Objective To observe the effect of transfer of immature mouse myeloid dendritic cells （DC） generated with low-dose granulocyte-macrophage colony-stimulating factor （GM-CSF） on cardiac allograft survival. Methods Mouse DC were generated with standard doses or low doses GM-CSF from bone marrow cells, the phenotype and functional properties of these DC were compared through fluorescence-activated cell sorting（FACS） analysis and mixed lymphocyte reaction（MLR）, 1. 0 × 106 DC generated with low doses GM-CSF were administered to the recipients 7 days before transplantation, and the cardiac allograft survival were observed. Results In contrast to DC generated with standard doses, DC generated with low doses were phenotypically immature DC （CD11c＋, CD80- , CD86- , MHCⅡlow）, and induced allogeneic T cell unresponsiveness, and administration of these DC to recipients prolonged cardiac allograft survival from 6.3±1.2 days to 14.3±1.9 days. Conclusions DC generated from mouse bone marrow progenitors in low doses of GM-CSF are phenotypically and functionally immature, and prolong cardiac allograft survival when they are administered 7 clays before transplantation.
Objectives To investigate the effects of the distribution of tumor associated macrophages (TAMs) on prognosis in the patients with non-small cell lung cancer. Methods The number of CD68+ macrophages in 136 lung cancer nest and stroma was counted simultaneously by labelled streptavidin biotin method(LSAB）,and its correlation with patient postoperation prognosis was analyzed. Results CD68 macrophas were observed in both inside and around the cancer tissue,The mean TAMs in cancer stroma (36.00/HFP) was higher than that in cancer nest (23.80/HFP,Plt;0.05). Mean TAMs in nest of stage Ⅰ＋Ⅱ cancer was significantly higher than that of stageⅢ＋Ⅳ cancer（32.60/HFP vs. 14.80/HFP,Plt;0.05）,and mean TAMs in stroma of stage Ⅰ＋Ⅱ cancer was significantly lower than that of stage Ⅲ＋Ⅳ cancer（24.30/HFP vs. 47.60/HFP,Plt;0.05）.The number of TAMs in cancer nest and the ratio of nest TAMs /stoma TAMs were both positively correlated with the patient survival time (rs=0.510, 0.633, respectively). Otherwise the number of TAMs in cancer stroma was negatively correlated with the patient survival time (rs=-0.187). Five-year survival rate in patients with high density TAMs in cancer nest was significantly higher than that in patients with low density TAMs (51.4% vs. 11.1%, Plt;0.05), while reverse correlation between TAMs in cancer stroma and patient 5-year survival rate was observed (18.9% vs. 44.4%,Plt;0.05). And 5 year suvival rate in patients with high ratio of nest/stroma TAMs was higher than that with low ratio (58.1% vs.4.2%,Plt;0.01). Conclusion Cox regressive prognostic analysis showed that the higher the nest/stroma TAMs ratio, the higher probability of the patients survival time. While the higher number of TAMs in the cancer stroma, the lower probability of the patients survival time. Our results showed that distribution pattern of TAMs in cancer nest and cancer stroma could possibly be used to estimate the prognosis of patients with non-small cell lung cancer.
Objective To explore the expression of myeloid differentiation protein2 ( MD-2) in rat lung and its role in acute lung injury ( ALI) induced by lipopolysaccharide ( LPS) . Methods Twenty male SD rats were randomly divided into a LPS group and a control group. The wet/dry ratios of lung tissues were measured and the histological changes of lung tissues were observed under microscope. Alveolar macrophages were collected from bronchial alveolar lavage fluid ( BALF) . The MD-2 mRNA and protein expressions were detected by RT-PCR, Western blot, and immunohistochemistry respectively. The MD2-siRNA oligo were transfected into NR8383 cells and 1 μg/mL LPS was used to stimulate the cells. The expressions of MD-2 mRNA and protein were detected by RT-PCR and Western blot. The levels of TNF-αin rat serum and cell culture supernatant were detected by ELISA. Results Compared with the control group, the expressions of MD-2 mRNA and protein in alveolar macrophages and lung tissue were elevated ( P lt;0. 01) , as well as the level of TNF-αin rat serum. The expressions of MD-2 mRNA and protein in NR8383 cell and the level ofTNF-αin supernatant increased obviously after LPS stimulation ( P lt;0. 01) . There were no changes of MD-2 mRNA and protein expressions and TNF-α of NR8383 cells treated by MD-2 siRNA with or without LPS stimulation ( P gt;0. 05) . Conclusions The expression of MD-2 in lung increases obviously after challengedby LPS. KnockdownMD-2 gene of NR8383 cell byMD-2 siRNA can inhibit TNF-αsecretion induced by LPS stimulation.MD-2 may play an important role in rat ALI induced by LPS.
Objective To explore the association of macrophages with carcinogenesis and development of gastric cancer. Method The related literatures at home and abroad were consulted and reviewed. Results The microenvironment of gastric cancer could induce the polarization of macrophages，and then the activated macrophages，especially the tumor associated macrophages，could in turn motivate the growth，invasion，and metastasis of tumor cells by secreting a series of active substances. Conclusions Macrophages，especially the tumor associated macrophages play an importantrole in the carcinogenesis and development of gastric cancer. Investigating the macrophages and their interaction with gastric cancer may lead to a profound understanding of carcinogenesis of gastric cancer as well as opening up a new prospectfor treatment.
ObjectiveTo explore the expression of senescence marker protein-30 (SMP-30) in human lung tissues and the significance in the pathogenesis of chronic obstructive pulmonary disease (COPD). MethodsLung tissue specimens ( > 5 cm away from cancerous tissues) obtained by surgery resection in 20 subjects with solitary peripheral carcinoma in Jiangsu Province Hospital were investagted. The subjects were divided into three groups according to lung function and smoking history, ie. a COPD group (6 cases), a healthy smoking group (7 cases) and a healthy control group (7 cases). Immunohistochemistry and Western blot were used to determine the distribution and expression of SMP-30 in human lung tissues. ResultsSMP-30 protein mainly expressed in the cytoplasm of alveolar macrophages (AM). The numbers of AM and SMP-30-positive AM were significantly increased in the COPD group. Western blot analysis confirmed a significant increase in SMP-30 expression in the healthy smokers compared with the non-smokers (2.16±0.23 vs. 1.10±0.14, P < 0.01) and further enhanced in the patients with COPD compared with the healthy smoking subjects (4.62±0.97 vs. 2.16±0.23, P < 0.05). The levels of the protein in different groups were: COPD group > smoking group > control group with significant difference. ConclusionThese results suggest that SMP-30 expression may be involved in the mechanism of prolonged survival and the increase in number of AM and may be involved in the pathogenesis of COPD.
Evidence from numerous animal models and clinical studies in recent years has demonstrated that macrophages play an important role in the regulation of liver fibrosis regression. The safety and efficacy of utilizing autologous macrophages for the treatment of liver fibrosis have been demonstrated in patients and shows promising application prospects, but the therapeutic effects need to be improved. Cirrhotic liver undergoes a process of marked extracellular matrix degradation after partial hepatectomy surgery, and single-cell sequencing identified multiple restorative macrophage subsets that express different matrix metalloproteinases (MMPs) at high levels. Future efforts to further characterize this population of macrophages and improve their enrichment in the liver may allow macrophage therapy to be a highly effective strategy to reverse liver fibrosis.
Objective To investigate the effect of M2-like macrophage/microglia-derived mitochondria transplantation in treatment of mouse spinal cord injury (SCI). Methods BV2 cells were classified into M1 (LPS treatment), M2 (IL-4 treatment), and M0 (no treatment) groups. After receiving M1 and M2 polarization, BV2 cells received microscopic observation, immunofluorescence staining [Arginase-1 (Arg-1)] and flow cytometry [inducible nitric oxide synthase (iNOS), Arg-1] to determine the result of polarization. MitoSox Red and 2, 7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) stainings were used to evaluate mitochondrial function difference. Mitochondria was isolated from M2-like BV2 cells through differential velocity centrifugation for following transplantation. Then Western blot was used to measure the expression levels of the relevant complexes (complexes Ⅱ, Ⅲ, Ⅳ, and Ⅴ) in the oxidative phosphorylation (OXPHOS), and compared with M2-like BV2 cells to evaluate whether the mitochondria were obtained. Thirty-six female C57BL/6 mice were randomly divided into 3 groups (n=12). Mice from sham group were only received the T10 laminectomy. After the T10 spinal cord injury (SCI) model was prepared in the SCI group and mitochondria transplantation (MT) group, mitochondrial storage solution and mitochondria (100 μg) derived from M2-like BV2 cells were injected into the injured segment, respectively. After operation, the Basso Mouse Scale (BMS) score was performed to evaluate the motor function recovery. And immunofluorescence staining, lycopersicon esculentum agglutinin (LEA)-FITC staining, and ELISA [vascular endothelial growth factor A (VEGFA)] were also performed. Results After polarization induction, BV2 cells in M1 and M2 groups showed specific morphological changes of M1-like and M2-like macrophages, respectively. Immunofluorescence staining showed that the positive expression of M2-like macrophages marker (Arg-1) was significantly higher in M2 group than in M0 group and M1 group (P<0.05). Flow cytometry showed that the expression of M1-like macrophage marker (iNOS) was significantly higher in M1 group than in M0 group and M2 group (P<0.05), and the expression of Arg-1 was significantly higher in M2 group than in M0 group and M1 group (P<0.05). MitoSox Red and DCFH-DA stainings showed that the fluorescence intensity of the M2 group was significantly lower than that of the M1 group (P<0.05), and there was no significant difference with the M0 group (P>0.05). The M2-like BV2 cells-derived mitochondria was identified through Western blot assay. Animal experiments showed that the BMS scores of MT group at 21 and 28 days after operation were significantly higher than those of SCI group (P<0.05). At 14 days after operation, the number of iNOS-positive cells in MT group was significantly lower than that in SCI group (P<0.05), but still higher than that in sham group (P<0.05); the number of LEA-positive cells and the expression of VEGFA in MT group were significantly more than those in the other two groups (P<0.05). Conclusion M2-like macrophage/microglia-derived mitochondria transplantation can promote angiogenesis and inhibit inflammatory M1-like macrophage/microglia polarization after mouse SCI to improve function recovery.
Acute respiratory distress syndrome (ARDS) is the most common cause of acute respiratory failure. Extensive researches have been conducted for the pathophysiology of this disease, but the mortality rate remains high. Previous studies have found that catecholamines play an important role in acute lung injury, and newly discover prompted that upregulation of phagocyte-derived catecholamines augmented the acute inflammatory response in acute lung injury which provides a new way of thinking. In the current review, we describe the mechanism of the phagocyte-derived catecholamines augmenting the acute lung injury.
Objective To evaluate therapeutic efficacy and safety of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) inhalation in patients with recurrent pulmonarv alveolar proteinosis (PAP). Methods Three cases of recurrent PAP were treated by GM-CSF inhalation after whole lung lavage. The clinical data of the pulmonary function and SpO 2, the clinical symptoms and pulmonary lesions were compared before and after treatment. Results The pulmonary function and manifestations were improved obviously after GM-CSF inhalation. Also the ground-glass opacity was improved in high-resolution CT. The pulmonary function and SpO 2 increased obviously after received GM-CSF inhalation. There were no any adverse reactions in 3 cases. Conclusion GM-CSF inhalation therapy is effective and safe in recurrent PAP, but the long-term effect remains to be seen.
ObjectiveTo establish a methodology for alveolar macrophages (AMs) phagocytosis of AlexaFluor 488 (AF488) labeled bacteria by flow cytometry.MethodsStaphylococcus aureus and Streptococcus pneumoniae were labeled with different concentrations of AF488. A flow cytometric assay was used to quantify in vivo bacterial uptake by AMs. AMs and different ratio of fluorescent-labeled bacteria were incubated at 37 ℃ for 2 hours, 4 hours, 6 hours and 8 hours, respectively. AMs were washed with DPBS and extracellular fluorescence was quenched with 1% (w/v) trypan blue. Trypan blue was aspirated and phagocytosis of fluorescent-labeled bacteria by AMs was measured using a flow cytometry. Confocal microscopy was performed to ensure that bacterial in positive AM had been internalized rather than bound to the cell surface.ResultsWhen the concentration of AF488 was more than 50 μg/mL, the labeling rates of Staphylococcus aureus and Streptococcus pneumoniae were higher than 92% (P<0.05), and has quickly reached the upper limit. With the prolongation of incubation time, the phagocytic rate of AMs increased from 20.4% at 2 hours to 76.5% at 8 hours. With the increase in the number of bacteria, the phagocytic rate of AMs increased from 7.7% by ratio of 1∶10 to 85.1% by ratio of 1∶300.ConclusionDetection of AMs phagocytosis of AF488 labeled bacteria by flow cytometry is an effective method, but the dye concentration, incubation time and the proportion of bacteria will influence the results.