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Find video protocols related to scientific articles indexed in Pubmed.
Mechanism responsible for the antitumor effect of BCG-CWS using the LEEL method in a mouse bladder cancer model.
J Control Release
PUBLISHED: 05-24-2014
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We previously reported on the development of a water soluble formulation of the cell wall skeleton of BCG (BCG-CWS), a major immune active center of BCG, by encapsulating it into a nanoparticle (CWS-NP). The CWS-NP allowed us to clarify the machinery associated with the BCG mediated anti-bladder tumor effect, especially the roles of bladder cancer cells and dendritic cells (DCs) in the initial step, which remains poorly understood. We show herein that the internalization of BCG-CWS by bladder cancer cells, but not DCs, is indispensable for the induction of an antitumor effect against bladder cancer. Tumor growth was significantly inhibited in mice that had been inoculated with mouse bladder cancer (MBT-2) cells containing internalized BCG-CWS. On the other hand, the internalization of BCG-CWS by DCs had only a minor effect on inducing an antitumor effect against MBT-2 tumors. This was clarified for the first time by using the CWS-NP. This finding provides insights into our understanding of the role of bladder cancer cells and DCs in BCG therapy against bladder cancer.
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Nanoparticulation of BCG-CWS for application to bladder cancer therapy.
J Control Release
PUBLISHED: 01-07-2014
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The Mycobacterium bovis Bacille Calmette-Guerin cell wall skeleton (BCG-CWS) could be used to replace live BCG as a bladder cancer drug. However, because BCG-CWS is poorly soluble, has a strong-negative charge, very high molecular weight and heterogeneity in size of tens of ?m, it cannot be used in such an application. We report herein on the development of a novel packaging method that permits BCG-CWS to be encapsulated into 166nm-sized lipid particles. The BCG-CWS encapsulated nano particle (CWS-NP) has a high uniformity and can be easily dispersed. Thus, it has the potential for use as a packaging method that would advance the scope of applications of BCG-CWS as a bladder cancer drug. In a functional evaluation, CWS-NP was efficiently taken up by mouse bladder tumor (MBT-2) cells in vitro and inhibited tumor growth in mice bearing MBT-2 tumors. Moreover, intravesically administered CWS-NP showed significant antitumor effects in a rat model with naturally developed bladder cancer. An enhancement in Th1 differentiation by CWS-NP was also confirmed in human T cells. In conclusion, CWS-NP represents a promising delivery system for BCG-CWS for clinical development as a potent bladder cancer drug.
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Bacterial sphingophospholipids containing non-hydroxy fatty acid activate murine macrophages via Toll-like receptor 4 and stimulate bacterial clearance.
Biochim. Biophys. Acta
PUBLISHED: 03-04-2013
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Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs). Our previous study determined the complete chemical structures of these SPLs. The compositions of the long-chain bases/fatty acids in the ceramide portion, isoheptadecasphingosine/isopentadecanoate or isoheptadecasphingosine/2-hydroxy isopentadecanoate, are characteristic. The immune response against bacterial lipid components is considered to play important roles in microbial infections. It is reported that several bacterial sphingolipids composed of ceramide are recognized by CD1-restricted T and NKT cells and that a non-peptide antigen is recognized by ?? T cells. In this study, we demonstrated that these bacterial SPLs activated murine bone marrow macrophages (BMMs) via Toll-like receptor (TLR) 4 but not TLR2, although they slightly activated CD1d-restricted NKT and ??T cells. Interestingly, this TLR 4-recognition pathway of bacterial SPLs involves the fatty acid composition of ceramide in addition to the sugar moiety. A non-hydroxy fatty acid composed of ceramide was necessary to activate murine BMMs. The bacterial survival was significantly higher in TLR4-KO mice than in TLR2-KO and wild-type mice. The results indicate that activation of the TLR4-dependent pathway of BMMs by SPLs induced an innate immune response and contributed to bacterial clearance.
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Lipid phenotype of two distinct subpopulations of Mycobacterium bovis Bacillus Calmette-Guerin Tokyo 172 substrain.
J. Biol. Chem.
PUBLISHED: 10-26-2011
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Bacillus Calmette-Guérin (BCG) Tokyo 172 is a predominant World Health Organization Reference Reagent for the BCG vaccine. Recently, the BCG Tokyo 172 substrain was reported to consist of two subpopulations with different colony morphologies, smooth and rough. Smooth colonies had a characteristic 22-bp deletion in Rv3405c of the region of difference (RD) 16 (type I), and rough colonies were complete in this region (type II). We hypothesized that the morphological difference is related to lipid phenotype and affects their antigenicity. We determined the lipid compositions and biosynthesis of types I and II. Scanning electron microscopy showed that type I was 1.5 times longer than type II. Phenolic glycolipid (PGL) and phthiocerol dimycocerosate (PDIM) were found only in type I. Although it has been reported that the RD16 is involved in the expression of PGL, type II did not possess PGL/PDIM. We examined the ppsA-E gene responsible for PGL/PDIM biosynthesis and found that the existence of PGL/PDIM in types I and II is caused by a ppsA gene mutation not regulated by the RD16. PGL suppressed the host recognition of total lipids via Toll-like receptor 2, and this suggests that PGL is antigenic and involved in host responses, acting as a cell wall component. This is the first report to show the difference between lipid phenotypes of types I and II. It is important to clarify the heterogeneity of BCG vaccine substrains to discuss and evaluate the quality, safety, and efficacy of the BCG vaccine.
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The therapeutic effects of R8-liposome-BCG-CWS on BBN-induced rat urinary bladder carcinoma.
Anticancer Res.
PUBLISHED: 07-09-2011
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The present gold standard for bladder cancer is Mycobacterium bovis bacillus Calmette-Guérin (BCG) immunotherapy, but serious side-effects are common. We previously reported that C3H/HeN mice vaccinated with a mixture of MBT-2 cells and artificial BCG, octaarginine-modified liposomes incorporating the cell wall of BCG (R8-liposome-BCG-CW), significantly inhibited growth of R8-liposome-BCG-CW pretreated MBT-2 cells. Our aim was to determine if a non-live bacterial agent could be as efficacious as live BCG in a model of bladder cancer. We investigated the suppressive effect of liposome-incorporating cell wall skeleton (BCG-CWS) on N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced urinary bladder carcinogenesis in rats.
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The liposome-incorporating cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guéin can directly enhance the susceptibility of cancer cells to lymphokine-activated killer cells through up-regulation of natural-killer group 2, member D ligands.
BJU Int.
PUBLISHED: 02-11-2011
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• To conduct a preclinical evaluation of the ability of natural killer cells to cytolyze bladder cancer cells that were modified to show enhanced expression of natural-killer group 2, member D (NKG2D) ligands by R8-liposome-bacillus Calmette-Guéin (BCG)-cell wall skeleton (CWS) treatment.
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Bacillus Calmette-Guerin and BCG cell wall skeleton suppressed viability of bladder cancer cells in vitro.
Anticancer Res.
PUBLISHED: 11-02-2010
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Bacillus Calmette-Guerin (BCG) is one of therapeutic options for urothelial carcinoma (UC). The objectives of this study were to determine the direct effect of viable or heat-killed BCG and BCG cell wall skeleton (BCG-CWS) on UC cells in vitro.
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Novel rhamnosyltransferase involved in biosynthesis of serovar 4-specific glycopeptidolipid from Mycobacterium avium complex.
J. Bacteriol.
PUBLISHED: 09-03-2010
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Glycopeptidolipids (GPLs) are one of the major glycolipid components present on the surface of Mycobacterium avium complex (MAC) that belong to opportunistic pathogens distributed in the natural environment. The serovars of MAC, up to around 30 types, are defined by the variable oligosaccharide portions of the GPLs. Epidemiological studies show that serovar 4 is the most prevalent type, and the prognosis of pulmonary disease caused by serovar 4 is significantly worse than that caused by other serovars. However, little is known about the biosynthesis of serovar 4-specific GPL, particularly the formation of the oligosaccharide portion that determines the properties of serovar 4. To investigate the biosynthesis of serovar 4-specific GPL, we focused on one segment that included functionally unknown genes in the GPL biosynthetic gene cluster of a serovar 4 strain. In this segment, a putative hemolytic protein gene, hlpA, and its downstream gene were found to be responsible for the formation of the 4-O-methyl-rhamnose residue, which is unique to serovar 4-specific GPL. Moreover, functional characterization of the hlpA gene revealed that it encodes a rhamnosyltransferase that transfers a rhamnose residue via 1?4 linkage to a fucose residue of serovar 2-specific GPL, which is a key pathway leading to the synthesis of oligosaccharide of serovar 4-specific GPL. These findings may provide clues to understanding the biological role of serovar 4-specific GPL in MAC pathogenicity and may also provide new insights into glycosyltransferase, which generates structural and functional diversity of GPLs.
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Effectiveness of BCG vaccination to aged mice.
Immun Ageing
PUBLISHED: 06-13-2010
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The tuberculosis (TB) still increases in the number of new cases, which is estimated to approach 10 million in 2010. The number of aged people has been growing all over the world. Ageing is one of risk factors in tuberculosis because of decreased immune responses in aged people. Mycobacterium bovis Bacillus Calmette Guérin (BCG) is a sole vaccine currently used for TB, however, the efficacy of BCG in adults is still a matter of debate. Emerging the multidrug resistant Mycobacterium tuberculosis (MDR-TB) make us to see the importance of vaccination against TB in new light. In this study, we evaluated the efficacy of BCG vaccination in aged mice.
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Biochemical characteristics among Mycobacterium bovis BCG substrains.
FEMS Microbiol. Lett.
PUBLISHED: 06-10-2010
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In order to evaluate the biochemical characteristics of 14 substrains of Mycobacterium bovis bacillus Calmette Guérin (BCG) - Russia, Moreau, Japan, Sweden, Birkhaug, Danish, Glaxo, Mexico, Tice, Connaught, Montreal, Phipps, Australia and Pasteur - we performed eight different biochemical tests, including those for nitrate reduction, catalase, niacin accumulation, urease, Tween 80 hydrolysis, pyrazinamidase, p-amino salicylate degradation and resistance to thiophene 2-carboxylic acid hydrazide. Catalase activities of the substrains were all low. Data for nitrate reduction, niacin accumulation, Tween 80 hydrolysis, susceptibility to hydrogen peroxide and nitrate, and optimal pH for growth were all variable among these substrains. These findings suggest that the heterogeneities of biochemical characteristics are relevant to the differences in resistance of BCG substrains to environmental stress. The study also contributes to the re-evaluation of BCG substrains for use as vaccines.
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Isolation and identification of arabinose mycolates of Cell Wall Skeleton (CWS) derived from Mycobacterium bovis BCG Tokyo 172 (SMP-105).
J. Microbiol. Methods
PUBLISHED: 01-04-2010
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A unique hydrolysis method using a two-layer solution, consisting of diluted hydrochloric acid and toluene was developed to isolate whole arabinose mycolates from the cell wall skeleton of Mycobacterium bovis BCG Tokyo 172 (SMP-105) in order to reveal its pivotal role in enhancing immune responses against tumors.
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Serodiagnostic contributions of antibody titers against mycobacterial lipid antigens in Mycobacterium avium complex pulmonary disease.
Clin. Infect. Dis.
PUBLISHED: 07-14-2009
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Although the incidence of pulmonary tuberculosis is decreasing, the number of immunocompetent patients with Mycobacterium avium complex (MAC) pulmonary disease is steadily increasing. Therefore, albeit not contagious, MAC pulmonary disease needs to be diagnosed rapidly and accurately. We examined the serodiagnostic contributions of serum immunoglobulin G antibody titers against the species-specific and -common mycobacterial lipid antigens in the diagnosis of MAC pulmonary disease.
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Comparable studies of immunostimulating activities in vitro among Mycobacterium bovis bacillus Calmette-Guérin (BCG) substrains.
FEMS Immunol. Med. Microbiol.
PUBLISHED: 04-27-2009
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During the serial passage of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in different countries after initial seed distribution from the Pasteur Institute, specific insertions and deletions in the genome among BCG substrains were observed and speculated to result in differences in immunological activities. Early-shared strains of BCG (Russia, Moreau, Japan, Sweden, Birkhaug), distributed by the Pasteur Institute, which conserve three types of mycolate (alpha, methoxy, keto) in cell wall, exhibited stronger activities of induction of nitric oxide, interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12 and tumor necrosis factor (TNF)-alpha, from human epithelial cell line A549, human myelomonocytic cell line THP-1 and mouse bone marrow cells in the presence of interferon-gamma (IFN-gamma) than did late-shared strains of BCG (Danish, Glaxo, Mexico, Tice, Connaught, Montreal, Phipps, Australia, Pasteur). The stronger induction of IL-12 and TNF-alpha in the presence of IFN-gamma was also observed by trehalose 6,6-dimycolate (TDM) extracted from BCG-Japan than by TDM from BCG-Connaught, which lacks the methoxymycolate residue. These results suggest that early-shared strains are more potent immunostimulating agents than late-shared strains, which could be attributed partially to methoxymycolate. Our study provides the basic information for immunological characterization of various BCG strains and may contribute to a re-evaluation of them as a reference strain for vaccination against tuberculosis.
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Immunoprotection against murine bladder carcinoma by octaarginine-modified liposomes incorporating cell wall of Mycobacterium bovis bacillus Calmette-Guérin.
BJU Int.
PUBLISHED: 03-27-2009
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To develop a prototype of a non-live bacterial agent that consists of a cell wall (CW) preparation from heat-killed bacillus Calmette-Guérin (BCG-CW) incorporated into octaarginine-modified cationized liposomes as a vector (R8-liposome-BCG-CW), and to evaluate its immunoprotective potentiation in mice, as although BCG is an established effective immunotherapy for nonmuscle-invasive bladder cancer, more active and less toxic treatments are needed.
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Separation and molecular characterization of mycolic acid from the cell wall skeleton of Mycobacterium bovis BCG Tokyo 172 (SMP-105) and BCG substrains by normal-phase high performance liquid chromatography and liquid chromatography/mass spectrometry.
J. Microbiol. Methods
PUBLISHED: 03-04-2009
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Since mycolic acids, the most characteristic major lipid component in mycobacterial cell envelopes, play pivotal roles in the cell surface-based host immune responses, normal-phase HPLC has been developed to quantify and identify mycolic acids of the cell wall skeleton from Mycobacterium bovis BCG Tokyo 172 (SMP-105).
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Whole genome sequence analysis of Mycobacterium bovis bacillus Calmette-Guérin (BCG) Tokyo 172: a comparative study of BCG vaccine substrains.
Vaccine
PUBLISHED: 01-13-2009
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To investigate the molecular characteristics of bacillus Calmette-Guérin (BCG) vaccines, the complete genomic sequence of Mycobacterium bovis BCG Tokyo 172 was determined, and the results were compared with those for BCG Pasteur and other M. tuberculosis complex. The genome of BCG Tokyo had a length of 4,371,711bp and contained 4033 genes, including 3950 genes coding for proteins (CDS). There were 18 regions of difference (showing differences of more than 20bp), 20 insertion or deletion (ins/del) mutations of less than 20bp, and 68 SNPs between the two BCG substrains. These findings are useful for better understanding of the genetic differences in BCG substrains due to in vitro evolution of BCG.
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Tonsilliphilus suis gen. nov., sp. nov., causing tonsil infections in pigs.
Int. J. Syst. Evol. Microbiol.
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A micro-organism resembling members of the genus Dermatophilus, strain W254(T), which was isolated from the submandibular lymph node of a pig, and an additional 16 strains isolated from swine tonsils, were studied to establish their taxonomic status. Although all 17 strains were isolated anaerobically under an atmosphere of 100 % CO2, all of them were aerotolerant anaerobes. The micro-organisms showed at least five cellular morphologies: (i) a radially protrusive thallus, which proliferated into tuber-like cells; (ii) segmentation in both tubers and thallus followed by multilocule formation, (iii) development of coccoid forms in the locules; (iv) a change from the coccoid forms to zoospores; (v) resting cells, which were able to develop into protrusive thalli again. The micro-organisms were positive for nitrate reduction, but negative for catalase, indole production, hydrolysis of urea and gelatin liquefaction. Milk was not decomposed and none of the strains was haemolytic. A total of 16 compounds, including glucose, were utilized as sole carbon sources and seven compounds, including l-arabinose, were not utilized. Three out of the 17 strains were subjected to further studies. The micro-organisms had meso-diaminopimelic acid in their peptidoglycan and galactose, glucose, madurose and a trace of mannose in their whole-cell sugar patterns. The major phospholipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylinositol.Cellular fatty acids were C15 : 0 (35.7-23.1 %), C16 : 0 (5.9-2.4 %) C17 : 0 (62.9-39.5 %), C17 : 1 (24.4-0 %) and C18 : 0 (3-1.6 %). The predominant menaquinone was MK-8 (H4). The G+C content of the DNA was 69.6-71.8 mol%. Analysis of the 16S rRNA gene sequences showed that the strains clustered with the type strains of members of the family Dermatophilaceae. Based on the polyphasic taxonomic characterization carried out, all 17 strains are considered to belong to a novel species in a new genus, for which the name Tonsilliphilus suis gen. nov., sp. nov. is proposed. The type strain of the type species is W254(T) ( = ATCC 35846(T) = CCM 3774(T) = DSM 21880(T) = JCM 15727(T)).
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Pulmonary TCR ?? T cells induce the early inflammation of granuloma formation by a glycolipid trehalose 6,6-dimycolate (TDM) isolated from Mycobacterium tuberculosis.
Immunopharmacol Immunotoxicol
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We previously showed that formation of pulmonary granulomas in mice in response to a mycobacterial glycolipid, trehalose 6,6-dimycolate (TDM) is due to the action of TNF-? and not of IFN-?. However, the mechanisms of formation and maintenance of pulmonary granulomas are not yet clear. The purpose of the present study is to evaluate the mechanisms of granuloma formation by TDM at the early phase. Histological analysis showed that inflammatory cells infiltrated the murine pulmonary interstitium on day 2 after an intravenous injection with TDM as a w/o/w emulsion. Clear granuloma formation was observed on day 7 after the injection. The mRNA expression of IL-17, IFN-? and macrophage inflammatory protein 2 was found in lung mononuclear cells at the day after TDM injection. The major IL-17-producing cells were T-cell receptor (TCR) ?? T cells expressing V?6. In mice depleted of ?? T cells by treatment with anti-TCR ?? monoclonal antibody, the number of TDM-induced granuloma was decreased, but the size of granuloma was not affected. Our results suggest that the mycobacterial glycolipid TDM causes activation of IL-17-producing TCR ?? T cells and stimulates chemotaxis of inflammatory cells including neutrophils in to lung.
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The effect of passages during Japanese BCG vaccine production on genetic stability and protective efficacy.
Vaccine
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Many genetic differences have been found among currently available BCG vaccines. To avoid continued accumulation of phenotypic or genotypic changes in the strains, WHO and most national regulatory authorities request that the vaccine should not be prepared by more than 12 passages from the master seed lot. However, it has recently been reported that genetic changes occur even during the passage for vaccine production. In this study, the genetic stability of Japanese BCG vaccine production using currently available PCR methods and protective efficacy using a guinea-pig model during the passages were examined. The results showed that there were no significant differences between the seed lot, the product manufactured by normal procedures, and the 20th passage product. These results indicate that the maximum number of passages as currently required by WHO for BCG vaccine production is adequate for the Japanese vaccine, and that new genetic tools may help to examine the quality control of the BCG vaccine.
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