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1.
Immune Network ; : e25-2020.
Article | WPRIM | ID: wpr-835460

ABSTRACT

Acinetobacter baumannii is known for its multidrug antibiotic resistance. New approaches to treating drug-resistant bacterial infections are urgently required. Cathelicidin-related antimicrobial peptide (CRAMP) is a murine antimicrobial peptide that exerts diverse immune functions, including both direct bacterial cell killing and immunomodulatory effects. In this study, we sought to identify the role of CRAMP in the host immune response to multidrug-resistant Acinetobacter baumannii. Wild-type (WT) and CRAMP knockout mice were infected intranasally with the bacteria. CRAMP−/− mice exhibited increased bacterial colony-forming units (CFUs) in bronchoalveolar lavage (BAL) fluid after A. baumannii infection compared to WT mice. The loss of CRAMP expression resulted in a significant decrease in the recruitment of immune cells, primarily neutrophils. The levels of IL-6 and CXCL1 were lower, whereas the levels of IL-10 were significantly higher in the BAL fluid of CRAMP−/− mice compared to WT mice 1 day after infection. In an in vitro assay using thioglycollate-induced peritoneal neutrophils, the ability of bacterial phagocytosis and killing was impaired in CRAMP−/− neutrophils compared to the WT cells. CRAMP was also essential for the production of cytokines and chemokines in response to A. baumannii in neutrophils. In addition, the A. baumannii-induced inhibitor of κB-α degradation and phosphorylation of p38 MAPK were impaired in CRAMP−/− neutrophils, whereas ERK and JNK phosphorylation was upregulated. Our results indicate that CRAMP plays an important role in the host defense against pulmonary infection with A. baumannii by promoting the antibacterial activity of neutrophils and regulating the innate immune responses.

2.
Article in English | WPRIM | ID: wpr-764240

ABSTRACT

Zika virus (ZIKV) is one of the pathogens which is transmitted world widely, but there are no effective drugs and vaccines. Whole genome sequencing (WGS) of viruses could be applied to viral pathogen characterization, diagnosis, molecular surveillance, and even finding novel pathogens. We established an improved method using direct RNA sequencing with Nanopore technology to obtain WGS of ZIKV, after adding poly (A) tails to viral RNA. This established method does not require specific primers, complimentary DNA (cDNA) synthesis, and polymerase chain reaction (PCR)-based enrichment, resulting in the reduction of biases as well as of the ability to find novel RNA viruses. Nanopore technology also allows to read long sequences. It makes WGS easier and faster with long-read assembly. In this study, we obtained WGS of two strains of ZIKV following the established protocol. The sequenced reads resulted in 99% and 100% genome coverage with 63.5X and 21,136X, for the ZIKV PRVABC59 and MR 766 strains, respectively. The sequence identities of the ZIKV PRVABC59 and MR 766 strains for each reference genomes were 98.76% and 99.72%, respectively. We also found that the maximum length of reads was 10,311 bp which is almost the whole genome size of ZIKV. These long-reads could make overall structure of whole genome easily, and WGS faster and easier. The protocol in this study could provide rapid and efficient WGS that could be applied to study the biology of RNA viruses including identification, characterization, and global surveillance.


Subject(s)
Bias , Biology , Diagnosis , DNA , Genome , Genome Size , Methods , Nanopores , Polymerase Chain Reaction , RNA Viruses , RNA , RNA, Viral , Sequence Analysis, RNA , Tail , Vaccines , Zika Virus
3.
Experimental Neurobiology ; : 458-473, 2019.
Article in English | WPRIM | ID: wpr-763781

ABSTRACT

The function of microglia/macrophages after ischemic stroke is poorly understood. This study examines the role of microglia/macrophages in the focal infarct area after transient middle cerebral artery occlusion (MCAO) in rhesus monkeys. We measured infarct volume and neurological function by magnetic resonance imaging (MRI) and non-human primate stroke scale (NHPSS), respectively, to assess temporal changes following MCAO. Activated phagocytic microglia/macrophages were examined by immunohistochemistry in post-mortem brains (n=6 MCAO, n=2 controls) at 3 and 24 hours (acute stage), 2 and 4 weeks (subacute stage), and 4, and 20 months (chronic stage) following MCAO. We found that the infarct volume progressively decreased between 1 and 4 weeks following MCAO, in parallel with the neurological recovery. Greater presence of cluster of differentiation 68 (CD68)-expressing microglia/macrophages was detected in the infarct lesion in the subacute and chronic stage, compared to the acute stage. Surprisingly, 98~99% of transforming growth factor beta (TGFβ) was found colocalized with CD68-expressing cells. CD68-expressing microglia/macrophages, rather than CD206⁺ cells, may exert anti-inflammatory effects by secreting TGFβ after the subacute stage of ischemic stroke. CD68⁺ microglia/macrophages can therefore be used as a potential therapeutic target.


Subject(s)
Brain , Haplorhini , Immunohistochemistry , Infarction, Middle Cerebral Artery , Inflammation , Macaca mulatta , Magnetic Resonance Imaging , Microglia , Middle Cerebral Artery , Primates , Stroke , Transforming Growth Factor beta
4.
Experimental Neurobiology ; : 414-424, 2019.
Article in English | WPRIM | ID: wpr-763764

ABSTRACT

Mitochondria continuously fuse and divide to maintain homeostasis. An impairment in the balance between the fusion and fission processes can trigger mitochondrial dysfunction. Accumulating evidence suggests that mitochondrial dysfunction is related to neurodegenerative diseases such as Parkinson's disease (PD), with excessive mitochondrial fission in dopaminergic neurons being one of the pathological mechanisms of PD. Here, we investigated the balance between mitochondrial fusion and fission in the substantia nigra of a non-human primate model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD. We found that MPTP induced shorter and abnormally distributed mitochondria. This phenomenon was accompanied by the activation of dynamin-related protein 1 (Drp1), a mitochondrial fission protein, through increased phosphorylation at S616. Thereafter, we assessed for activation of the components of the cyclin-dependent kinase 5 (CDK5) and extracellular signal-regulated kinase (ERK) signaling cascades, which are known regulators of Drp1(S616) phosphorylation. MPTP induced an increase in p25 and p35, which are required for CDK5 activation. Together, these findings suggest that the phosphorylation of Drp1(S616) by CDK5 is involved in mitochondrial fission in the substantia nigra of a non-human primate model of MPTP-induced PD.


Subject(s)
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine , Cyclin-Dependent Kinase 5 , Cyclin-Dependent Kinases , Dopaminergic Neurons , Homeostasis , Mitochondria , Mitochondrial Dynamics , Neurodegenerative Diseases , Parkinson Disease , Phosphorylation , Phosphotransferases , Primates , Substantia Nigra
5.
Article in English | WPRIM | ID: wpr-758919

ABSTRACT

Microorganisms play important roles in obesity; however, the role of the gut microbiomes in obesity is controversial because of the inconsistent findings. This study investigated the gut microbiome communities in obese and lean groups of captive healthy cynomolgus monkeys reared under strict identical environmental conditions, including their diet. No significant differences in the relative abundance of Firmicutes, Bacteroidetes and Prevotella were observed between the obese and lean groups, but a significant difference in Spirochetes (p < 0.05) was noted. Microbial diversity and richness were similar, but highly variable results in microbial composition, diversity, and richness were observed in individuals, irrespective of their state of obesity. Distinct clustering between the groups was not observed by principal coordinate analysis using an unweighted pair group method. Higher sharedness values (95.81% ± 2.28% at the genus level, and 79.54% ± 5.88% at the species level) were identified among individual monkeys. This paper reports the association between the gut microbiome and obesity in captive non-human primate models reared under controlled environments. The relative proportion of Firmicutes and Bacteroidetes as well as the microbial diversity known to affect obesity were similar in the obese and lean groups of monkeys reared under identical conditions. Therefore, obesity-associated microbial changes reported previously appear to be associated directly with environmental factors, particularly diet, rather than obesity.


Subject(s)
Bacteroidetes , Diet , Environment, Controlled , Firmicutes , Gastrointestinal Microbiome , Haplorhini , Macaca fascicularis , Methods , Microbiota , Obesity , Prevotella , Primates , Spirochaetales
6.
Immune Network ; : e13-2019.
Article in English | WPRIM | ID: wpr-740215

ABSTRACT

6-kDa early secretory antigenic target (ESAT6), a virulent factor of Mycobacterium tuberculosis, is involved in immune regulation. However, the underlying mechanism behind the activation and maturation of dendritic cells (DCs) by ESAT6 remains unclear. In this study, we investigated the effect on TLRs signaling on the regulation of ESAT6-induced activation and maturation of DCs. ESAT6 induced production of IL-6, TNF-α, and IL-12p40 in bone marrow-derived dendritic cells (BMDCs) from wild-type and TLR2-deficient mice, with this induction abolished in TLR4-deficient cells. NF-κB is essential for the ESAT6-induced production of the cytokines in BMDCs. TLR4 was also required for ESAT6-induced activation of NF-κB and MAPKs in BMDCs. ESAT6 additionally upregulated the expression of surface molecules CD80, CD86, and MHC-II, and also promoted the ability of CD4⁺ T cells to secrete IFN-γ via the TLR4-dependent pathway. Our findings suggest that TLR4 is critical in the activation and maturation of DCs in response to ESAT6.


Subject(s)
Animals , Cytokines , Dendritic Cells , Interleukin-12 Subunit p40 , Interleukin-6 , Mice , Mycobacterium tuberculosis , Mycobacterium , T-Lymphocytes , Toll-Like Receptor 4
7.
Article in Korean | WPRIM | ID: wpr-648761

ABSTRACT

BACKGROUND AND OBJECTIVES: Unlike the normal skin, cholesteatomas characterized by hyperproliferative keratinocytes exhibits up-regulation of connexins (Cxs) and gap junctional intercellular communication (GJIC). Currently, there are no appropriate clinical methods that can inhibit cholesteatoma progression nor are there available optimal in vitro models of cholesteatomas. The objectives of this study were to identify the regulating materials that control GJIC using human keratinocyte cells (HaCaT) and to get preliminary information about how to inhibit cholesteatoma progression with an aim to make in vitro models. MATERIALS AND METHOD: Acetic acid (AA), H2O2, dexamethasone, retinoic acid (RA), or green tea extracts-epicatechin (EC) and epigallocatechin gallate (EGCG) were used for this study. After HaCaT cells were cultured with chemicals for 24 hours, cytotoxicity was quantitatively analyzed by cell counting and Neutral-red uptake test. Reverse transcriptase-polymerase chain reaction, Western blot and immunocytochemistry were performed to analyze the change of Cx expression. GJIC was functionally evaluated with scrape-loading dye transfer (SLDT). RESULTS: After the 24-hour culture, H2O2 or EGCG (100 microM) were observed to have interfered with cell growth. In the Western blot, Cx26 and Cx30 showed higher up-regulation by EGCG or dexamethasone, but less down-regulation by AA or H2O2 than the control. In comparison with the control, immunocytochemistry (Cx26, Cx43) showed less expression and abnormal location of Cxs under AA, H2O2, or 50 microM EGCG than the control, and increased up-regulation or equal expression under 5microM EGCG, EC, RA, or dexamethasone was greater than the control. In SLDT, dye transfer was significantly lower in AA-, H2O2-, dexamethasone-, or RA-treated cells than in the control cells. EC showed higher dye transfer than the control cells. CONCLUSION: The expression of Cxs and GJIC on human HaCaT keratinocytes can be up- or down-regulated by chemicals such as AA, H2O2, dexamethasone, or EC. These results may be useful information in understanding the progression or inhibition mechanisms of cholesteatomas.


Subject(s)
Acetic Acid , Blotting, Western , Catechin , Cell Count , Cholesteatoma , Connexins , Dexamethasone , Down-Regulation , Gap Junctions , Humans , Immunohistochemistry , Keratinocytes , Skin , Tea , Tretinoin , Up-Regulation
8.
Article in English | WPRIM | ID: wpr-172834

ABSTRACT

This experiment was conducted to assess the efficacy of typhoid vaccine newly produced by purifying Vi antigen of Salmonella typhi. With Karber method, LD50 of challenging organism (S. typhi ty2) was determined as 6.31 CFU/mouse, and then the organism was used for the study. With Probits method, ED50 of the vaccine was determined as 0.016 microgram / 0.5 ml / mouse. The ELISA titer (0.5097+/-0.0606) was 4 times in the group treated with high dose (0.25 microgram/0.5ml) as in control (0.1113+/-0.0110). Six major protein bands of 66, 55, 35, 33, 18, and 9 kd were detected in Western blot analysis with serum of a vaccine treated mouse, whereas only one weak band of about 35 kd was detected with serum of a control mouse. We concluded that typhoid vaccine produced by purifying Vi antigen of S. typhi very effectively prevent S. typhi infection in mice.


Subject(s)
Animals , Antibodies, Bacterial/immunology , Antigens, Bacterial/immunology , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Lethal Dose 50 , Logistic Models , Male , Mice , Mice, Inbred BALB C , Polysaccharides, Bacterial/immunology , Salmonella typhi/chemistry , Typhoid Fever/immunology , Typhoid-Paratyphoid Vaccines/administration & dosage
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