With high throughput screening, novel therapeutic agents can be efficiently identified. Unfortunately, researchers only resort to in vitro cell viability assays for screening of anticancer drugs for retinoblastoma, the most common intraocular cancer in the childhood. Current available animal models of retinoblastoma require more than 2 weeks for tumour formation and the investigation of the efficacy of therapeutic agents. In this study, we established a novel orthotopic transplantation model of retinoblastoma in zebrafish as an in vivo animal model for screening of anticancer drugs.
We injected retinoblastoma cells into the vitreous cavity of zebrafish at 48 hours after fertilization. Eyeballs of zebrafish were scanned daily under the confocal laser microscope, and the tumor population was quantitatively analyzed by measuring the mean intensity of green fluorescent protein (GFP). Transplanted retinoblastoma cells were isolated to perform further analyses including Western blotting and reverse transcriptase-polymerase chain reaction to confirm that retinoblastoma cells maintained their characteristics as tumor cells even after transplantation and further isolation. To figure out the potential of this model for screening of anticancer drugs, zebrafish were cultured in Ringer’s solution containing carboplatin and melphalan after the injection of retinoblastoma cells.
The degree of the tumor population was dependent on the number of retinoblastoma cells injected and maintained stably for at least 4 days. Transplanted retinoblastoma cells maintain their proliferative potential and characteristics as retinoblastoma cells after isolation. Interestingly, systemic application of carboplatin and melphalan demonstrated significant reduction in the tumor population, which could be quantitatively analyzed by the estimation of the mean intensity of GFP.
This orthotopic retinoblastoma model in zebrafish is expected to be utilized for the screening of anticancer drugs for the treatment of retinoblastoma.
Anticancer drug screen; Orthotopic transplantation; Retinoblastoma; Zebrafish
Recently, it has been demonstrated that high cholesterol diet induced hypercholesterolemia and vascular lipid oxidation and accumulation in zebrafish larvae, suggesting that zebrafish is a new promising atherosclerosis model in addition to mouse models. However, up to date, there was no report regarding inflammatory cytokine expression during the lipid accumulation in zebrafish larva and adult fish. In this study, we first demonstrated the expression levels of IL-1β and TNF-α in high cholesterol diet (HCD)-fed zebrafish larvae, and found that although HCD induced vascular lipid accumulation, the cytokine expressions in the larvae were not changed by HCD. Furthermore, there was no significant difference in leukocyte accumulation in vessels between control and HCD fed group. But prolonged HCD induced IL-1β expression in spleen and liver compared to those of control zebrafish, and produced very early stage of fatty streak lesion in dorsal aorta of 19 week HCD-fed zebrafish. These results indicate that HCD induced hypercholesterolemia and atherosclerotic changes in zebrafish are very early stage, and suggest the necessity of the generation of mutant zebrafish having a disruption in a lipid metabolism-related gene leading to severe hypercholesterolemia and advanced atherosclerosis.
After skin grafting, to prevent hematoma or seroma collection at the graft site, a tie-over dressing has been commonly used. However, although the conventional tie-over dressing by suture is a useful method for securing a graft site, refixation is difficult when repeated tie-over dressing is needed. Therefore, we recommend a redoable tie-over dressing technique with multiple loops threads and connecting silk threads. After the raw surface of each of our cases was covered with a skin graft, multiple loop silk thread attached with nylon at the skin graft margin. We applied the ointment gauze and wet cotton/fluffy gauze over the skin graft, then fixed the dressing by connecting cross-counter multiple loop thread with connecting silk threads. When we opened the tie-over dressing by cutting the connecting silk threads, we repeated the tie-over dressing with the same method. The skin graft was taken successfully without hematoma or seroma collection or any other complications. In conclusion, we report a novel tie-over dressing enabling simple fixation of the dressing to maintain proper tension for wounds that require repetitive fixation. Further, with this reliable method, the skin grafts were well taken.
Skin transplantation; Reconstructive surgical procedure; Surgical fixation devices
KR-31543, (2S, 3R, 4S)-6-amino-4-[N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl) amino]-3,4-dihydro-2-dimethyoxymethyl-3-hydroxy-2-methyl-2H-1-benz opyran is a new neuroprotective agent for ischemia-reperfusion damage. It has also been reported that KR-31543 has protective effects on lipid peroxidation and H2O2-induced reactive oxygen species production. In this study, we investigated the anti-inflammatory and anti-atherogenic properties of KR-31543. We observed that KR-31543 treatment reduced the production of MCP-1, IL-8, and VCAM-1 in HUVECs, and of MCP-1 and IL-6 in THP-1 human monocytes. We also examined the effect of KR-31543 on monocytes migration in vitro. KR-31543 treatment effectively reduced the migration of THP-1 human monocytes to the HUVEC monolayer in a dose-dependent manner. We next examined the effects of this compound on atherogenesis in LDL receptor deficient (Ldlr-/-) mice. After 10 weeks of western diet, the formation of atherosclerotic lesion in aorta was reduced in the KR-31543-treated group compared to the control group. The accumulation of macrophages in lesion was also reduced in KR-31543 treated group. However, the plasma levels of total cholesterol, HDL, LDL, and triglyceride were not affected by KR-31543 treatment. Taken together, these results show that KR-31543 has anti-inflammatory properties on human monocytes and endothelial cells, and inhibits fatty streak lesion formation in mouse model of atherosclerosis, suggesting the potential of KR-31543 for the treatment for atherosclerosis.
atherosclerosis; endothelial cells; KR-31543; monocytes; transendothelial and transepithelial migration
Dendritic cells (DCs), critical antigen presenting cells for immune control, normally derive from bone marrow precursors distinct from monocytes. It is not yet established if the large reservoir of monocytes can develop into cells with critical features of DCs in vivo. We now show that fully differentiated Mo-DCs develop in mice and DC-SIGN/CD209a marks the cells. Mo-DCs are recruited from blood monocytes into lymph nodes by lipopolysaccharide and live or dead gram negative bacteria. Mobilization requires TLR4 and its CD14 coreceptor and Trif. When tested for antigen presenting function, Mo-DCs are as active as classical DCs, including cross presentation of proteins and live gram negative bacteria on MHC I in vivo. Fully differentiated Mo-DCs acquire DC morphology and localize to T cell areas via L-selectin and CCR7. Thus the blood monocyte reservoir becomes the dominant presenting cell in response to select microbes, yielding DC-SIGN+ cells with critical functions of DCs.
Mouse DC-SIGN CD209a is a type II transmembrane protein, one of a family of C-type lectin genes syntenic and homologous to human DC-SIGN. Current anti-mouse DC-SIGN monoclonal antibodies (MAbs) are unable to react with DC-SIGN in acetone fixed cells, limiting the chance to visualize DC-SIGN in tissue sections. We first produced rabbit polyclonal PAb-DSCYT14 against a 14-aa peptide in the cytosolic domain of mouse DC-SIGN, and it specifically detected DC-SIGN and not the related lectins, SIGN-R1 and SIGN-R3 expressed in transfected CHO cells. MAbs were generated by immunizing rats and DC-SIGN knockout mice with the extracellular region of mouse DC-SIGN.. Five rat IgG2a or IgM MAbs, named BMD10, 11, 24, 25, and 30, were selected and each MAb specifically detected DC-SIGN by FACS and Western blots, although BMD25 was cross-reactive to SIGN-R1. Two mouse IgG2c MAbs MMD2 and MMD3 interestingly bound mouse DC-SIGN but at 10 fold higher levels than the rat MAbs. When the binding epitopes of the new BMD and two other commercial rat anti-DC-SIGN MAbs, 5H10 and LWC06, were examined by competition assays, the epitopes of BMD11, 24, and LWC06 were identical or closely overlapping while BMD10, 30, and 5H10 were shown to bind different epitopes. MMD2 and MMD3 epitopes were on a 3rd noncompeting region of mouse DC-SIGN. DC-SIGN expressed on the cell surface was sensitive to collagenase treatment, as monitored by polyclonal and MAb. These new reagents should be helpful to probe the biology of DC-SIGN in vivo.
Monoclonal Antibody; Polyclonal Antibody; DC-SIGN; CD209a; Dendritic Cells
A variety of benzylidenethiazole analogs have been demonstrated to inhibit 5-lipoxygenase (5-LOX). Here we report the anti-atherogenic potential of 5-(4-hydroxy-2,3,5-trimethylbenzylidene) thiazolidin-2,4-dione (HMB-TZD), a benzylidenethiazole analog, and its potential mechanism of action in LDL receptor-deficient (Ldlr-/-) mice. HMB-TZD Treatment reduced leukotriene B4 (LTB4) production significantly in RAW264.7 macrophages and SVEC4-10 endothelial cells. Macrophages or endothelial cells pre-incubated with HMB-TZD for 2 h and then stimulated with lipopolysaccharide or tumor necrosis factor-alpha (TNF-α) displayed reduced cytokine production. Also, HMB-TZD reduced cell migration and adhesion in accordance with decreased proinflammatory molecule production in vitro and ex vivo. HMB-TZD treatment of 8-week-old male Ldlr-/- mice resulted in significantly reduced atherosclerotic lesions without a change to plasma lipid profiles. Moreover, aortic expression of pro-atherogenic molecules involved in the recruitment of monocytes to the aortic wall, including TNF-α , MCP-1, and VCAM-1, was downregulated. HMB-TZD also reduced macrophage infiltration into atherosclerotic lesions. In conclusion, HMB-TZD ameliorates atherosclerotic lesion formation possibly by reducing the expression of proinflammatory molecules and monocyte/macrophage recruitment to the lesion. These results suggest that HMB-TZD, and benzylidenethiazole analogs in general, may have therapeutic potential as treatments for atherosclerosis.
antioxidants; arachidonate 5-lipoxygenase; atherosclerosis; endothelial cells; macrophages
Previously, we prepared monoclonal antibodies (mAbs) by immunizing rats with the recombinant fusion proteins of mouse Langerin/CD207, which contained a flexible linker sequence from E. coli OmpF and a FLAG epitope. We found many of new rat mAbs were not reactive to mouse Langerin, and here we identify the epitopes of two of these IgG mAbs, L2 and L5, and assess their efficacy in various immunodetection methods. MAb L5 is a rat IgG mAb against the FLAG epitope, which detected both N-terminal and C-terminal FLAG tagged protein 2 to 8 times better than the conventional anti-FLAG mAb M2 by Western blot. For mAb L2, we found its epitope to be a 14 amino acid sequence SGFANELGPRLMGK which consisted of both sequences from the OmpF derived linker and mouse Langerin. This epitope sequence was named OLLAS (E. coli OmpF Linker and mouse Langerin fusion Sequence), and mAb L2 as mAb OLLA-2. When the OLLAS sequence was inserted into recombinant proteins at N-terminal, C-terminal, or internal sites, the OLLAS tag was detected by mAb OLLA-2 with very high sensitivity compared to other conventional epitope tags and anti-tag mAbs. MAb OLLA-2 recognized OLLAS tagged proteins with at least 100-fold more sensitivity than anti-FLAG M2 and anti-V5 mAbs in Western blot analyses. We also find the OLLAS epitope to be superior in immunoprecipitation and other immunodetection methods, such as fluorescent immunohistochemistry and flow cytometry. In the process, we successfully utilized the OLLAS epitope sequence as an internal linker for fusion between the engineered mAb and the antigen, and thus achieved improved immunodetection.
Monoclonal Antibody; Protein Tagging; FLAG Tag; OLLAS Tag
Vascular endothelial growth factor A (VEGFA), a critical mediator of tumor angiogenesis, is a well-characterized target of hypoxia-inducible factor 1 (HIF-1). Murine arrest-defective protein 1A (mARD1A225) acetylates HIF-1α, triggering its degradation, and thus may play a role in decreased expression of VEGFA.
We generated ApcMin/+/mARD1A225 transgenic mice and quantified growth of intestinal polyps. Human gastric MKN74 and murine melanoma B16F10 cells overexpressing mARD1A225 were injected into mice, and tumor growth and metastasis were measured. VEGFA expression and microvessel density in tumors were assessed using immunohistochemistry. To evaluate the role of mARD1A225 acetylation of Lys532 in HIF-1α, we injected B16F10-mARD1A225 cell lines stably expressing mutant HIF-1α/K532R into mice and measured metastasis. All statistical tests were two-sided, and P values less than .05 were considered statistically significant.
ApcMin/+/mARD1A225 transgenic mice (n = 25) had statistically significantly fewer intestinal polyps than ApcMin/+ mice (n = 21) (number of intestinal polyps per mouse: ApcMin/+ mice vs ApcMin/+/mARD1A225 transgenic mice, mean = 83.4 vs 38.0 polyps, difference = 45.4 polyps, 95% confidence interval [CI] = 41.8 to 48.6; P < .001). The growth and metastases of transplanted tumors were also statistically significantly reduced in mice injected with mARD1A225-overexpressing cells than in mice injected with control cells (P < .01). Moreover, overexpression of mARD1A225 decreased VEGFA expression and microvessel density in tumor xenografts (P < .04) and ApcMin/+ intestinal polyps (P = .001). Mutation of lysine 532 of HIF-1α in B16F10-mARD1A225 cells prevented HIF-1α degradation and inhibited the antimetastatic effect of mARD1A225 (P < .001).
mARD1A225 may be a novel upstream target that blocks VEGFA expression and tumor-related angiogenesis.
Presumptive dendritic cells (DCs) bearing the CD11c integrin and other markers have previously been identified in normal mouse and human aorta. We used CD11c promoter–enhanced yellow fluorescent protein (EYFP) transgenic mice to visualize aortic DCs and study their antigen-presenting capacity. Stellate EYFP+ cells were readily identified in the aorta and could be double labeled with antibodies to CD11c and antigen-presenting major histocompatability complex (MHC) II products. The DCs proved to be particularly abundant in the cardiac valves and aortic sinus. In all aortic locations, the CD11c+ cells localized to the subintimal space with occasional processes probing the vascular lumen. Aortic DCs expressed little CD40 but expressed low levels of CD1d, CD80, and CD86. In studies of antigen presentation, DCs selected on the basis of EYFP expression or binding of anti-CD11c antibody were as effective as DCs similarly selected from the spleen. In particular, the aortic DCs could cross-present two different protein antigens on MHC class I to CD8+ TCR transgenic T cells. In addition, after intravenous injection, aortic DCs could capture anti-CD11c antibody and cross-present ovalbumin to T cells. These results indicate that bona fide DCs are a constituent of the normal aorta and cardiac valves.
Langerin CD207 is a type II transmembrane protein. It is responsible for the formation of Birbeck granules, which are intracellular organelles within Langerhans cells, the dendritic cells of stratified squamous epithelia like the epidermis. Because current anti-CD207 antibodies have limitations, we prepared new monoclonals by immunizing rats with the extracellular region of mouse Langerin followed by a boost with enriched Langerhans cells (LCs). We secured a large panel of mAbs, most of which reacted with the carboxy terminal carbohydrate recognition domain. These mAbs could be used to immunoblot and immunoprecipitate mouse Langerin and to stain the cell surface and intracellular pools of CD207 by FACS analysis. Labeling of Birbeck granules was also achieved by immunoelectron microscopy. Anti-CD207 identified LCs in the epidermis and skin draining lymph nodes of BALB/c and C57BL/6 mice, but BALB/c mice had an additional Langerin+ population in spleen, thymus and mesenteric lymph node. This additional subset had higher levels of CD8 and CD205 than epidermal LCs, and also had a less mature phenotype, i.e., lower MHC II, CD40 and CD86. Subcutaneous injection of IgG but not IgM forms of these new anti-CD207 mAbs led to rapid and selective labeling of the Langerin+ cells in skin draining lymph nodes as well as spleen. The new IgG anti-CD207 mAbs should be useful for further research on LCs and dendritic cells including an evaluation of the consequences of antigen delivery within anti-CD207 mAbs in vivo.
Monoclonal Antibody; Langerhans Cells; Langerin; CD207; Dendritic Cells
Pulse-induced permeabilization of cellular membranes, generally referred to as electroporation (EP), has been used for years as a tool to increase macromolecule uptake in tissues, including nucleic acids, for gene therapeutic applications, and this technique has been shown to result in improved immunogenicity. In this study, we assessed the utility of EP as a tool to improve the efficacy of HB-110, a novel therapeutic DNA vaccine against chronic hepatitis B, now in phase 1 of clinical study in South Korea. The potency of HB-110 in mice was shown to be improved by EP. The rapid onset of antigen expression and higher magnitude of humoral and cellular responses in electric pulse-treated mice revealed that EP may enable a substantial reduction in the dosage of DNA vaccine required to elicit a response similar in magnitude to that achievable via conventional administration. This study also showed that EP-based vaccination at 4-week-intervals elicited a cellular immune response which was about two-fold higher than the response elicited by conventional vaccination at 2-week intervals. These results may provide a rationale to reduce the clinical dose and increase the interval between the doses in the multidose vaccination schedule. Electric pulsing also elicited a more balanced immune response against four antigens expressed by HB-110: S, preS, Core, and Pol.
electroporation; hepatitis B virus; vaccination; vaccines, DNA
We examined the ischemia-modified albumin (IMA) level during exercise in patients with coronary artery disease (CAD).
Forty patients with a history of chest pain underwent both symptom-limited treadmill exercise stress testing and coronary angiography within one week. During the treadmill tests, blood samples were obtained at baseline and 5 min after exercise to measure the serum IMA level.
Of the 40 patients, fourteen (35%, CAD group) had significant coronary artery stenosis, while the other 26 (65%, non-CAD group) did not. The baseline and post-exercise IMA levels in the two groups did not differ significantly (105.2±7.2 vs. 107.7±6.7 U/mL at baseline and 93.1±10.1 vs. 94.8±5.7 U/mL at post-exercise in the CAD and non-CAD groups, p=0.29 and 0.57, respectively). The changes in IMA after exercise did not differ either (-10.4±7.5 vs. -14.0±7.6 U/mL in the CAD and non-CAD groups, respectively, p=0.10). Similarly, the change in IMA between the exercise ECG test positive (TMT positive, n=9) and negative (TMT negative, n=20) groups did not differ (-14.63±5.19, vs -8.50±9.01 U/mL, p=0.15, in the TMT positive and negative groups, respectively).
Our results suggest that IMA has limitation in detecting myocardial ischemia during symptom-limited exercise stress tests.
Ischemia-modified albumin; Exercise ECG test; Ischemic heart disease