enhancer-binding protein-β, interferon regulatory factor 4, and peroxisome proliferator–activated receptor-γ in macrophages. Altogether, our results indicate that therapeutic strategies targeting A2B ARs hold promise for preventing adipose tissue inflammation and insulin resistance.

Chronic Treatment With Electroconvulsive Shock May Modulate the Immune Function of Macrophages

To determine the effect of single and chronic electroconvulsive shock (ECS) administration on the immunoregulatory functions of macrophages. Male Wistar rats received single or chronic treatment with ECS (150 mA, 50 Hz, 0.5 seconds) delivered through ear clips, once a day for 10 consecutive days, or sham ECS administered likewise. The rats were killed 24 hours after the last treatment, and peritoneal macrophages were cultured in vitro for 3 or 36 hours for a subsequent determination of their metabolic activity. The ability of macrophages to reduce Alamar Blue, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and nitrotetrazolium blue chloride and pinocytosis, adherence, and vitality, as well as synthesis of nitric oxide and arginase activity, was assessed. We found statistically significant changes in the biological properties of macrophages which occurred after 36 hours of incubation, especially in cultures stimulated with lipopolysaccharide; in contrast, no differences were observed between groups assessed after 3 hours of incubation. Rats receiving chronic 10-fold ECS showed a substantial increase in the metabolic activity of macrophages, reflected as their ability to reduce Alamar Blue and MTT and to increase arginase activity, accompanied with a marked but statistically insignificant decrease in nitric oxide synthesis compared with respective controls. Our results suggest that chronic treatment with ECS may induce long-lasting changes in the activity of peritoneal macrophages. Attenuation of their proinflammatory properties indicates that ECS can change the primarily immunoregulatory functions of macrophages.

Abstract 5825: Association between Patterns of FDG Uptake and Arterial Wall Calcification on PET/CT and Atherogenic Risk Factors in Healthy Subjects

Background: Augmented metabolic activity of macrophages leads to enough F-18 Fluorodeoxyglucose (FDG) uptake to allow visualization by positron emission tomography (PET). A large body of data, based on computed tomography (CT), has also accumulated concerning the relevance of vascular calcification to the atherosclerotic process. FDG PET/CT can localize both inflammatory changes and vascular calcification. The purpose of this study was to investigate risk factors contributing to these changes in the aorta in healthy subjects. Materials and Methods: A total of 66 consecutive healthy subjects (44 men, 22 women; age range, 30–82 years, mean age, 55.8 years) participating in a health check protocol including FDG PET/CT were evaluated retrospectively. We placed regions of interest on the arterial wall to measure FDG uptake by PET images. To assess arterial calcification, the calcium score of the aorta was measured on CT images. Results: FDG uptake was observed most commonly in proximal, followed by descending, thoracic, and abdominal segments. On the other hand, the most common site of vascular calcification was the descending thoracic aorta, followed by abdominal and, proximal segment. Whole aortic calcification (total calcium score of the whole aorta) was significantly correlated with age (r= 0.353, P= 0.004). On the other hand, FDG uptake (total SUV max of the whole aorta) was significantly correlated with systolic blood pressure (SBP) (r= 0.303, P= 0.013), triglyceride (TG) (r= 0.281, P= 0.022), fasting plasma glucose (FPG) (r= 0.317, P= 0.010), HbA1c (r= 0.433, P&lt; 0.001), visceral abdominal fat area (r= 0.319, P= 0.005), and was negatively correlated with high density lipoprotein (HDL) (r= −0.317, P= 0.010), and adiponectin (r= −0.273, P= 0.029). Conclusions: Aortic calcification was significantly correlated with age. On the other hand, FDG uptake was significantly correlated with the components of metabolic syndrome such as SBP, TG, FPG, HbA1c, visceral adipose fat area and negatively correlated with HDL and adiponectin, but not with age. Our results may suggest that the components of metabolic syndrome and aging affect the progression of atherosclerosis differently.

Changes in the metabolic activity of macrophages under the influence of tick-borne encephalitis virus

The metabolic activity of macrophages infected with tick-borne encephalitis virus (TBEV) affecting the human nervous system has been studied for the first time. The penetration and reproduction of TBEV in the macrophages stimulated their oxygen metabolism, increasing the activity of NADPH-oxidase complex, as well as the mitochondrial enzymes lactate dehydrogenase, succinate dehydrogenase, and cytochrome oxidase. A wave-like change in the activity of these enzymes in the macrophages reflected the reaction of the cells to the penetration of the virus in the first period (within 3 h) and to the synthesis of the virus particles and their exit into the extracellular space in the second period (from 5 to 48 h). In the macrophages infected with TBEV, accumulation of NO metabolites was observed. In the late period of the examination (1-4 days), the activities of superoxide dismutase and lysosomal enzymes (nonspecific esterase and acid phosphatase) were detected. Thus, the early increase in the activity of the cell enzymes indicates the activation of the macrophages, and the subsequent increase in their activity corresponds to the enhanced synthetic activity of the macrophages.

Fluorodeoxyglucose and calcium uptake in the vascular wall: Clinically relevant or sugar-coated pill?

As nuclear cardiology has developed a broader ceptual base, moving from classical precepts of ems physiology to molecular and cell biology, there been increasing interest in the study of the vasculain addition to classical studies that have focused on myocyte. Experiments in laboratory animals and an beings have used a variety of new biologically vant targets for imaging the blood vessel wall, such ow-density lipoprotein cholesterol, oxidized lowsity lipoprotein cholesterol, macrophages, cellular ptosis, integrins such as v 3, matrix metalloproes, and cathepsins. There has been substantial rest in the macrophage as an imaging target, based on reness of this cell’s fundamental importance in the ogy and progression of atherosclerosis. Macroges clearly play an important role in the development laque vulnerability and hence are a real risk factor for ue rupture, erosion, and resultant intravascular mbosis. Fluorodeoxyglucose (FDG) accumulation in the vessel wall has been shown to correspond to of macrophage infiltration. Presumably, the augted metabolic activity of macrophages leads to local uptake sufficient to allow visualization with tron emission tomography (PET). In the smaller nary arteries FDG uptake has been noted with ially designed intravascular catheter-based radiation ctors. A large body of data has accumulated cerning the relevance of vascular calcification by puted tomography (CT) to the atherosclerotic pro. Visualized vascular calcification generally signifies ore advanced or mature stage of the atherosclerotic ess.

Forced retraction of spinal root injury enhances activation of p38 MAPK cascade in infiltrating macrophages

Root-rupture injury is a type of preganglionic brachial plexus injury resulting from traction force, where a small section of the spinal root is usually left behind. We have established experimental models of both root-rupture injury with traction force and rhizotomy without traction force in rats and we examined the activation of microglia/ macrophages in both conditions. LGP107 and LGP96, which are rat homologs of lysosome-associated membrane proteins, were most useful as immunohistochemical markers of mononuclear phagocytes. The metabolic activation of macrophages was analyzed by immunohistochemistry with a series of antibodies against tumor necrosis factor-alpha (TNF-alpha), cathepsin B, p38 mitogen-activated protein kinase (MAPK), and mitogen-activated kinase kinase 3 (MKK3). Both root-rupture injury and rhizotomy rapidly induced the aggregation of numerous macrophages from the injured dorsal root to the dorsal funiculus and TNF-alpha was highly expressed by the macrophages in the injured dorsal root at 48 h. Activation of p38 MAPK was preferentially observed in the macrophages at the ruptured dorsal root; however, only slight activation of p38 MAPK was observed at the rhizotomized dorsal root. These findings suggest that traction injury of the spinal root might induce activation of the p38 MAPK cascade and production of TNF-alpha in the infiltrating macrophages, both of which might participate in aggravation of the root injury.

The state of the immune system in thyroidectomized rats.

Disorders in the immune system manifesting in decreased weight and cellularity of lymphoid organs, decreased count of large granular lymphocytes, suppression of the immune response and metabolic activity of macrophages were observed in experimental animals 1-6 months after thyroidectomy. The intensity of these disorders depends on the period elapsed after the surgery. These disorders can result from not only changes in the levels of the pituitary-thyroid hormones, but also disturbances in the endocrine function of the thymus in thyroidectomized animals.

Immunomodulative effect of muramyldipeptide in mice with larval toxocarosis

The phagocytic ability of polymorphonuclear leukocytes, the metabolic activity of peritoneal macrophages, the proliferative response of T- and B-cells, and the production of specific anti-Toxocara antibodies were studied in paratenic hosts with experimental larval toxocarosis after treatment with the immunomodulator muramyldipeptide for 119 days. Peroral infection of mice with 2,500 Toxocara canis eggs partially reduced the numbers of cells and inhibited the activity of all cellular immune-response parameters studied. During most of the experiment the greatest suppression was recorded for the peritoneal-macrophage metabolic activity. Parenteral administration of muramyldipeptide to mice at two doses prior to and after infection restored and significantly stimulated the parasite-inhibited phagocytic ability of blood polymorphonuclear leukocytes, metabolic activity of macrophages, and T-lymphocyte proliferative response. It also elevated the production of specific circulating anti-Toxocara antibodies. The immunomodulator significantly reduced the count of migrating T. canis larvae in the host organism (30.6%) and decreased by half the percentage of larvae in the brain.

The effect of cadmium on the immune behaviour of guinea pigs with experimental ascariasis

The subchronic effect of cadmium on selected immunological parameters was studied in guinea pigs with experimental ascariasis. Cadmium chloride given orally for 28 days caused a considerable suppression of T- and B-cells in the lymphoid organs of intoxicated animals, of the metabolic activity of their peritoneal macrophages and a moderate decline in the level of complement CH50 and AH50 from day 1 to day 28 of the experiment, in comparison with the initial value. After a subsequent infection of the subchronically intoxicated guinea pigs the values for both the cell populations and the macrophage metabolic activity remained considerably suppressed, compared with infected control animals. The phagocytic and metabolic activity of macrophages as well as the CH50 and AH50 level, however, increased conspicuously for a short time after application of heavy metal and after infection but it did not reach the level of the infected group. The level of specific circulating antibodies was not affected by intoxication. Compared with the controls, the mean intensity of infection with Ascaris larvae migrating in the lungs of intoxicated animals increased by 20%. The results have established the negative effect of cadmium on the immunological parameters of the cellular immunity and humoral immunity studied and the impairment of the organism's response to the antigenic stimulus after an A. suum infection.