Objectives miR-207 has been identified as being expressed in natural killer (NK) cell exosomes that play a role in disease progression; however, to date, there are no studies specifically linking miR-207 to tuberculosis (TB). Methods Bioinformatics methods employed for prediction, followed by a dual luciferase reporter assay to determine whether lysosome-associated membrane protein 2 (LAMP2) is targeted by miR-207. The experiments were divided into four groups using the liposome transfection method (OP-LAMP2 group: co-transfected with miR-207 mimics and LAMP2 overexpression plasmid; EP group: co-transfected with mimics NC and null-loaded plasmid; siLAMP2 group: transfected with siLAMP2; and siLAMP2-NC group: transfected with siLAMP2-NC). TB infection was modeled using H37Ra-infected Ana-1 cells. The impact of LAMP2 on intracellular mycobacterial load and clearance of extracellular residual mycobacteria were assessed by tuberculosis colony-forming unit counting. Flow cytometry was used to assess the total apoptosis rate. Real-time fluorescent quantitative PCR was conducted to determine the relative expression of LAMP2, apoptosis genes, pyroptosis genes, and autophagy genes. Western blot analysis was performed to measure the relative expression of LAMP2 proteins, apoptosis proteins, pyroptosis proteins, and autophagy proteins. Results Dual luciferase reporter assay test showed that there was a targeting relationship between LAMP2 and miR-207. The transfection model was successfully constructed under real-time fluorescent quantitative PCR and Western blot statistical analysis, and microscopic observation. The infection model was successfully established under microscopic observation. Colony forming unit counting revealed that the number of colonies in the OP-LAMP2 group was lower than that in the EP group, while the number of colonies in the siLAMP2 group was higher than that in the siLAMP2-NC group. Flow cytometry assay revealed that the total apoptosis in OP-LAMP2 group was lower than that in EP group, and the total apoptosis in siLAMP2 group was higher than that in siLAMP2-NC group. Real-time fluorescence quantitative PCR and Western blot analysis revealed that the relative expression of apoptosis and pyroptosis-related proteins and genes in the control group was lower in the OP-LAMP2 group compared to the EP group, and higher in the siLAMP2 group compared to the siLAMP2-NC group. Real-time fluorescence quantitative PCR detected that the relative expression of autophagy positively regulated genes Microtubule-associated protein 1 light chain 3(LC3)and Beclin1 in the OP-LAMP2 group was higher in the OP-LAMP2 group compared to the EP group, and lower in the siLAMP2 group compared to the siLAMP2-NC group, while the relative expression of negatively regulated autophagy genes followed the opposite trend to that of autophagy positively regulated genes. The relative expression of autophagy-related proteins was consistent with the trend of autophagy genes. Conclusions miR-207 enhances macrophage apoptosis, cellular pyroptosis and inhibits autophagy, promoting survival of Mycobacterium tuberculosis by targeting the autophagy-related protein LAMP2, thus offering a novel therapeutic direction for tuberculosis.
Lysosomal-Associated Membrane Protein 2/metabolism* ; MicroRNAs/metabolism* ; Mycobacterium tuberculosis/physiology* ; Autophagy/genetics* ; Humans ; Macrophages/metabolism* ; Apoptosis/genetics* ; Tuberculosis/metabolism* ; Cell Line ; Pyroptosis/genetics*

This study investigated the effects of Agrimoniae Herba-Coptidis Rhizoma(XHC-HL)-medicated serum on the proliferation, migration, invasion, and apoptosis of human colorectal cancer HT29 and HCT116 cells via the autophagy mediated by lysosome-associated membrane protein type 2A(LAMP2A). Bioinformatics analysis was conducted to explore the role of LAMP2A in the development and progression of colorectal cancer. Western blot(WB) was used to detect the expression of LAMP2A protein in colorectal cancer cell lines. Lentiviral transfection was utilized to construct LAMP2A knockdown in HT29 and overexpression in HCT116 colorectal cancer cell models. Real-time fluorescence quantitative polymerase chain reaction(real-time qPCR) was performed to assess transfection efficiency. HT29 and HCT116 cells were treated with different concentrations of XHC-HL-medicated serum. The cell counting kit-8(CCK-8) assay was used to detect cell proliferation and determine the optimal concentration and duration of medicated serum intervention. HT29 cells were divided into a normal control(NC) group, an XHC-HL(medicated serum treatment) group, and an XHC-HL+shLAMP2A(medicated serum treatment+LAMP2A knockdown) group. HCT116 cells were divided into a NC group, an XHC-HL group, and an XHC-HL+LAMP2A(medicated serum treatment+LAMP2A overexpression) group. CCK-8 was used to measure cell viability. Colony formation assay was employed to assess cell proliferation ability. Scratch and Transwell migration assays were conducted to evaluate cell migration ability, and Transwell invasion assay was used to detect cell invasion ability. Flow cytometry was adopted to determine apoptosis rates. WB and real-time qPCR were employed to detect the effect of XHC-HL on the protein and mRNA expression of LAMP2A, heat shock cognate protein 70(HSC70), heat shock protein 90(HSP90), and glyceraldehyde-3-phosphate dehydrogenase(GAPDH) in colorectal cancer cells. Differential expression analysis revealed that LAMP2A expression was significantly higher in colorectal cancer patients compared to that in normal controls. Survival analysis indicated that the key molecule of chaperone-mediated autophagy(CMA), LAMP2A, was closely associated with colorectal cancer progression. Gene set enrichment analysis showed that patients with high LAMP2A expression significantly upregulated tumor progression-related signaling pathways such as angiogenesis and immune suppression. Immune infiltration analysis found that patients with high LAMP2A expression had fewer CD8 T cell infiltrations in their tumor microenvironment. XHC-HL-medicated serum inhibited the viability of HT29 and HCT116 cells, with the optimal intervention concentration and duration being 20% and 48 hours, respectively. Compared to the NC group, XHC-HL inhibited the proliferation, migration, and invasion of HT29 and HCT116 cells, and induced apoptosis. The medicated serum treatment with LAMP2A knockdown further inhibited colorectal cancer cell proliferation, invasion, and migration, and promoted apoptosis, whereas overexpression of LAMP2A reversed the inhibitory effects of the medicated serum on proliferation, migration, and invasion, and reduced apoptosis rates. XHC-HL-medicated serum inhibited CMA by upregulating the protein and mRNA expression of LAMP2A, HSC70, and HSP90 and downregulating substrate protein GAPDH expression via the autophagy mediated by LAMP2A. In conclusion, XHC-HL-medicated serum inhibits the proliferation, migration, and invasion of colorectal cancer cells and induces apoptosis by downregulating the expression of the key CMA molecule LAMP2A and inhibiting CMA activity.
Humans ; Colorectal Neoplasms/pathology* ; Drugs, Chinese Herbal/pharmacology* ; Lysosomal-Associated Membrane Protein 2/metabolism* ; Cell Proliferation/drug effects* ; Autophagy/drug effects* ; HCT116 Cells ; Cell Movement/drug effects* ; Apoptosis/drug effects* ; HT29 Cells ; Serum/chemistry* ; Coptis chinensis

Objective: To review the clinical data of 7 patients with Danon disease and analyze their clinical characteristics. Methods: The medical records of 7 patients with Danon disease, who were hospitalized in Peking Union Medical College Hospital of Chinese Academy of Medical Sciences from April 2008 to July 2021, were reviewed and summarized, of which 6 cases were diagnosed as Danon disease by lysosomal-associated membrane protein-2 (LAMP-2) gene mutation detection and 1 case was diagnosed by clinicopathological features. Clinical manifestations, biochemical indexes, electrocardiogram, echocardiography, skeletal muscle and myocardial biopsy and gene detection results were analyzed, and patients received clinical follow-up after discharge. Results: Six patients were male and average age was (15.4±3.5) years and the average follow-up time was (27.7±17.0) months. The main clinical manifestations were myocardial hypertrophy (6/7), decreased myodynamia (2/7) and poor academic performance (3/7). Electrocardiogram features included pre-excitation syndrome (6/7) and left ventricular hypertrophy (7/7). Echocardiography examination evidenced myocardial hypertrophy (6/7), and left ventricular dilatation and systolic dysfunction during the disease course (1/7). The results of skeletal muscle biopsy in 6 patients were consistent with autophagy vacuolar myopathy. Subendocardial myocardial biopsy was performed in 3 patients, and a large amount of glycogen deposition with autophagosome formation was found in cardiomyocytes. LAMP-2 gene was detected in 6 patients, and missense mutations were found in all these patients. During the follow-up period, implantable cardioverter defibrillator implantation was performed in 1 patient because of high atrioventricular block 4 years after diagnosis, and there was no death or hospitalization for cardiovascular events in the other patients. Conclusion: The main clinical manifestations of Danon disease are cardiomyopathy, myopathy and mental retardation. Pre-excitation syndrome is a common electrocardiographic manifestation. Autophagy vacuoles can be seen in skeletal muscle and myocardial pathological biopsies. LAMP-2 gene mutation analysis is helpful in the diagnose of this disease.
Adolescent ; Child ; Female ; Humans ; Male ; Cardiomyopathies/etiology* ; Glycogen Storage Disease Type IIb/complications* ; Hypertrophy, Left Ventricular/etiology* ; Lysosomal-Associated Membrane Protein 2/genetics* ; Pre-Excitation Syndromes/genetics*

X-linked dominant mutations in lysosome-associated membrane protein 2 (LAMP2) gene have been shown to be the cause of Danon disease, which is a rare disease associated with clinical triad of cardiomyopathy, skeletal myopathy, and mental retardation. Cardiac involvement is a common manifestation and is the leading cause of death in Danon disease. We report a case of a 24-month-old boy with hemizygous LAMP2 mutation who presented with failure to thrive and early-onset hypertrophic cardiomyopathy. We applied targeted exome sequencing and found a novel hemizygous c.692del variant in exon 5 of the LAMP2 gene, resulting a frameshift mutation p.Thr231Ilefs*11. Our study indicates that target next-generation sequencing can be used as a fast and highly sensitive screening method for inherited cardiomyopathy.
Cardiomyopathies ; Cardiomyopathy, Hypertrophic* ; Cause of Death ; Child, Preschool ; Exome* ; Exons ; Failure to Thrive ; Frameshift Mutation ; Glycogen Storage Disease Type IIb ; Humans ; Intellectual Disability ; Lysosomal-Associated Membrane Protein 2 ; Male ; Mass Screening ; Membrane Proteins ; Methods ; Muscular Diseases ; Rare Diseases

OBJECTIVETo study the protective effect of puerarin on MPP(+) -induced SH-SY5Y cells by chaperone-mediated autophagy (CMA).

METHODThe Parkinson's disease cell model was established by injuring SH-SY5Y cells with 1 mmol x L(-1) MPP+. The CCK-8 staining was adopted to detect the effect the puerarin of different concentrations on the survival rate of MPP(+)-induced SH-SYSY cells. The autophagosome formation was observed under transmission electron microscope. The AO staining showed the changes in the lysosome activity. RT-PCR was used to detect the changes in Lamp2a and Hsc70 mRNA expressions. The western blotting was adopted to test the expressions of Lamp2a, Hsc70 and alpha-synuclein protein in cells.

RESULTWithin the concentration range of 12. 5-50.0 micromol x L(-1), the pretreatment with puerain for 30 minutes could protect the injury of MPP+ in SH-SY5Y cells, and showed a certain dose-effect relationship. The AO staining and electron microscope showed the effect of puerain within the concentration range of 12.5-50.0 micromol x L(-1) on 1 mmol x L(-1) MPP(+)-induced SH-SY5Y cells; autophagosomes emerged in cells, and increased along with the rise in the puerarin dose. The results of the flow cytometry revealed that 50.0 micromol x L(-1) of puerarin could protect against the increase of the ROS level in 1 mmol x L(-1) MPP(+) -induced SH-SY5Y cells and prevent the oxidative injury. The results of RT-PCR and western blotting indicated that puerain within the concentration range of 12.5-50.0 micromol x L(-1) alleviated the MPP(+)-induced SH-SY5Y cell injury, and inhibited the accumulation of alpha-synuclein proteins in MPP(+) -induced SH-SY5Y cells by up-regulating Hsc70, Lamp2a mRNA and protein level.

CONCLUSIONPuerarin could protect against the MPP(+) -induced cell injury, whose protective mechanism may be related to the chaperone-mediated autophagy pathway of interventional molecules.

Autophagy ; drug effects ; genetics ; HSC70 Heat-Shock Proteins ; genetics ; Humans ; Isoflavones ; pharmacology ; Lysosomal-Associated Membrane Protein 2 ; genetics ; Molecular Chaperones ; genetics ; Parkinson Disease ; drug therapy ; genetics ; Phagosomes ; drug effects ; genetics ; Piperidines ; pharmacology ; Pyrazoles ; pharmacology ; Tumor Cells, Cultured ; Up-Regulation ; drug effects ; genetics

OBJECTIVETo investigate the effects of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus.

METHODSSD rats were randomly divided into three groups: control group was given distilled water, lead-exposed groups were given 0.5 g/L (low-dose) or 2.0 g/L(high-dose) lead acetate solution in drinking water. The rat pups started to drink the lead content water until 60 d maturity. The lead contents in blood and brain samples were analyzed by graphite furnace atomic absorption spectrophotometry. The expressions of Beclin 1, LC3, LAMP2 and cathepsin B proteins were detected by Western blot and immunohistochemistry.

RESULTSCompared with control group, the contents of lead were significantly higher in blood and hippocampus samples in chronic lead-exposed rats (P<0.01). Western blot showed that the expression of Beclin 1 and LC3-II/LC3-I increased significantly in high dose lead-exposed group compared with control group (P<0.05 or P<0.001). The confocal laser immunostaining results demonstrated that increased immunofluorescence staining of cathepsin B in hippocampal neurons compared with control animals.

CONCLUSIONThe disturbance of autophagy-lysosome signaling molecules might be partially contribute to neurotoxicity of chronic lead exposure.

Animals ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; drug effects ; physiology ; Beclin-1 ; Cathepsin B ; metabolism ; Chronic Disease ; Disease Models, Animal ; Female ; Hippocampus ; drug effects ; metabolism ; pathology ; Lead Poisoning ; metabolism ; pathology ; Lysosomal-Associated Membrane Protein 2 ; metabolism ; Male ; Microtubule-Associated Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects

OBJECTIVETo establish a novel flow cytometry-based assay for measuring the expression of lysosomal-associated membrane protein 1 (LAMP-1, CD107α) on the cell surface of natural killer (NK) cells and cytotoxic T lymphocyte (CTL) and evaluate the screening value of this assay for cytotoxic defects-related diseases such as familial hemophagocytic lymphopro-liferative (FHL) syndrome.

METHODThree suspected Chediak-Higashi Syndrome (CHS) patients, three suspected FHL patients and 10 healthy children were enrolled in the study from October 2010 to June 2011. Their PBMCs were separated and activated overnight with IL-2. After the granule release of NK cells activated by phytohemagglutinin (PHA) and CD8+T cells by anti-CD3, the CD107α expression were analyzed by flow cytometry. The peripheral blood DNA and RNA of the patients were extracted to analyze the pathogenic genes via DNA-PCR/RT-PCR and direct sequencing.

RESULTThe CD107α expression on CTL in the ten healthy children significantly increased after activation by anti-CD3 [(0.18 ± 0.07)% vs. (4.47 ± 2.36)%, P < 0.05] and NK cells after activation by PHA [(0.27 ± 0.07)% vs. (5.80 ± 2.83)%, P < 0.05]. The frequency of CD107α-expression NK cells in three suspected CHS after activation was significantly elevated when compared with the healthy control [0.5%, 0.6% vs. (5.80 ± 2.83)%] except patient 2. After the anti-CD3 activation, the frequency of CD107α expression on CTL cells also showed no significant difference [0.3%, 0.9%, 0.2% vs. (4.47 ± 2.36)%] in three patients. All of their mean fluorescence intensity (MFI) showed the same trend. Patient 1 and 3 were identified to have LYST mutations (Patient 1: c.5411-5414 del TTTC, L1741fsX1758 and c.7975 C > T, R2596X; Patient 3: c.4863G > A, R1563H and c.5392-5393delAA, E1739fsX1756). There was no mutation identified in the LYST gene for patient 2. CD107α expression of NK cells and CTL in the suspected FHL patients and in mirror of these findings, no underlying gene variation of PRF, MUNC13-4 and STX11 were identified.

CONCLUSIONWe developed a method to quantitatively assess cytotoxicity of the NK cells and CTL by measuring the expression of CD107α on the cell membrane, which appeared to be an effective and rapid screening test for cytotoxic defects-related diseases such as FHL and other HLH secondary to primary immunodeficiency.

Case-Control Studies ; Cell Degranulation ; immunology ; Cell Membrane ; metabolism ; Chediak-Higashi Syndrome ; diagnosis ; genetics ; immunology ; metabolism ; Child, Preschool ; Cytotoxicity, Immunologic ; Female ; Flow Cytometry ; methods ; Humans ; Infant ; Interleukin-2 ; metabolism ; Killer Cells, Natural ; immunology ; metabolism ; Lymphohistiocytosis, Hemophagocytic ; diagnosis ; genetics ; immunology ; metabolism ; Lysosomal-Associated Membrane Protein 1 ; metabolism ; Male ; Mutation ; Phytohemagglutinins ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism

OBJECTIVETo break immune tolerance to prion (PrP) proteins using DNA vaccines.

METHODSFour different human prion DNA vaccine candidates were constructed based on the pcDNA3.1 vector: PrP-WT expressing wild-type PrP, Ubiq-PrP expressing PrP fused to ubiquitin, PrP-LII expressing PrP fused to the lysosomal integral membrane protein type II lysosome-targeting signal, and PrP-ER expressing PrP locating the ER. Using a prime-boost strategy, three-doses of DNA vaccine were injected intramuscularly into Balb/c mice, followed by two doses of PrP protein. Two weeks after the last immunization, sera and spleens were collected and PrP-specific humoral and cellular immune responses evaluated by ELISA and ELISPOT tests.

RESULTSHigher levels of serum PrP antibodies were detected in mice vaccinated using the strategy of DNA priming followed by protein boosting. Of these, WT-PrP, Ubiq-PrP, and PrP-LII induced significantly higher humoral responses. ELISPOT tests showed markedly increased numbers of IFN-γ-secreting T cells in mice vaccinated using the strategy of DNA priming followed by protein boosting after stimulation with recombinant PrP23-90 and PrP23-231. PrP-ER induced the strongest T-cell response.

CONCLUSIONPrion vaccines can break tolerance to PrP proteins and induce PrP-specific humoral and cellular immune responses.

Animals ; Antibodies ; immunology ; CHO Cells ; COS Cells ; Cercopithecus aethiops ; Cricetinae ; Cricetulus ; Enzyme-Linked Immunosorbent Assay ; Female ; HeLa Cells ; Humans ; Immune Tolerance ; Interferon-gamma ; immunology ; Lysosomal-Associated Membrane Protein 2 ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Peptide Fragments ; immunology ; Prions ; genetics ; immunology ; Receptors, Peptide ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; Recombinant Proteins ; immunology ; Transfection ; Ubiquitin ; genetics ; immunology ; Vaccines, DNA