Number Of CD68-positive Macrophages Research Articles

Relationship Between Clinical Parameters and Histological Features of Epicardial Adipose Tissue and Aortic Valve Calcification Assessed on Computed Tomography.

The relationships of the clinical and biological attributes of epicardial adipose tissue (EAT) with aortic valve calcification (AVC) have not been characterized. We evaluated the relationships of the clinical and histological features of EAT with AVC assessed using computed tomography (CT).Methods and Results: We enrolled 43 patients undergoing cardiac CT examination prior to elective cardiac surgery in whom AVC was identified on CT. EAT volume and density, coronary calcium score (CCS), AVC score (AVCS), and coronary atherosclerosis on CT angiography were evaluated in each patient. During cardiac surgery, 2 EAT samples were obtained for immunohistochemistry. The number of CD68- and CD11c-positive macrophages and osteocalcin-positive cells was counted in 6 random high-power fields of EAT sections. EAT density, but not EAT volume normalized to body surface area, was positively correlated with the number of macrophages and osteocalcin-positive cells in EAT. There was a positive correlation between ln(AVCS), but not ln(CCS+1), and the number of macrophages and osteocalcin-positive cells in EAT. Multivariate analysis revealed significant positive correlations for ln(AVCS) with EAT density (β=0.42; P=0.0072) and the number of CD68-positive macrophages (β=0.57; P=0.0022), CD11c-positive macrophages (β=0.62; P=0.0003), and osteocalcin-positive cells (β=0.52; P=0.0021) in EAT. Inflammation and osteogenesis in EAT, reflected by high CT density, are associated with the severity of AVC representing aortic valve degeneration.

Abstract PO4-25-06: SerMa (Seroma of the Mammary gland) pilot - Is there a possible link between macrophage-based immune response and age?

Abstract Background The primary objective of the SerMa pilot study was to identify possible immunological or inflammatory factors related to seroma formation after mastectomy in cases with primary breast cancer. Preliminary data suggest a distinct association between age and seroma formation. The work presented here investigates therefore a possible link between age and differences in the microenvironment represented by tumor-associated macrophages. The latter have been characterized by the expression of CD68 and CD163 receptors, that are markers of cells from the monocyte/macrophage lineage with known clinical implications. Methods Tumor tissue of 80 primary breast cancer cases of the SerMa pilot study was available for further analyses. Immunohistochemistry based on antibody staining against CD68 and CD163 was used and due to the mean age of 63 years two groups were formed and compared: patients aged under 63 and patients aged 63 and older. The number of macrophages (standardized manually quantified) was compared in these two groups regarding seroma formation. Results Seroma formation occurred significantly more frequently with older age of the patient (p < 0.001). For the overall cohort, the number of CD68-positive macrophages was significantly increased (p=0.036) in patients with seroma formation, as well as for CD163 (p=0.027). The macrophage polarization was shown to be independent of tumor biological characteristics (p > 0.130). However, closer examination revealed an age-dependent effect of the macrophage polarization. In the group 63 years and older, there was no significant difference in the number of CD68- or CD163-positive macrophages (p = 0.610 and p = 0.425, respectively) in relation to seroma formation. A significant effect was seen in this analysis only in patients younger than 63 regarding CD163 (p < 0.001) and a non-significant trend for CD68 (p = 0.065). Conclusions These data demonstrate an age-dependent significant correlation of CD163 positive macrophages measured in the tumor environment with seroma formation in the breast after mastectomy. For CD68 positive macrophages a trend was seen, but didn’t reach statistical significance due to the small number of cases. These data show a different immunological response in tumor tissue depending on age and therefore support the thesis that seroma formation is primarily related to immunological/inflammatory processes. In the future, detection of CD163-positive macrophages in the tumor microenvironment could therefore be considered a marker for patients under 63 years at increased risk of seroma. The planned international SerMa study (EUBREAST 5) will have to show whether these results can be transferred to a prospective design with higher case number. Citation Format: Melitta Koepke, Felicitas Schneider, Christina Kuhn, Mathis Wild, Mariella Schneider, Nicole Pochert, Claudia Traidl-Hoffmann, Jacqueline Sagasser, Christian Dannecker, Christian Hinske, Maggie Banys-Paluchowski, Michael Untch, Thorsten Kühn, Udo Jeschke, Nina Ditsch. SerMa (Seroma of the Mammary gland) pilot - Is there a possible link between macrophage-based immune response and age? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-06.

The efficacy of using metformin and/or quercetin for amelioration of gamma-irradiation induced tongue toxicity in diabetic rats

BackgroundDiabetes is a common disease that cancer patients may suffer from and may aggravate side effects of radiotherapy. This study aimed to detect whether metformin and/or quercetin will improve gamma-irradiation induced tongue toxicity in diabetic rats.Methods35 male albino rats were divided into five groups; NOR no streptozotocin, no radiation and no treatment was given, DR rats were subjected to streptozotocin then gamma-irradiation, DRM rats were subjected to streptozotocin then gamma-irradiation then metformin, DRQ rats were subjected to streptozotocin then gamma-irradiation then quercetin, DRMQ rats were subjected to streptozotocin then gamma-irradiation then metformin and quercetin. Rats were euthanized 24 h after last treatment dose. Mean blood glucose level was recorded. Tongue specimens were stained with H&E and CD68. Histomorphometric analysis of length, diameter and taste buds of lingual papillae and epithelial, keratin and lamina propria thickness and CD68 positive cells were calculated.ResultsBlood glucose level of DRMQ was significantly lower than DR, DRM and DRQ, whereas higher than NOR. Metformin or quercetin partially restored tongue structure, papillae length and diameter and tongue layers thickness. The ameliorative effect was superior when metformin and quercetin were used together. Diabetes and irradiation significantly increased number of CD68 positive macrophages in submucosa and muscles. Metformin or quercetin significantly reduced number of lingual macrophages with more noticeable effect for quercetin. Treatment with metformin and quercetin significantly decreased number of macrophages.ConclusionsCombined use of metformin and quercetin might help mitigate the harmful effects of radiotherapy and diabetes on lingual tissues.

VNS improves VSMC metabolism and arteriogenesis in infarcted hearts through m/n-AChR-Akt-SDF-1α in adult male rats

Vagal nerve stimulation (VNS) provides a novel therapeutic strategy for injured hearts by activating cholinergic anti-inflammatory pathways. However, little information is available on the metabolic pattern and arteriogenesis of VSMCs after MI. VNS has been shown to stimulate the expression of CPT1α, CPT1β, Glut1, Glut4 and SDF-1α in coronary VSMCs, decreasing the number of CD68-positive macrophages while increasing CD206-positive macrophages in the infarcted hearts, leading to a decrease in TNF-α and IL-1β accompanied by a reduced ratio of CD68- and CD206-positive cells, which were dramatically abolished by atropine and mecamylamine in vivo. Knockdown of SDF-1α substantially abrogated the effect of VNS on macrophagecell alteration and inflammatory factors in infarcted hearts. Mechanistically, ACh induced SDF-1α expression in VSMCs in a dose-dependent manner. Conversely, atropine, mecamylamine, and a PI3K/Akt inhibitor completely eliminated the effect of ACh on SDF-1α expression. Functionally, VNS promoted arteriogenesis and improved left ventricular performance, which could be abolished by Ad-shSDF-1α. Thus, VNS altered the VSMC metabolism pattern and arteriogenesis to repair the infarcted heart by inducing SDF-1α expression, which was associated with the m/nAChR-Akt signaling pathway.

Toxic effect of carpet dust on the biochemical indices and histological structure of the lung in rats: the potential role of cytochrome P450 2E1 and extracellular signal-regulated kinase/mitogen-activated protein kinase pathways

Background: Carpet dust exposure in the carpet industry causes various respiratory hazards that lead to permanent loss of lung function. This study investigated the potentially toxic effects of knotted and tufted carpet dust on rat lungs and the possible involvement of cytochrome P450 2E1 (CYP2E1) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathways in the induced toxicity, as well as histological changes in the lung induced by carpet dust. Methods: This study divided 48 adult rats into six groups: group I was the control group, group II (vehicle group) received phosphate buffer saline (50 µL/rat), groups III and IV received knotted dust (2.5 and 5 mg/kg, respectively), and groups V and VI received tufted dust (2.5 and 5 mg/kg, respectively). All treatments were intranasally administered once a day for 7 days. Results: Both dust types significantly decreased the lung content of GSH compared with the control. Significantly elevated malondialdehyde (MDA) and nitric oxide (NO) lung contents were observed with an increased CYP2E1, interleukin (IL)-6, nuclear factor kappa B (NF-κβ), and ERK/MAPK. The histological lung structure was moderately affected with a moderately increased number of CD68-positive macrophages in the lung parenchyma of knotted dust-exposed rats, whereas tufted dust exposure severely affected the lung tissue with significantly increased CD68-positive macrophages. Conclusions: Carpet dust exposure could induce oxidative stress and inflammatory response in the lung tissue via induction of CYP2E1 that stimulates ERK/MAPK signalling pathway proteins, resulting in elevated MDA, NO and IL-6 levels in the lung tissue with suppressed GSH content. Tufted dust could possess a more toxic response than knotted ones.

Liver-adipose tissue crosstalk in alcohol-associated liver disease: The role of mTOR

BackgroundAlcohol-associated liver disease (ALD) is a major chronic liver disease around the world without successful treatment. Acute alcoholic hepatitis is one of the most severe forms of ALD with high mortality, which is often associated with binge drinking. Alcohol drinking dysregulates lipid metabolism, increases adipose tissue lipolysis, and induces liver steatosis and adipose tissue atrophy. Increasing evidence implicates that crosstalk of liver and adipose tissue in the pathogenesis of ALD. Mechanistic target of rapamycin (mTOR) is a phosphatidylinositol 3-kinase (PI3K)-like serine/threonine protein kinase that regulates lipid metabolism, cell proliferation and autophagy. However, the role of mTOR in regulating adipose-liver crosstalk in binge drinking-induced organ damage remains unclear. MethodsWe generated liver-specific and adipocyte-specific regulatory-associated protein of mTOR (Rptor) knockout (RptorLKO and RptorAKO) as well as Mtor knockout (MtorLKO and MtorAKO) mice, by crossing Rptorflox and Mtorflox mice with albumin Cre or adiponectin Cre mice, respectively. In addition, we generated liver and adipocyte double deletion of Rptor or Mtor (MtorLAKO and RptorLAKO) mice. The knockout mice with their matched wild-type littermates (RptorWT and MtorWT) were subjected to acute gavage of 7 g/kg ethanol. ResultsMice with adipocyte deletion of Rptor or Mtor developed hepatomegaly and adipose tissue atrophy. Alcohol gavage increased liver injury, hepatic steatosis and inflammation in mouse livers as demonstrated by elevated serum alanine aminotransferase activities, increased hepatic levels of triglyceride and increased hepatic numbers of CD68 positive macrophages in mouse livers after alcohol gavage. Liver injury was further exacerbated by deletion of adipocyte Rptor or Mtor. Serum adipokine array analysis revealed that increased levels of pro-inflammatory cytokines IL-6 and TNFα as well as chemokine MCP-1 following acute alcohol gavage in wild-type mice, which were further increased in adipocyte-specific Mtor or Rptor knockout mice. Conversely, levels of anti-inflammatory cytokine IL-10 decreased in adipocyte-specific Mtor or Rptor knockout mice. The levels of circulating fibroblast growth factor 21 (FGF21) increased whereas levels of circulating adiponectin and fetuin A decreased in wild-type mice after alcohol gavage. Intriguingly, adipocyte-specific Mtor or Rptor knockout mice already had decreased basal level of FGF21 which increased by alcohol gavage. Moreover, adipocyte-specific Mtor or Rptor knockout mice already had increased basal level of adiponectin and decreased fetuin A which were not further changed by alcohol gavage. ConclusionsAdipocyte but not hepatocyte ablation of Mtor pathway contributes to acute alcohol-induced liver injury with increased inflammation. Our results demonstrate the critical role of adipocyte mTOR in regulating the adipose-liver crosstalk in ALD.

Long-term volume retention after fat processing with cotton gauze rolling and centrifugation: A comparative study in nude mice.

Autologous fat grafting for correcting soft-tissue defects in cosmetic and reconstructive procedures has grown in popularity. Fat processing is implicated as a variable affecting quality, viability, and subsequent graft retention. This study aimed to identify a better fat processing technique for optimal outcomes. Fresh human aspirated fat was processed with cotton gauze rolling or centrifugation and named rolled fat (RF) and centrifuged fat (CF), respectively. Processed fat grafts were analyzed in vitro to determine yield, stromal vascular fraction (SVF) content, and viability. Then, RF and CF were transplanted subcutaneously to different flanks of every nude mouse. Fat samples were weighed to evaluate the volume retention 3 months post-transplantation. Tissue structure, densities of vessels, and CD68-positive macrophages were examined by histological staining. The compression rate of lipoaspirate by cotton gauze rolling was 25%, which was more effective than the rate of 50% by centrifugation. The numbers of SVF cells per gram of RF and CF were (1.02 ± 0.14) ×106 and (0.65 ± 0.26)×106, respectively (P < 0.05). Long-term graft retention was significantly higher in the RF group than in the CF group. Histological analysis of all implants revealed intact adipose tissue and equivalent vascularity. The number of CD68-positive macrophages in the RF group was much less than in the CF group on day 7. The results of this animal experiment showed that, compared with centrifugation, processing with cotton gauze rolling produces more condensed fat, higher SVF content, and decreased inflammatory response, thereby improving long-term volume retention. Further explorations are required to verify the superiority of cotton gauze rolling in clinical settings.

Prognostic significance of CD163- and CD68-expressing macrophages in nodular sclerosis Hodgkin lymphoma

The objective of the study was to determine the clinical and morphological criteria and prognostic significance of CD163- and CD68-expressing macrophages in formalin-fixed paraffin-embedded (FFPE) lymph node samples of nodular sclerosis Hodgkin lymphoma.Materials and methods. We have studied clinical and laboratory characteristics and treatment results of 52 patients with newly diagnosed nodular sclerosis Hodgkin lymphoma. The treatment efficacy was assessed after 2–4 courses of chemotherapy by positron emission tomography combined with computed tomography scan (PET-CT) according to the Deauville scale.Results. In FFPE lymph node samples, differences in the localization and relative number of CD163-positive macrophages in patients of both study groups were determined. Morphometric counting of CD68-positive cells did not reveal statistically significant intergroup differences. The threshold of CD163-positive cells has been established. The relationship between the CD68 expression level and the response to frontline therapy was not revealed. The ratio of tumorassociated macrophages in each patient was assessed by the CD163/CD68 index. The threshold of the index was determined, which allows predicting the course of nodular sclerosis Hodgkin lymphoma.Conclusion. The following parameters could be used as additional morphological criteria for predicting the response to frontline therapy in patients with nodular sclerosis Hodgkin lymphoma: tissue distribution pattern and quantitative calculation of macrophage-histiocytic microenvironment subpopulation composition. The results could be used to risk group stratification of patients with nodular sclerosis Hodgkin lymphoma in order to predict the response to initial phase of therapy, as well as to determine personalized treatment approaches.

Iron oxide nanoparticles exert inhibitory effects on N-Bis(2-hydroxypropyl)nitrosamine (DHPN)-induced lung tumorigenesis in rats

Iron oxide nanoparticles (magnetite) have been widely used in industry and medicine. However, the safety assessment of magnetite has not been fully completed. The present study was conducted to assess effects of magnetite on carcinogenic activity, using a medium-term bioassay protocol. A total of 100 male Fischer 344 rats, 6 weeks old, were randomly divided into 5 groups of 20 animals each, and given a basal diet and drinking water containing 0 or 0.1% of N-bis(2-hydroxypropyl)nitrosamine (DHPN) for 2 weeks. Two weeks later, the rats were intratracheally instilled magnetite 7 times at an interval of 4 weeks, at the doses of 0, 1.0 or 5.0 mg/kg body weight, and sacrificed at the end of the experimental period of 30 weeks. The multiplicities of macroscopic lung nodules and histopathologically diagnosed bronchiolo-alveolar hyperplasia, induced by DHPN, were both significantly decreased by the high dose of magnetite. The expression of minichromosome maintenance (MCM) protein 7 in non-tumoral alveolar epithelial cells, and the number of CD163-positive macrophages in tumor nodules were both significantly reduced by magnetite. It is suggested that magnetite exerts inhibitory effects against DHPN-induced lung tumorigenesis, by the reduction of alveolar epithelial proliferation and the M2 polarization of tumor-associated macrophages.

Tongue Cancer Cell-Derived CCL20 Induced by Interaction With Macrophages Promotes CD163 Expression on Macrophages.

BackgroundCD163-positive macrophages contribute to the aggressiveness of oral squamous cell carcinoma. We showed in a previous report that CD163-positive macrophages infiltrated not only to the cancer nest but also to its surrounding epithelium, depending on the presence of stromal invasion in tongue carcinogenesis. However, the role of intraepithelial macrophages in tongue carcinogenesis remains unclear. In this study, we assessed the biological behavior of intraepithelial macrophages on their interaction with cancer cells.Materials and MethodsWe established the indirect coculture system (intraepithelial neoplasia model) and direct coculture system (invasive cancer model) of human monocytic leukemia cell line THP-1-derived CD163-positive macrophages with SCC25, a tongue squamous cell carcinoma (TSCC) cell line. Conditioned media (CM) harvested from these systems were analyzed using cytokine array and enzyme-linked immunosorbent assay and extracted a specific upregulated cytokine in CM from the direct coculture system (direct CM). The correlation of both this cytokine and its receptor with various clinicopathological factors were evaluated based on immunohistochemistry using clinical samples from 59 patients with TSCC. Moreover, the effect of this cytokine in direct CM on the phenotypic alterations of THP-1 was confirmed by real-time polymerase chain reaction, western blotting, immunofluorescence, and transwell migration assay.ResultsIt was shown that CCL20 was induced in the direct CM specifically. Interestingly, CCL20 was produced primarily in SCC25. The expression level of CCR6, which is a sole receptor of CCL20, was higher than the expression level of SCC25. Our immunohistochemical investigation showed that CCL20 and CCR6 expression was associated with lymphatic vessel invasion and the number of CD163-positive macrophages. Recombinant human CCL20 induced the CD163 expression and promoted migration of THP-1. We also confirmed that a neutralizing anti-CCL20 antibody blocked the induction of CD163 expression by direct CM in THP-1. Moreover, ERK1/2 phosphorylation was associated with the CCL20-driven induction of CD163 expression in THP-1.ConclusionsTongue cancer cell-derived CCL20 that was induced by interaction with macrophages promotes CD163 expression on macrophages.

Induction of CCN1 in Growing Saccular Aneurysms: A Potential Marker Predicting Unstable Lesions.

Growing evidence has suggested that inflammatory responses promote the progression of saccular intracranial aneurysms (IAs). However, a biomarker predicting the progression has yet to be established. This study aimed to identify novel molecules upregulated during the progression using a previously established rat aneurysm model. In this model, aneurysms are induced at the surgically created common carotid artery (CCA) bifurcation. Based on sequential morphological data, the observation periods after the surgical manipulations were defined as the growing phase (on the 10th day) or the stable phase (on the 30th day). Total cell lysates from the CCA with or without an aneurysm lesion were prepared to perform protein array analysis. The protein array analysis revealed that the matricellular protein cellular communication network factor 1 (CCN1) is induced in lesions during the growing phase. Immunohistochemistry corroborated the significant upregulation of CCN1 in the growing phase compared with the stable phase. Simultaneously with the induction of CCN1, significant increases in the number of CD68-positive macrophages, myeloperoxidase-positive cells, and proliferating smooth muscle cells in lesions were observed. Immunohistochemistry of human IA specimens reproduced the induction of CCN1 in some lesions. These findings imply a potential role of CCN1 as a marker predicting the progression of saccular aneurysms.

Berberine Mediated CD47-Blocking As a Potential Immunotherapy in Diffuse Large B-Cell Lymphoma

Keywords diffuse large B-cell lymphoma; CD47; berberine; immunotherapy; macrophage Introduction Cluster of differentiation 47 (CD47) is a tetraspanin that expresses widely in human tissue and overexpresses in several malignance cells. The interaction between CD47 overexpressing on tumor cells and signal regulatory protein alpha (SIRP-α) on macrophages triggers an inhibitory signal cascade that suppresses the phagocytosis function. Berberine is a quaternary amine isoquinoline alkaloid which is traditionally used in intestinal infection and found to have anti-tumor activities in several tumors. However, the function and mechanism of berberine in immunoregulation targeting CD47 is undefined. Herein, we investigated the functional significance of CD47 in diffuse large B-cell lymphoma (DLBCL) progression and proposed an adjuvant agent for DLBCL treatment. Methods Paraffin imbedding tissue sections of 45 newly diagnosed DLBCL patients and 25 reactive hyperplasia (RHL) patients were obtained after informed consents. CD47 expression profile was analyzed in DLBCL sections and DLBCL cell lines. CD68, a biomarker of macrophages, was stained in DLBCL sections and the number of CD68 positive macrophages per high power field (HPF) was counted. The phagocytosis suppressing function of CD47 was detected in a co-culture system of macrophages induced from peripheral blood mononuclear cells (PBMCs) with DLBCL cells pretreated with RNAi-mediated CD47 knockdown lentivirus or anti-CD47 antibody (clone B6H12). CD47 level was analyzed after berberine treatment, and related regulatory pathway was explored. Results Firstly, we analyzed the CD47 expression in tissue sections form DLBCL and RHL patients (Figure 1A). Compared to RHL, CD47 was overexpressed in DLBCL tissues (31/45 vs 10/25, p=0.016, Figure 1B). CD47 overexpression was correlation with higher Ann-Arbor stage(p=0.028), but not with age, gender, NCCN-IPI score, serum LDH and DLBCL subtype. CD68 positive macrophages infiltration was detected in DLBCL tissue sections (Figure 1C, red arrow), and CD47 overexpression was correlation with higher macrophages infiltration (Figure 1D). Further, aberrantly increased expression of CD47 was also detected in some DLBCL cell lines (Figure 1E-F). Interestingly, total CD47 protein level was detected to be overexpressed in LY3 and Val cells, however, no statistical significance was found when the cytomembrane CD47 level was compared. To investigate the biofunction of CD47 in DLBCL, expression of CD47 was down-regulated by RNAi-mediated knockdown lentivirus in LY1 cells. ShCD47 or shNC LY1 cells was co-cultured with PBMC-derived macrophages, and the phagocytosis ratio was significantly elevated in shCD47 cells (Figure 2A). Consistently, the anti-phagocytosis effect of CD47 was also proved by blocking CD47 on LY1 cells via anti-CD47 antibody (clone B6H12) in the co-culture system (Figure 2B). To validate the impact of berberine on CD47 expression, DLBCL cells were treated with 30μM berberine for 0, 24 and 48 hours. The results revealed that CD47 was downregulated by berberine at a time-dependent manner (Figure 3A and B). To explore the upstream of CD47 in DLBCL, inhibitors of CD47-related upstream protein were applied to LY1 cells. The results showed that inhibition of ERK1/2 phosphorylation and c-Myc expression could suppress CD47 expression (Figure 3C). The expression level of c-Myc and p-ERK1/2 was also decreased after berberine treatment, which was consistent with the downregulation of CD47, indicating that berberine restrained CD47 expression through the ERK/c-Myc pathway (Figure 3D). Engulfment of LY1 cells by macrophages was improved when LY1 cells were pretreated with 30μM berberine for 48 hours (Figure 3E). Combination of berberine and B6H12 showed a more potent enhancement of macrophage function than single agent (Figure 3F). Moreover, rituximab-mediated phagocytosis was observed and proved to be synergized by berberine (Figure 3G). Conclusion Our results highlighted that CD47 was a significant feature and played an anti-phagocytosis function in DLBCL progression. We provided evidence for the first time that berberine could enhance rituximab-mediated phagocytosis and restore macrophage function in tumor microenvironment via suppressing CD47 expression, which provided novel insights into the rituximab based immunochemotherapy and CD47-targeted therapy in DLBCL. Disclosures No relevant conflicts of interest to declare.

Phenotypic Changes in Macrophage Activation in a Model of Nonalcoholic Fatty Liver Disease using Microminipigs.

Aim: Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver disorders associated with metabolic syndrome, and its prevalence has been on the rise. The pathogenesis of NAFLD has not yet been sufficiently elucidated due to the multifactorial nature of the disease, although the activation of macrophages/Kupffer cells is considered to be involved. We previously reported an animal model of NAFLD using Microminipigs TM (µMPs) fed high-fat diets containing cholesterol with or without cholic acid. The aim of this study was to investigate the phenotypic changes of macrophages that occur during the development of NAFLD. Methods: Immunohistochemistry of macrophages, lymphocytes, and stellate cells was performed using liver samples, and the density of positive cells was analyzed. Results: The number of Iba-1-positive macrophages increased with increasing cholesterol content in the diet. The numbers of CD163-positive macrophages and CD204-positive macrophages also increased with increasing cholesterol content in the diet; however, the proportion of CD204-positive macrophages among Iba-1-positive macrophages was significantly reduced by cholic acid supplementation. Conclusion: The results suggest that lipid accumulation induced macrophage recruitment in swine livers, and that the number of M2-like macrophages increased at the early stage of NAFLD, while the number of M1-like macrophages increased at the late stage of NAFLD, resulting in a liver condition like non-alcoholic steatohepatitis. We provide evidence of the phenotypic changes that occur in macrophages during the development of NAFLD that has never been reported before using µMPs.

Lack of RAC1 in macrophages protects against atherosclerosis.

The Rho GTPase RAC1 is an important regulator of cytoskeletal dynamics, but the role of macrophage-specific RAC1 has not been explored during atherogenesis. We analyzed RAC1 expression in human carotid atherosclerotic plaques using immunofluorescence and found higher macrophage RAC1 expression in advanced plaques compared with intermediate human atherosclerotic plaques. We then produced mice with Rac1-deficient macrophages by breeding conditional floxed Rac1 mice (Rac1fl/fl) with mice expressing Cre from the macrophage-specific lysosome M promoter (LC). Atherosclerosis was studied in vivo by infecting Rac1fl/fl and Rac1fl/fl/LC mice with AdPCSK9 (adenoviral vector overexpressing proprotein convertase subtilisin/kexin type 9). Rac1fl/fl/LC macrophages secreted lower levels of IL-6 and TNF-α and exhibited reduced foam cell formation and lipid uptake. The deficiency of Rac1 in macrophages reduced the size of aortic atherosclerotic plaques in AdPCSK9-infected Rac1fl/fl/LC mice. Compare with controls, intima/media ratios, the size of necrotic cores, and numbers of CD68-positive macrophages in atherosclerotic plaques were reduced in Rac1-deficient mice. Moreover, we found that RAC1 interacts with actin-binding filamin A. Macrophages expressed increased RAC1 levels in advanced human atherosclerosis. Genetic inactivation of RAC1 impaired macrophage function and reduced atherosclerosis in mice, suggesting that drugs targeting RAC1 may be useful in the treatment of atherosclerosis.

Bioactive Elastic Scaffolds Loaded with Neural Stem Cells Promote Rapid Spinal Cord Regeneration.

Despite decades of research, spinal cord injury (SCI) still causes irreparable damage to the human body. Key challenges that hinder the regeneration and extension of neurons following SCI must be overcome, including the overexpressed glial scar formation and strong inflammatory responses in lesion tissue. Transplantation of neural stem cells (NSCs) represents a promising therapeutic method due to its beneficial roles like growth factor secretion and anti-inflammation. However, NSCs usually differentiate into astrocytes, which is considered as one potential limitation of current NSC therapy. Herein, we fabricate an elastic poly(sebacoyl diglyceride) (PSeD) scaffold to mimic the mechanical properties of the natural spinal cord. The PSeD scaffold is coated with poly(sebacoyl diglyceride)-isoleucine-lysine-valine-alanine-valine-serine (PSeD-IKVAVS) to create a bioactive interface. The core point of this topic is divided into two parts. First, PSeD is a bioelastomer and its mechanical properties are similar to those of the natural spinal cord. This feature reduces the direct stimulation to the spinal cord tissue by the elastomer and then reduces the immune response or resistance caused by the host spinal cord tissue. Second, the IKVAVS peptide modifies PSeD to create a bioactive interface to support NSC growth and differentiation. In the in vivo study, the number of CD68-positive macrophages decreased in the PSeD-IKVAVS/NSC group compared to that in the SCI group (20% vs 60%). The low inflammation induced by the scaffold was beneficial to NSCs, resulting in increased locomotor recovery, as indicated by the increased Basso-Beattie-Bresnahan score (5, the average score in the PSeD-IKVAVS/NSC group, vs 2, the average score in the SCI group). Based on the above two characteristics, a PSeD-IKVAVS bioelastomer is fabricated, which provides a beneficial and bioactive microenvironment for NSCs after transplantation.

Repository corticotropin injection attenuates collagen-induced arthritic joint structural damage and has enhanced effects in combination with etanercept

BackgroundMelanocortin receptor (MCR) agonists have anti-inflammatory and immunomodulatory properties mediated by receptors expressed on cells relevant to arthritis. Repository corticotropin injection (RCI; Acthar® Gel), an MCR agonist preparation, is approved as adjunctive therapy for rheumatoid arthritis (RA), but its mechanism of action in RA is unclear. This study explored the efficacy of RCI as monotherapy or adjunctive therapy with etanercept (ETN) in an established animal model of collagen-induced arthritis (CIA).MethodsAfter induction of CIA, rats (n = 10 per group) were randomized to receive subcutaneous RCI (40, 160, or 400 U/kg twice daily) alone or in combination with ETN (10 mg/kg 3 times daily), ETN alone, or vehicle (on days 13 through 19). Inflammation was assessed via changes in paw edema. Bone damage was determined by microfocal computed tomography histopathology, and immunohistochemistry. Statistical analyses were performed using a 2-way analysis of variance (ANOVA) followed by the Newman-Keuls, Dunn’s, or Dunnett’s multiple comparisons test or a 1-way ANOVA followed by the Dunnett’s or Holm-Sidak multiple comparisons test.ResultsRCI administration resulted in dose-dependent decreases in ankle edema and histopathologic measures of inflammation, pannus formation, cartilage damage, bone resorption, and periosteal bone formation. RCI and ETN showed combined benefits on all parameters measured. Radiographic evidence of bone damage was significantly reduced in rats that received RCI alone or in combination with ETN. This reduction in bone density loss correlated with decreases in the number of CD68-positive macrophages and cathepsin K–positive osteoclasts within the lesions.ConclusionsAs monotherapy or adjunctive therapy with ETN, RCI attenuated CIA-induced joint structural damage in rats. These data support the clinical efficacy of RCI as adjunctive therapy for patients with RA.

Preoperative oral immunonutrition in gastrointestinal surgical patients: How the tumour microenvironment can be modified.

This study is focused on the impact of enteral immunonutrition on the cell-mediated immune response in the microenvironment of gastric and colorectal cancers. This is a prospective pilot study approved by the local Ethics Committee. The immunophenotypic structure of the immune cells before (on the biopsy) and after (on the surgical sample) the administration of the immunonutrition in 16 patients is compared with 8 patients receiving regular diet. The samples of non-tumour tissue from sleeve-gastrectomy are used as non-neoplastic control. Antibodies were tested: CD4, CD8, PD-1, FOX-P3, CD68, CD163, CD80, CD21, CD56, PD-L1. We applied already well-known scoring systems for the evaluation of the immunohistochemistry and compared our data in the different groups by statistical analysis. In treated patients, we detected a modulation of the immune response with higher number of cytotoxic and helper T-lymphocytes in the tumour microenvironment of the surgical specimens compared to the pre-operative biopsy, and a lower number of lymphocytes presenting an exhausted (i.e. double positive CD8 and PD-1 lymphocytes) and regulatory (i.e. double positive CD4 and FOX-P3 lymphocytes) phenotype. Moreover we observed the M1 polarization with a lower number of CD163 positive macrophages and the inhibition of the PD-1/PD-L1 pathway in treated patients. The immunonutrition impacts on the tumoral microenvironment of gastric and colorectal cancer activating the inflammatory pathway, in terms of humoral and cellular response.

Histological features of endomyocardial biopsies in patients undergoing hemodialysis: Comparison with dilated cardiomyopathy and hypertensive heart disease

Heart failure is a frequently occurring complication in patients on maintenance hemodialysis (HD). However, the histological features of right ventricular endomyocardial biopsy (RVEMB) samples remain unclear. The clinical characteristics and histological findings of consecutive patients undergoing HD with available RVEMB samples (HD group; n=28) were retrospectively compared with those of patients with dilated cardiomyopathy (n=56) and hypertensive heart disease (n=15). The mean myocyte diameter was significantly larger in the HD group than in the other groups (P<.001), whereas the mean percent area of fibrosis did not differ among the three groups. Immunohistochemical analysis revealed that the capillary density was significantly lower in the HD group compared with the other groups (P<.001), and it was positively associated with left ventricular ejection fraction (P=.014). The number of CD68-positive macrophages, which was significantly higher in the HD group compared with the other two groups (P<.001), was associated with cardiovascular mortality (P=.020; log-rank test). Myocyte hypertrophy, macrophage infiltration, and reduced capillary density were characteristic histological features of the RVEMB samples in patients undergoing HD, which may be related to the pathogenesis of cardiac dysfunction.

Evaluation of the relationship between CD163 positive macrophages and prognostic factors in serous ovarian tumors

Introduction: Ovarian cancer is the seventh most common cancer in women and is the fifth most common cause of mortality from malignancy. Objectives: The present study was performed to evaluate the number of CD163 positive macrophages and prognostic factors in serous ovarian tumors. Materials and Methods: In the present cross-sectional study, 43 tissue samples obtained from patients with serous ovarian tumors were selected from the pathology ward of Al-Zahra hospital, Isfahan, Iran. Then, the patients’ demographic information including age, pathological results, clinical stage of the disease, degree of tissue differentiation, the number of CD163 positive macrophages, and the five-year survival rate after the surgery were recorded and evaluated. Results: Patient age, International Federation of Gynecology and Obstetrics (FIGO) stage, histological grade, lymphatic metastasis, and the five-year survival rate were positively and significantly associated with the number of CD163 positive macrophages (P &lt;0.05). In addition, the number of CD163 positive macrophages increased the odds of the five-year survival rate in these patients by 1.490 times (P = 0.026). Conclusion: According to the results of this study, the number of CD163 positive macrophages had a significant association with the progression of ovarian cancer. Moreover, the increased number of CD163 positive macrophages increased the chance of mortality in these patients.

CD163 deficiency facilitates lipopolysaccharide‐induced inflammatory responses and endotoxin shock in mice

ObjectivesSeptic (or endotoxin) shock is a severe systemic inflammatory disease caused by bacteraemia or endotoxaemia. Although it is known that increased serum levels of CD163 are observed in septic/endotoxin shock patients, the exact function and significance of CD163 in macrophage activation remain unclear. Therefore, in the current study, we tested whether CD163 contributes to the pathogenesis of endotoxin shock in mice.Methods and resultsIn samples obtained from autopsy, the number of CD163‐positive macrophages was increased in the kidney, liver, heart, bone marrow and spleen of patients who had died from septic/endotoxin shock when compared to patients who had died from other causes. The animal study revealed a significantly lower survival rate in CD163‐deficient mice after lipopolysaccharide (LPS) injection. Several cytokines and oxidative stress‐related molecules were significantly elevated in the sera of LPS‐induced endotoxin shock mice models. Higher concentrations of IL‐6, TNF‐α, IL‐1β, nitrite (NO2‐) and nitrate (NO3‐) and a lower concentration of IL‐10 were observed in CD163‐deficient mice treated with LPS. Similar results were observed in CD163‐deficient LPS‐stimulated macrophages. Furthermore, in an antitype II collagen antibody‐induced arthritis (CAIA), rheumatoid arthritis model, inflammation and bone erosion scores as well as the expression of IL‐6 and IL‐1β were significantly increased in CD163‐deficient mice.ConclusionsCD163 was suggested to be involved in the regulation of inflammatory cytokine expression in septic/endotoxin shock and CAIA.