• IMMUNE NETWORK
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• 제11권1호
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• pp.79-94
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• 2011

Background: Dendritic cell (DC)-based vaccines are currently being evaluated as a novel strategy for tumor vaccination and immunotherapy. However, inducing long-term regression in established tumor-implanted mice is difficult. Here, we show that deoxypohophyllotoxin (DPT) induces maturation and activation of bone marrow-derived DCs via Toll-like receptor (TLR) 4 activation of MAPK and NF-${\kappa}B$. Methods: The phenotypic and functional maturation of DPT-treated DCs was assessed by flow cytometric analysis and cytokine production, respectively. DPT-treated DCs was also used for mixed leukocyte reaction to evaluate T cell-priming capacity and for tumor regression against melanoma. Results: DPT promoted the activation of $CD8^+$ T cells and the Th1 immune response by inducing IL-12 production in DCs. In a B16F10 melanoma-implanted mouse model, we demonstrated that DPT-treated DCs (DPT-DCs) enhance immune priming and regression of an established tumor in vivo. Furthermore, migration of DPT-DCs to the draining lymph nodes was induced via CCR7 upregulation. Mice that received DPT-DCs displayed enhanced antitumor therapeutic efficacy, which was associated with increased IFN-${\gamma}$ production and induction of cytotoxic T lymphocyte activity. Conclusion: These findings strongly suggest that the adjuvant effect of DPT in DC vaccination is associated with the polarization of T effector cells toward a Th1 phenotype and provides a potential therapeutic antitumor immunity.

• IMMUNE NETWORK
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• 제15권2호
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• pp.83-90
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• 2015

Evaluation and screening of vaccines against tuberculosis depends on development of proper cost effective disease models along with identification of different immune markers that can be used as surrogate endpoints of protection in preclinical and clinical studies. The objective of the present study was therefore evaluation of subcutaneous model of M.tuberculosis infection along with investigation of different immune biomarkers of tuberculosis infection in BALB/c mice. Groups of mice were infected subcutaneously with two different doses : high ($2{\times}10^6CFU$) and low doses ($2{\times}10^2CFU$) of M.tuberculosis and immune markers including humoral and cellular markers were evaluated 30 days post M.tuberculosis infections. Based on results, we found that high dose of subcutaneous infection produced chronic disease with significant (p<0.001) production of immune markers of infection like $IFN{\gamma}$, heat shock antigens (65, 71) and antibody titres against panel of M.tuberculosis antigens (ESAT-6, CFP-10, Ag85B, 45kDa, GroES, Hsp-16) all of which correlated with high bacterial burden in lungs and spleen. To conclude high dose of subcutaneous infection produces chronic TB infection in mice and can be used as convenient alternative to aerosol models in resource limited settings. Moreover assessment of immune markers namely mycobacterial antigens and antibodies can provide us valuable insights on modulation of immune response post infection. However further investigations along with optimization of study protocols are needed to justify the outcome of present study and establish such markers as surrogate endpoints of vaccine protection in preclinical and clinical studies in future.

• Asian-Australasian Journal of Animal Sciences
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• 제19권4호
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• pp.500-506
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• 2006

An experiment was conducted with twenty crossbred male calves (7-15 days old) divided into 4 different experimental groups on the basis of body weights to study the effect of vitamin E supplementation on the serum ${\alpha}$-tocopherol concentration and immune response of the calves. All the calves were fed on milk and calf starter up to 13 weeks and afterwards, they were fed on concentrate mixture and oat hay up to 32 weeks of age. In addition, the calves in groups I, II, III and IV were supplemented with 0, 125, 250 and 500 IU feed grade DL-${\alpha}$-tocopheryl acetate, respectively. Blood samples were collected at 0 day and subsequently at 1, 2, 3, 4, 6 and 8 months of age to monitor the serum ${\alpha}$-tocopherol concentration in crossbred calves. After 24 weeks of experimental feeding, 4 animals from each group were intramuscularly inoculated with single dose (3 ml) of Haemorrhagic septiceaemia (Pasteurella multocida P52 strain) oil adjuvant vaccine. The cumulative group mean serum ${\alpha}$-tocopherol concentration (${\mu}g/100ml$) was 88.12, 210.11, 235.21 and 294.02 in-groups I, II, III and IV, respectively and differed significantly (p<0.001) among the four groups. Lymphocyte stimulation indices (LSI) did not differ among the groups significantly. The pooled mean ELISA antibody titer against Pasteurella multocida (P52 strain) was 788.02, 926.85, 1,214.00 and 1,109.51 for group I, II, III and IV, respectively, which indicated higher antibody titer in groups supplemented with vitamin E as compared to the control group. It may be concluded that vitamin E supplementation increased the ${\alpha}$-tocopherol concentration in serum and dietary supplementation of vitamin E at higher level has a humoral immune enhancing effect against killed bacterial antigen.

• Journal of Periodontal and Implant Science
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• 제38권4호
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• pp.565-578
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• 2008

Purpose: Porphyromonas gingivalis(P. gingivalis) heat shock protein (HSP)60 may play a role in the immunopathogenesis of periodontitis as well as atherosclerosis by modulating autoimmune reaction due to its high level of sequence homology between bacteria and human counterpart. The purpose of this study was to identify immunodomiant epitope of P. gingivalis HSP60 that is reactive exclusively to the homologous bacteria without reacting with human HSP. Materials and methods: The present study was performed to identify the peptide specifically recognized by anti-P. gingivalis HSP60 monoclonal antibodies mono-reactive to P. gingivalis HSP60. Results: Four different hybridomas were cloned producing monoclonal IgG antibodies exclusively to P. gingivalis HSP60. Thirty seven synthetic peptides (20-mer with 5-amino acid overlapping) were synthesized. All of these peptide were subject to SDS-PAGE for immunblot analysis. One peptide (TVPGGGTTYIRAIAALEGLK) and the other peptide (TLVVNRLRGSLKICAVKAPG) were recognized by all and one of the four monoclonal antibodies, respectively, that reacted solely with P. gingivalis HSP60. Immunohistochemistry to identify the localization of the HSP60 in the diseased gingival tissues revealed that all of the four monoclonal antibodies were highly reacted with the diseased gingival tissue than normal gingival tissue. Conclusion: The P. gingivalis HSP60 peptides (TVPGGGTTYIRAIAALEGLK and TLVVNRLRGSLKICAVKAPG, respectively) are positively involved in the immunopathologic process of periodontal disease. The peptide may potentially be developed as vaccine candidates. Further investigations are under way to identify more clones producing monoclonal antibodies reactive to P. gingivalis HSP and to other periodontopathogenic bacteria as well, while maintaining specificities to human counterpart.

• Journal of Dairy Science and Biotechnology
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• 제24권1호
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• pp.53-63
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• 2006

Nano and micro capsulation (NM capsulation) involve the incorporation for nanofood materials, enzymes, cells or other materials in small capsules. Since Kim D. M. (2001) showed that a new type of food called firstly the name of nanofood, which means nanotechnology for food, and the encapsulated materials can be protected from moisture, heat or other extreme conditions, thus enhancing their stability and maintaining viability applications for this nanofood technique have increased in the food. NM capsules for nanofood is also utilized to mask odours or tastes. Various techniques are employed to form the capsules, including spray drying, spray chilling or spray cooling, extrusion coating, fluidized bed coating, liposome entrapment, coacervation, inclusion complexation, centrifugal extrusion and rotational suspension separation. Each of these techniques is discussed in this review. A wide variety of nanofood is NM capsulated - flavouring agents, acids, bases, artificial sweeteners, colourants, preservatives, leavening agents, antioxidants, agents with undesirable flavours, odours and nutrients, among others. The use of NM capsulation for sweeteners such as aspartame and flavors in chewing gum is well known. Fats, starches, dextrins, alginates, protein and lipid materials can be employed as encapsulating materials. Various methods exist to release the ingredients from the capsules. Release can be site-specific, stage-specific or signaled by changes in pH, temperature, irradiation or osmotic shock. NM capsulation for the nanofood, the most common method is by solvent-activated release. The addition of water to dry beverages or cake mixes is an example. Liposomes have been applied in cheese-making, and its use in the preparation of nanofood emulsions such as spreads, margarine and mayonnaise is a developing area. Most recent developments include the NM capsulation for nanofood in the areas of controlled release, carrier materials, preparation methods and sweetener immobilization. New markets are being developed and current research is underway to reduce the high production costs and lack of food-grade materials.

• Journal of Microbiology and Biotechnology
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• 제12권3호
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• pp.463-469
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• 2002

Rotaviruses are the world-wide leading causative agents of severe dehydrating gastroenteritis in young children and animals. The outer capsid glycoprotein VP7 and inner capsid glycoprotein VP6 of rotaviruses are highly antigenic and immunogenic. An SFV-based expression system has recently emerged as a useful tool for heterologous protein production in mammalian cells, exhibiting a much more efficient performance compared to other gene expression systems. Accordingly, the current study adopted an SFV-based expression system to express the VP7 of a group A human rotavirus from a Korean isolate, and the VP6 of a group B bovine rotavirus from a Korean isolate, in mammalian cells. The genes of the VP6 and VP7 were inserted into the SFV expression vector pSFV-1. The RNA was transcribed in vitro from pSFV-VP6 and pSFV-VP7 using SP6 polymerase. Each RNA was then electroporated into BHK-21 cells along with pSFV-helper RNA containing the structural protein gene without the packaging signal. The expression of VP6 and VP7 in the cytoplasm was then detected by immunocytochemistry. The recombinant virus was harvested by ultracentrifugation and examined under electron microscopy. After infecting BHK-21 cells with the defective viruses, the expressed proteins were separated by SDS-PAGE and analyzed by a Western blot. The results indicate that an SFV-based expression system fur the VP6 and VP7 of rotaviruses is an efficient tool for developing a diagnostic kit and/or preventive vaccine.

• Asian-Australasian Journal of Animal Sciences
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• 제21권10호
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• pp.1495-1500
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• 2008

In an attempt to develop a probiotic formulation for poultry feed, a number of lactic acid bacteria (LAB) were isolated from chicken intestinal specimens and a series of in vitro experiments were performed to evaluate their efficacy as a potential probiotic candidate. A total of 650 LAB strains were isolated and screened for their antagonistic potential against each other. Among all the isolates only three isolates (TMU121, 094 and 457) demonstrated a wide spectrum of inhibition and were thus selected for detailed investigations. All three selected isolates were able to inhibit the growth of E. coli and Salmonella species, although to variable extent. The nature of the inhibitory substance produced by the isolates TMU121 and 094 appeared to be associated with bacteriocin, as their activity was completely lost after treatment with proteolytic enzymes, while pH neutralization and catalase enzyme had no effect on the residual activity. In contrast, isolate TMU457 was able to resist the effect of proteolytic enzymes while pH neutralization completely destroyed its activity. Attempts were made to study the acid, bile tolerance and cell surface hydrophobicity of these isolates. TMU121 showed high bile salt tolerance (0.3%) and high cell surface hydrophobicity compared to the other two strains studied, while TMU094 appeared the most pH resistant strain. Based on these results, the three selected LAB isolates were considered as potential ingredients for a chicken probiotic feed formulation and were identified to species level based on their carbohydrate fermentation pattern by using API 50CH test kits. The three strains were identified as Lactobacillus fermentum TMU121, Lactobacillus rhamnosus TMU094, and Pediococcus pentosaceous TMU457.

• Asian-Australasian Journal of Animal Sciences
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• 제28권9호
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• pp.1354-1361
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• 2015

The complement system is a part of the natural immune regulation mechanism against invading pathogens. Complement activation from three different pathways (classical, lectin, and alternative) leads to the formation of C5-convertase, an enzyme for cleavage of C5 into C5a and C5b, followed by C6, C7, C8, and C9 in membrane attack complex. The C9 is the last complement component of the terminal lytic pathway, which plays an important role in lysis of the target cells depending on its self-polymerization to form transmembrane channels. To address the association of C9 with traits related to disease resistance, the complete porcine C9 cDNA was comparatively sequenced to detect single nucleotide polymorphisms (SNPs) in pigs of the breeds Hampshire (HS), Duroc (DU), Berlin miniature pig (BMP), German Landrace (LR), Pietrain (PIE), and Muong Khuong (Vietnamese potbelly pig). Genotyping was performed in 417 $F_2$ animals of a resource population (DUMI: $DU{\times}BMP$) that were vaccinated with Mycoplasma hyopneumoniae, Aujeszky diseases virus and porcine respiratory and reproductive syndrome virus at 6, 14 and 16 weeks of age, respectively. Two SNPs were detected within the third exon. One of them has an amino acid substitution. The European porcine breeds (LR and PIE) show higher allele frequency of these SNPs than Vietnamese porcine breed (MK). Association of the substitution SNP with hemolytic complement activity indicated statistically significant differences between genotypes in the classical pathway but not in the alternative pathway. The interactions between eight time points of measurement of complement activity before and after vaccinations and genotypes were significantly different. The difference in hemolytic complement activity in the both pathways depends on genotype, kind of vaccine, age and the interaction to the other complement components. These results promote the porcine C9 (pC9) as a candidate gene to improve general animal health in the future.

• 대한약침학회지
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• 제18권4호
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• pp.7-11
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• 2015

Objectives: Bee venom (BV) is a complex mixture of proteins and contains proteins such as phospholipase and melittin, which have an effect on blood clotting and blood clots. The mechanism of action of honey bee venom (HBV, Apis mellifera) on human plasma proteins and its anti-thrombotic effect were studied. The purpose of this study was to investigate the anti-coagulation effect of BV and its effects on blood coagulation and purification. Methods: Crude venom obtained from Apis mellifera was selected. The anti-coagulation factor of the crude venom from this species was purified by using gel filtration chromatography (sephadex G-50), and the molecular weights of the anti-coagulants in this venom estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blood samples were obtained from 10 rabbits, and the prothrombin time (PT) and the partial thromboplastin time (PTT) tests were conducted. The approximate lethal dose (LD) values of BV were determined. Results: Crude BV increased the blood clotting time. For BV concentrations from 1 to 4 mg/mL, clotting was not observed even at more than 300 seconds, standard deviations $(SDs)={\pm}0.71$; however, clotting was observed in the control group 13.8 s, $SDs={\pm}0.52$. Thus, BV can be considered as containing anti-coagulation factors. Crude BV is composed 4 protein bands with molecular weights of 3, 15, 20 and 41 kilodalton (kDa), respectively. The $LD_{50}$ of the crude BV was found to be $177.8{\mu}g/mouse$. Conclusion: BV contains anti-coagulation factors. The fraction extracted from the Iranian bees contains proteins that are similar to anti-coagulation proteins, such as phospholipase $A_2(PLA_2)$ and melittin, and that can increase the blood clotting times in vitro.

• IMMUNE NETWORK
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• 제5권3호
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• pp.172-178
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• 2005

Background: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. However, it is known that the induction of immune response to CEA is very difficult because CEA is a self-antigen expressed in fetal cells and weakly expressed in normal colorectal epithelial cells. To enhance anti-tumor immunity specific for CEA, recombinant CEA protein was modified using listeriolysin O (LLO) for endosomal lysis and trans activator of transcription (Tat) domain for transducing extracellular proteins into cytoplasm. Methods: After immunization using dendritic cells pulsed with Tat-CEA, both Tat-CEA and LLO, and both Tat-CEA and Tat-LLO, antibody titer to CEA and LLO, cytotoxic T lymphocyte activity and the frequency of IFN-${\gamma}$ producing T lymphocytes were measured. Results: Immunization using DC pulsed with both Tat-CEA and Tat-LLO protein showed the increasement of production of CEA-specific antibody in serum, cytotoxic T lymphocyte activity, the frequency of IFN-${\gamma}$ secreting T cells, compared with DC pulsed with both Tat-CEA and LLO. Furthermore the ratio of CD8+T cell to $CD4^+$ cell among CEA-specific T cells was increased in group pulsed with both Tat-CEA and Tat-LLO. Conclusion: These results suggested that DC vaccine using Tat-LLO could be used for the development of effective immunotherapy for the treatment of tumor.