• Title/Summary/Keyword: in vitro infection method

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CD7-Specific Single Chain Antibody Mediated Delivery of siRNA to T Cells Inhibits HIV Replication in a Humanized Mouse Model

  • Ban, Hong-Seok;Kumar, Priti;Kim, Na-Hyun;Choi, Chang-Son;Shankar, Premlata;Lee, Sang-Kyung
    • Proceedings of the Microbiological Society of Korea Conference
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    • 2008.05a
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    • pp.62-64
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    • 2008
  • A major hurdle to the development of RNA interference as therapy for HIV infection is the delivery of siRNA to T lymphocytes which are difficult cells to transfect even in vitro. We have employed a single chain antibody to the pan T cell surface antigen CD7 was conjugated to an oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2${\gamma}$-/- mice reconstituted with human peripheral blood lymphocytes (Hu-PBL). Using a novel delivery, we first show that scFvCD7-9R efficiently delivered CD4 siRNA into human T cells in vitro. In vivo administration to Hu-PBL mice resulted in reduced levels of surface CD4 expression on T cells. Mice infected with HIV-1 and treated on a weekly basis with scFvCD7-9R-siRNA complexes targeting a combination of viral genes and the host coreceptor molecule CCR5 successfully maintained CD4/CD3 T cell ratios up to 4 weeks after infection in contrast to control mice that displayed a marked reduction in CD4 T cell numbers. p24 antigen levels were undetectable in 3 of the 4 protected mice. scFvCD7-9R/antiviral siRNA treatment also helped maintain CD4 T cell numbers with reduced plasma viral loads in Hu-PBL mice reconstituted with PBMC from donors seropositive for HIV, indicating that this method can contain viral replication even in established HIV infections. Our results show that scFvCD7-9R could be further developed as a potential therapeutic for HIV-1 infection.

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In vitro and In vivo Anti-Helicobacter pylori Activities of Centella asiatica Leaf Extract

  • Zheng, Hong-Mei;Choi, Myung-Joo;Kim, Jae Min;Lee, Kye Wan;Park, Yu Hwa;Lee, Don Haeng
    • Preventive Nutrition and Food Science
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    • v.21 no.3
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    • pp.197-201
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    • 2016
  • Helicobacter pylori infection is associated with an increased risk of developing upper gastrointestinal tract diseases. However, treatment failure is a major cause of concern mainly due to possible recurrence of infection, the side effects, and resistance to antibiotics. The aim of this study was to investigate the activities of Centella asiatica leaf extract (CAE) against H. pylori both in vitro and in vivo. The minimum inhibitory concentrations (MICs) against 55 clinically isolated strains of H. pylori were tested using an agar dilution method. The MICs of CAE ranged from 0.125 mg/mL to 8 mg/mL, effectiveness in inhibiting H. pylori growth was 2 mg/mL. The anti-H. pylori effects of CAE in vivo were also examined in H. pylori-infected C57BL/6 mice. CAE was orally administrated once daily for 3 weeks at doses of 50 mg/kg and 250 mg/kg. CAE at the 50 mg/kg dose significantly reduced H. pylori colonization in mice gastric mucosa. Our study provides novel insights into the therapeutic effects of CAE against H. pylori infection, and it suggests that CAE may be useful as an alternative therapy.

The Effect of Injinchunggan-tang on Hepatitis C Virus Infection (인진청간탕이 C형 간염 바이러스의 임파구계 세포감염에 미치는 영향)

  • 김선민;이장훈;우홍정
    • The Journal of Korean Medicine
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    • v.22 no.1
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    • pp.53-62
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    • 2001
  • Objective : The aim of this study was to evaluate the efficacy of Injinchunggan-tang on Hepatitis C virus infection, and to clarify the mechanism of treatment by indentifying the effect of Injinchunggan-tang on cytokine secretion. Methods : In vitro model of HCV infection in MOLT 4 cell was used. The effect of Injinchunggan-tang on the attachment of HCV on MOLT 4 cell was studied by PCR method. The change of cytokine secretion according to Injinchunggan-tang treatment was investigated by ELISA. Results : Injinchunggan-tang inhibited the attachment of HCV on MOLT 4 in the concentration of $10-2{\mu\textrm{g}}/\mu\textrm{\ell}$ and $10-1{\mu\textrm{g}}/\mu\textrm{\ell}$. In cytokine assay, Injinchunggan-tang increased the secretion of IL-4 of mouse splenocytes and PBMC in 48 hour culture as well as the secretion of IL-12 of mouse splenocytes and PBMC in 48 hour culture, whereas it decreased the secretion of $IFN-{\gamma}$ of mouse splenocytes in 24 and 48 hour culture. Conclusion : The results of this study show that Injinchunggan-tang has an inhibitory effect on the attachment on HCV on Mo1t4 Cell, and that it increases the secretion of IL-4 and IL-12 of mouse splenocyte and PBMC.

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In Vitro and in Vivo Antifungal Activities of 6-[(N-4-bromophenyl)amino]-7-chloro-5,8-quinolinediones

  • Ryu, Chung-Kyu;Kim, Dong-Hyun;Kwon, Sang-Mee;Jung, Sung-Hee;Kim, Sung-Hee
    • Archives of Pharmacal Research
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    • v.20 no.6
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    • pp.586-589
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    • 1997
  • Antifungal activities of 6-[(N-4-bromophenyl)amino]-7-chloro-5,8-quinolinedione (RCK7) were tested. The MIC values of RCK7 were determined for antifungal suceptibility, in vitro against Aspergillus niger, Cryptococcus neoformans and Trichophyton mentagrophyte by standard agar streak method. In vitro, RCK7 showed more potent antifungal activity than fluconazole and ketoconazole. Also, RCK7 was tested for in vivo antifungal activity in the treatment of systemic infection with Candida albicans in normal mice. The therapeutic potential of RCK7 had been assessed by evaluating their survival rate against systemic infections compared with that of ketoconazole. $ED_{50}$ of intraperitoneally administered RCK7 ws $2.05{\pm}0.30mg/kg$ but that of ketoconazole was $8.00{\pm}0.73 mg/kg$, respectively. When RCK7 was administered intravenously at the $ED_{50}$(2.05 mg/kg). the colony counts of Candida albicans in the liver after 7 days and 14 days were reduced as likely as ketoconazole at the $ED_{50}(8.00 mg/kg)$, and the better survival rates than ketoconazole's were achieved after 14 days. The results suggest that RCK7 may be a potent antifungal agent.

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Antiviral effect of fucoxanthin obtained from Sargassum siliquastrum (Fucales, Phaeophyceae) against severe acute respiratory syndrome coronavirus 2

  • Nalae Kang;Seong-Yeong Heo;Eun-A Kim;Seon-Heui Cha;Bomi Ryu;Soo-Jin Heo
    • ALGAE
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    • v.38 no.4
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    • pp.295-306
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    • 2023
  • Human coronavirus diseases, particularly severe acute respiratory syndrome coronavirus 2, still remain a persistent public health issue, and many recent studies are focusing on the quest for new leads against coronaviruses. To contribute to this growing pool of knowledge and explore the available marine natural products against coronaviruses, this study investigated the antiviral effects of fucoxanthin isolated from Sargassum siliquastrum-a brown alga found on Jeju Island, South Korea. The antiviral effects of fucoxanthin were confirmed in severe acute respiratory syndrome coronavirus 2-infected Vero cells, and its structural characteristics were verified in silico using molecular docking and molecular dynamic simulations and in vitro colorimetric method. Fucoxanthin inhibited the infection in a concentration-dependent manner, without showing cytotoxicity. Molecular docking simulations revealed that fucoxanthin binds to the angiotensinconverting enzyme 2-spike protein (binding energy -318.306 kcal mol-1) and main protease (binding energy -205.118 kcal mol-1). Moreover, molecular dynamic simulations showed that fucoxanthin remains docked to angiotensin-converting enzyme 2-spike protein for 20 ns, whereas it breaks away from main protease after 3 ns. Also, the in silico prediction of the fucoxanthin was verified through the in vitro colorimetric method by inhibiting the binding between angiotensinconverting enzyme 2 and spike protein in a concentration-dependent manner. These results indicate that fucoxanthin exhibits antiviral effects against severe acute respiratory syndrome coronavirus 2 by blocking the entry of the virus. Therefore, fucoxanthin from S. siliquastrum can be a potential candidate for treating coronavirus infection.

In vitro and In vivo Activity of DW286, an Experimental Fluoronaphthyridione, against Gram-Positive Bacteria (새로운 Fluoronaphthyridinone계 항균제 DW286의 그람 양성세균에 대한 항균 활성)

  • Kwak, Jin-Hwan;Park, Hee Soo;Jung, Sung Ji;Jeong, Ji-Woong;Kim, Joseph;Choi, Dong-Rack;Choi, Eung-Chil
    • YAKHAK HOEJI
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    • v.56 no.5
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    • pp.304-308
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    • 2012
  • In vitro antibacterial activity of DW286 was tested against recently collected clinical isolates of Gram-positive strains by the two fold agar dilution method. In vivo activity of DW286 was also determined against systemic infections in mice caused by Staphylococcus aureus. DW286 showed 16~64-fold more potent in vitro activities than ciprofloxacin against Gram-positive bacteria. Against systemic infection model caused by two S. aureus strains, one being methicillin-susceptible and the other methicillin-resistant, DW286 ($ED_{50}s$, 0.16 mg/kg and 4.36 mg/kg, respectively) was more potent than gemifloxacin (1.37 mg/kg, 26.58 mg/kg, respectively).

Use of In Vivo-Induced Antigen Technology to Identify In Vivo-Expressed Genes of Campylobacter jejuni During Human Infection

  • Hu, Yuanqing;Huang, Jinlin;Li, Qiuchun;Shang, Yuwei;Ren, Fangzhe;Jiao, Yang;Liu, Zhicheng;Pan, Zhiming;Jiao, Xin-An
    • Journal of Microbiology and Biotechnology
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    • v.24 no.3
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    • pp.363-370
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    • 2014
  • Campylobacter jejuni is a prevalent foodborne pathogen worldwide. Human infection by C. jejuni primarily arises from contaminated poultry meats. Genes expressed in vivo may play an important role in the pathogenicity of C. jejuni. We applied an immunoscreening method, in vivo-induced antigen technology (IVIAT), to identify in vivo-induced genes during human infection by C. jejuni. An inducible expression library of genomic proteins was constructed from sequenced C. jejuni NCTC 11168 and was then screened using adsorbed, pooled human sera obtained from clinical patients. We successfully identified 24 unique genes expressed in vivo. These genes were implicated in metabolism, molecular biosynthesis, genetic information processing, transport, and other processes. We selected six genes with different functions to compare their expression levels in vivo and in vitro using real-time RT-PCR. The results showed that the selected six genes were significantly upregulated in vivo but not in vitro. In short, these identified in vivo-induced genes may contribute to human infection of C. jejuni, some of which may be meaningful vaccine candidate antigens or diagnosis serologic markers for campylobacteriosis. IVIAT may present a significant and efficient method for understanding the pathogenicity mechanism of Campylobacter and for finding targets for its prevention and control.

Anti-protozoal effect of organic acids against Azumiobodo hoyamushi that causes soft tunic syndrome to Halocynthia roretzi (멍게 물렁증의 원인충인 Azumiobodo hoyamushi에 대한 유기산의 살충효과 연구)

  • Lee, Ji Hoon;Park, Kyung Il;Park, Kwan Ha
    • Journal of fish pathology
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    • v.28 no.3
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    • pp.117-123
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    • 2015
  • Economic loss by soft tunic syndrome of edible ascidian, Halocynthia roretzi has become a serious problem. Recently, it has discovered that the cause of this syndrome is infection by a protozoan parasite Azumiobodo hoyamushi. However, only a few studies have been conducted to control this parasitic disease. In a previous research, non-specific disinfectants have been found to be effective in controling the causative parasite. In an attempt to eradicate this causative parasite, organic acids were tested in this study to evaluate their in vitro and in vivo efficacy. In vitro tests showed that 8 different organic acids used in this study were moderately or highly effective with protozoan-killing effects ($EC_{50}=153{\sim}275{\mu}g/ml$). Despite weak in vivo penetration of organic acids into the tunic tissues, treatment with high concentration reduced the mortality of ascidian caused by infection the parasite, indicating that we might be able to develop a disinfection method using environmentally-friendly organic acids.

A Homogeneous Immunoassay Method for Detecting Interferon-Gamma in Patients with Latent Tuberculosis Infection

  • Wu, Fei;Wang, Lin;Guo, Qiaomei;Zhao, Mingna;Gu, Hongchen;Xu, Hong;Lou, Jiatao
    • Journal of Microbiology and Biotechnology
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    • v.26 no.3
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    • pp.588-595
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    • 2016
  • IFN-γ release assays (IGRAs) have been developed as viable alternative diagnostic tools for detecting latent tuberculosis infection (LTBI). A customized homogeneous sandwich luminescent oxygen channeling immunoassay (LOCI) was used to quantify IFN-γ levels in IGRAs. Samples were collected from healthy volunteers (n = 40) who were T-Spot-negative and T-Spot-positive patients (n = 32) at rest. Then the amount of IFN-γ in the supernatant of IGRAs was measured by LOCI. The results demonstrated a low background, and high sensitivity, specificity, accuracy, and reproducibility, and a short assay time (only 30 min) with LOCI for IFN-γ. The recovery range was 81.63-102.06%, the coefficients of variation were below 5%, and the limit of detection was 19.0 mIU/ml. Excellent agreement between LOCI IFN-γ and the T-SPOT.TB test was obtained (97.2% agreement, κ = 0.94). The LOCI IFN-γ concentrations were significantly higher in T-Spot-positive patients than in the healthy group (p < 0.001). Moreover, as observed for the comparative LOCI IFN-γ assay, IFN-γ concentrations were related to the numbers of T-SPOT.TB spots. We have established an in vitro blood test for LTBI diagnosis, defined as LOCI IFN-γ. A high level of agreement between the LOCI IFN-γ method and T-SPOT.TB assay was observed in clinical studies that showed the LOCI IFN-γ method could determine LTBI. This study shows acceptable performance characteristics of the LOCI IFN-γ assay to diagnose LTBI.

Infection and Pathogenesis Mechanisms of Marek's Disease Virus (마렉병 바이러스 감염과 병원성 발현 기전)

  • Jang, H.K.;Park, Y.M.;Cha, S.Y.;Park, J.B.
    • Korean Journal of Poultry Science
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    • v.35 no.1
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    • pp.39-55
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    • 2008
  • Like the other herpesviruses, the virion of MDV consists of an envelope, which surrounds an amorphous tegument. Within the tegument, and icosahedral capsid encloses a linear double-stranded DNA core. Although the genome structure of MDV indicates that it is an ${\alpha}-herpesvirus$ like herpes simplex and varicella-zoster viruses, biological properties indicate MDV is more akin to the ${\gamma}-herpesvirus$ group, which includes Epstein-Barr and Kaposi's sarcoma herpesviruses. These herpesviruses replicate lytically in lymphocytes, epithelial and fibroblastic cells, and persist in lymphoblastoid cells. MDV has a complex life cycle and uses two means of replication, productive and non-productive, to exist and propagate. The method of reproduction changes according to a defined pattern depending on changes in virus-cell interactions at different stages of the disease, and in different tissues. Productive (lytic) interactions involve active invasion and take-over of the host cell, resulting in the production of infectious progeny virions. However, some herpesviruses, including MDV, can also establish a non-productive (abortive) infection in certain cell types, resulting in production of cell-associated progeny virus. Non-productive interactions represent persistent infection, in which the viral genome is present but gene expression is limited, there is no structural or regulatory gene translation, no replication, no release of progeny virions and no cell death. Reactivation of the virus is rare, and usually the infectious virus can be re-isolated only after cultivation in vitro. MDV establishes latency in lymphoid cells, some of which are subsequently transformed. In this review article, recent knowledges of the pathogenesis mechanisms followed by MDV infection to sensitive cells and chickens are discussed precisely.