In order to research immunogenicity of the recombinant rVP2-IL-2 fusion protein, we obtained the rVP2-IL-2 fusion protein using Pichia pastoris expression system, and then evaluated its potential to induce immune responses in chicken. The effect was determined in the form of protective anti-IBDV VP2 titers, antibodies (IgG1 and IgG2a), lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and challenge experiment. Antibody titers and proliferation lymphocyte level suggested that the fusion protein could elicit specific humoral immune and cellular immune responses, antibody sub-type results indicated that the rVP2-IL-2 fusion protein induced secretion both of IgG1 and IgG2a. The seem result elicited from cytokines ELISA test, secretion of both of Th1 (gamma-IFN) and Th2 (IL-4) were induced by the rVP2-IL-2 fusion protein. Challenge experiment result shown that chicken immunized the rVP2-IL-2 fusion protein obtained 85% protection. These results confirm that the fusion protein enhances the protection against IBDV through both humoral and cell-mediated immunity, and thus could serve as a candidate for the development of IBDV subunit vaccine.
Animals ; Antibodies, Viral ; biosynthesis ; blood ; Chickens ; immunology ; Immunoglobulin G ; blood ; Interleukin-2 ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Vaccines, Subunit ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

OBJECTIVETo investigate the effects of glutamine enriched enteral feeding on immunoregulation in burn patients.

METHODSTwenty burn patients were randomly divided into enteral nutrition (EN) group and enteral immune nutrition (EIN) group, with 12 patients in each group. Patients in EN group received a standard enteral formula, while those in EIN group received an enteral formula enriched with glutamine after hospital admission. Nutritional support was continued for 10 days. Blood samples were obtained to determine plasma level of total protein (TP), albumin (ALB), prealbumin (PAB) and transferrin (TF) at 1, 4, 7, 10 post-burn days (PBD). At the same time the concentration of immunoglobulin (IgA, IgG and IgM) were determined, the percentage of CD3+, CD4+, CD8+ subpopulations of T lymphocytes, and the ratio of CD4+/CD8+ were determined by FCM.

RESULTS(1) There were no obvious difference of the plasma level of TP, ALB, TF, CD3+, IgM between the two groups at each time-point (P > 0.05). (2) The plasma PAB contents in EIN group were significant higher than that in EN group on 4 PBD [(90 +/- 14 vs 60 +/- 15) mg/L, P < 0.05], 7 PBD [(92 +/- 16 vs 64 +/- 13) mg/L, P < 0.05] and 10 PBD [(106 +/- 21 vs 72 +/-16) mg/L, P < 0.05]. (3) The percentage of CD4+ subpopulation and ratio of CD4+/CD8+ in EIN group were obviously higher than those in EN group on 7 PBD [CD4+ (55 +/- 5 vs 45 +/- 5)%, CD4+/CD8+ (1.92 +/- 0.31 vs 1.53 +/- 0.27)%, P < 0.05] and 10 PBD [CD4+ (56 +/- 5 vs 49 +/- 5)%, CD4+/CD8+ (2.36 +/- 0.36 vs 1.72 +/- 0.42), P < 0.05]. (4) The concentration of IgA and IgG in EIN group were markedly higher than that in EN group on 7 PBD [IgA (2.8 +/- 0.6 vs 2.2 +/- 0.5) g/L, IgG (12.1 +/- 1.3 vs 9.8 +/- 1.2) g/L, P < 0.05] and 10 PBD [IgA (3.1 +/- 0.6 vs 2.5 +/- 0.5) g/L, IgG (14.2 +/- 1.3 vs 10.4 +/- 1.3) g/L, P < 0.05].

CONCLUSIONThese findings suggest that glutamine enriched enteral feeding can improve nutritional status by promoting the synthesis of IgA, IgG, and increasing the PAB concentration, and corrected immunologic dysfunction in burn patients.

Adolescent ; Adult ; Burns ; blood ; immunology ; therapy ; Enteral Nutrition ; Female ; Glutamine ; therapeutic use ; Humans ; Immunoglobulin A ; biosynthesis ; Immunoglobulin G ; biosynthesis ; Male ; Middle Aged ; Prealbumin ; metabolism ; T-Lymphocyte Subsets ; immunology ; Young Adult

The experiment was conducted to prepare chitosan nanoparticles (CNP), to entrap VRMIL-2 with CNP, the eukaryotic VR1020 expression plasmid containing murine IL-2 gene (mlL-2), and to investigate the expression in vivo and the regulatory effect of mIL-2 on immune-response and immuno-protection in mice inoculated muscularly with CNP entrapped VMIL-2 at 21 days old. The results showed that IgG, IgM and IgA contents increased to different degrees in the sera from the inoculated mice, which were remarkably higher than those of the controls inoculated VR1020 packed with CNP (P<0.05); so were the IL-2, IL-4 and IL-6 contents in the sera of the immunized mice. The number of white blood cells and lymphocytes significantly increased respectively in the vaccinated mice, compared with those of controls. These mice were orally challenged with virulent E. coli 35 days post-inoculation, and all the immune responses were significantly higher than those of the control except the number of neutrophils. The mice inoculated with VRMIL-2 survived healthily, while the mice of control group were ill with the evident lesions. Although there are no remarkable differences between the cellular and humoral immune indexes of mice inoculated with CNP-VRMIL-2 and nude VRMIL-2 (P>0.05), the dosage of CNP-VRMIL-2 is only one fifth of the VRMIL-2. These indicated that entrapment of mIL-2 gene with chitosan nanoparticles could remarkably enhance the expression of mIL-2 in vivo, and significantly raise the levels of cellular and humoral immune, and increase the resistance of mice against E. coli infection. The results suggested that chitosan nanoparticles and IL-2 gene could be used as an effective immunoenhancer to increase the immunity of animals against infection.
Adjuvants, Immunologic ; pharmacology ; Animals ; Chitosan ; pharmacology ; Escherichia coli Infections ; immunology ; prevention & control ; Female ; Immunoglobulin G ; blood ; Interleukin-2 ; biosynthesis ; genetics ; pharmacology ; Mice ; Nanostructures

BACKGROUNDTo find a rapid and sensitive method for early diagnosis of nasopharyngeal carcinoma by using EBV TK kinase.

METHODSProkaryotic expression plasmid pRSETTK was constructed. EBV TK kinase was highly expressed in E.coil BL21 (DE3). The authors identified specificity of TK kinase by Western blot, then used purified TK kinase in ELISA to detect the IgG antibody in the serum of NPC patients.

RESULTSSpecific IgG antibody against TK kinase was found in the serum of NPC patients. The specificity and sensitivity of TK kinase were both 100% in Western blot and were 98.0% and 93.4% respectively in ELISA.

CONCLUSIONSThe EBV TK kinase showed high specificity and sensitivity in ELISA, therefore it can be used for early diagnosis of NPC

Antibodies, Viral ; blood ; Enzyme-Linked Immunosorbent Assay ; methods ; Herpesvirus 4, Human ; enzymology ; Humans ; Immunoglobulin G ; blood ; Nasopharyngeal Neoplasms ; diagnosis ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Sensitivity and Specificity ; Thymidine Kinase ; biosynthesis ; isolation & purification

We observed the time gap between oocyst shedding and antibody responses in mice (3-week-old C57BL/6J females) infected with Cryptosporidium parvum. Oocyst shedding was verified by modified acid-fast staining. The individually collected mouse sera were assessed for C. parvum IgM and IgG antibodies by enzyme-linked immunosorbent assay from 5 to 25 weeks after infection. The results showed that C. parvum oocysts were shed from day 5 to 51 post-infection (PI). The IgM antibody titers to C. parvum peaked at week 5 PI, whereas the IgG antibody titers achieved maximum levels at week 25 PI. The results revealed that IgM responses to C. parvum infection occurred during the early stage of infection and overlapped with the oocyst shedding period, whereas IgG responses occurred during the late stage and was not correlated with oocyst shedding. Hence, IgM antibody detection may prove helpful for the diagnosis of acute cryptosporidiosis, and IgG antibody detection may prove effective for the detection of past infection and endemicity.
Animals ; Antibodies, Protozoan/*biosynthesis/blood ; Cryptosporidiosis/*immunology ; Cryptosporidium parvum/*immunology/isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Feces/parasitology ; Female ; Immunocompromised Host ; Immunoglobulin G/biosynthesis/blood ; Immunoglobulin M/biosynthesis/blood ; Mice ; Mice, Inbred C57BL ; Oocysts/immunology ; Specific Pathogen-Free Organisms ; Time Factors

OBJECTIVETo express and purify Hap protein of nontypeable Haemophilus influenzae (NTHi) in prokaryotic system, and study its immunogenicity and adhesive activity.

METHODHap protein was expressed in E.coli BL21 with pET32a (+)-Hap and purified by affinity chromatography. The adhesive activity of the recombinant Hap protein was observed in competitive adhesion assay using scanning electron microscope and bacterial counting. BALB/C mice were immunized intranasally with the purified recombinant Hap protein and cholera toxin B subunit (CT-B), and anti-Hap IgA and IgG were detected by enzyme-linked immunosorbent assay.

RESULTSSDS-PAGE analysis showed a single band of the target protein, whose purity reached 85% according to the result of Gel analysis software. The concentration of the protein was 3.2 g/L after ultrafiltration and condensation. Competitive adhesion assay showed that compared with control group, the recombinant Hap protein significantly inhibited the adhesion of NTHi to ECM (P<0.01). Compared with Hap immunization alone, immunization with Hap combined with CT-B resulted in significantly higher titers of anti-Hap IgG and IgA in mice (P<0.05).

CONCLUSIONHighly purified recombinant Hap protein has been obtained in a prokaryotic system and shows good immunogenic and adhesive activities. These results will establish the basis for further study of NTHi vaccine.

Adhesiveness ; Animals ; Bacterial Outer Membrane Proteins ; biosynthesis ; genetics ; immunology ; Cholera Toxin ; immunology ; Escherichia coli ; genetics ; metabolism ; Haemophilus Infections ; prevention & control ; Haemophilus influenzae ; metabolism ; Immunization ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Serine Endopeptidases ; biosynthesis ; genetics ; immunology

BACKGROUND: Without appropriate culture systems for hepatitis E virus (HEV), sufficient natural viral proteins are difficult to generate for use in serological tests. Therefore, it is important to produce large amounts of HEV recombinant proteins in an economical way. The present study developed ELISAs using 2 truncated forms of the HEV open reading frame (ORF) 2 protein in order to detect anti-HEV IgG in serum samples. METHODS: Two truncated forms of the ORF2 protein were expressed in Escherichia coli and were purified by Ni2+-chelate-affinity chromatography (Qiagen, Germany). Two ELISAs were developed using these proteins and were compared with DIA.PRO HEV IgG ELISA kit (DIA.PRO. Italy) in 220 serum samples. RESULTS: High yields of the target proteins were obtained through codon optimization. The concentration and purity of the proteins were improved with Amicon filters (EMD Millipore, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis of the resultant proteins showed a protein band of approximately 60 kDa corresponding to ORF2.1 (amino acids 112-660) and a protein band of approximately 55 kDa corresponding to ORF2.2 (amino acids 112-607). Positive agreement, negative agreement, and concordance of the 2 in-house ELISAs compared with DIA.PRO HEV IgG ELISA kit were 87%, 99.5%, and 98.1%, respectively (kappa=0.899, P=0.625). CONCLUSIONS: The newly developed ELISAs are useful for detecting anti-HEV IgG in serum samples and are highly concordant with DIA.PRO HEV IgG ELISA kit.
Amino Acid Sequence ; Antibodies/*blood ; *Enzyme-Linked Immunosorbent Assay ; Escherichia coli/metabolism ; Hepatitis E virus/*metabolism ; Humans ; Immunoglobulin G/*blood ; Molecular Sequence Data ; Recombinant Proteins/biosynthesis/immunology/isolation & purification ; Sequence Alignment ; Viral Proteins/chemistry/*immunology/metabolism

OBJECTIVETo characterize the immune response induced by SAG1 encoding plasmid combined with IL-2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis.

METHODSMice were co-injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL-2 expression vector at a dose of 100 microg. Booster immunizations were employed 2 more times at 3-week interval. As controls, mice were inoculated with PBS or empty plasmid pcDNA3. Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN-gamma, as well as IL-4. To detect the integration and dissemination of DNA in the injected mice, PCR and in situ hybridization were performed. All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally.

RESULTSSignificant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid. With respect to the IgG isotype, co-inoculation of IL-2 expression plasmid enhanced the level of IgG2a and the production of IFN-gamma. Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival.

CONCLUSIONHumoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co-inoculation with IL-2 expression plasmid. The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T. gondii infection warrants further investigation.

Animals ; Antibodies, Protozoan ; blood ; Antigens, Protozoan ; Cytokines ; biosynthesis ; Female ; Immunization ; Immunoglobulin G ; blood ; classification ; Interleukin-2 ; genetics ; Mice ; Protozoan Proteins ; genetics ; Protozoan Vaccines ; immunology ; Toxoplasma ; immunology ; Toxoplasmosis, Animal ; prevention & control ; Vaccines, DNA ; immunology

BACKGROUNDDNA immunization is a promising novel type of immunotherapy against allergy. An estimated 79.2% patients with asthma, wheezing and/or rhinitis suffer from Dermatophagoides pteronyssinus group 2 (Der p 2) allegen. The aim of the present study was to determine whether DNA vaccine encoding Der p 2 could generate immunologic protection in recombinant Der p 2 (rDer p 2) allergen-induced allergic airway inflammation mice model and to understand the role of DNA vaccination in specific-allergen immunotherapy for asthma.

METHODSAfter DNA vaccination, BALB/c mice were sensitized by intraperitoneal injection (i.p) and challenged by intranasal instillation of rDer p 2. The lung tissues were assessed using hematoxylin and eosin. Mucus-producing goblet cells were identifed using periodic acid-Schiff (PAS)/alcian blue. The total cell number and composition of bronchoalveolar lavage samples were determined. The levels of the cytokines IL-4 and IFN-gamma, as well as IgE and IgG2a in the serum were determined by enzyme-linked immunosorbent assay. Allergen-specific IL-4 and IFN-gamma production by spleen cells were also measured by enzyme-linked immunosorbent assay. Expression of signal transducer and activator of transcription 6 (STAT6) in splenocytes were determined by Western blot.

RESULTSDNA vaccine encoding Der p 2 allergen inhibited extensive infiltration of inflammatory cells and production of mucin induced by allergen. The influx of eosinophils into the lung interstitium was significantly reduced after administration of DNA vaccine. Significant reductions of IL-4 and increase in levels of IFN-gamma in bronchoalveolar lavage fluid were observed. The allergen-specific IgE was markedly decreased in mice receiving DNA vaccination. Allergen could induce higher IFN-gamma, weaker IL-4 in cultured spleen cells from mice receiving DNA vaccine. DNA vaccination inhibited STAT6 expression of spleen cells induced by allergen.

CONCLUSIONThese results indicated that DNA vaccine encoding Der p 2 allergen generates immunologic protection in recombinant Der p 2 allergen-induced allergic airway inflammation mice model with regulating the immune response towards a Th1-type reaction.

Animals ; Antigens, Dermatophagoides ; genetics ; immunology ; Arthropod Proteins ; Asthma ; immunology ; therapy ; Eosinophilia ; prevention & control ; Humans ; Immunoglobulin E ; blood ; Immunoglobulin G ; blood ; Interferon-gamma ; biosynthesis ; Interleukin-4 ; biosynthesis ; Mice ; Mice, Inbred BALB C ; STAT6 Transcription Factor ; Th1 Cells ; immunology ; Trans-Activators ; analysis ; Vaccination ; Vaccines, DNA ; immunology

Food allergies affect about 4% of the Korean population, and buckwheat allergy is one of the most severe food allergies in Korea. The purpose of the present study was to develop a murine model of IgE-mediated buckwheat hypersensitivity induced by intragastric sensitization. Young female C3H/HeJ mice were sensitized and challenged intragastricly with fresh buckwheat flour (1, 5, 25 mg/dose of proteins) mixed in cholera toxin, followed by intragastric challenge. Anaphylactic reactions, antigen-specific antibodies, splenocytes proliferation assays and cytokine productions were evaluated. Oral buckwheat challenges of sensitized mice provoked anaphylactic reactions such as severe scratch, perioral/periorbital swellings, or decreased activity. Reactions were associated with elevated levels of buckwheatspecific IgE antibodies. Splenocytes from buckwheat allergic mice exhibited significantly greater proliferative responses to buckwheat than non-allergic mice. Buckwheat-stimulated IL-4, IL-5, and INF-gamma productions were associated with elevated levels of buckwheat-specific IgE in sensitized mice. In this model, 1 mg and 5 mg dose of sensitization produced almost the same degree of Th2-directed immune response, however, a 25 mg dose showed blunted antibody responses. In conclusion, we developed IgE-mediated buckwheat allergy by intragastric sensitization and challenge, and this model could provide a good tool for future studies.
Anaphylaxis/blood/immunology ; Animals ; Cell Proliferation/drug effects ; Comparative Study ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Enzyme-Linked Immunosorbent Assay ; Fagopyrum/*immunology ; Female ; *Flour ; Food Hypersensitivity/blood/*immunology ; Immunoglobulin E/blood/immunology ; Immunoglobulin G/blood/immunology ; Interferon Type II/biosynthesis ; Interleukin-4/biosynthesis ; Interleukin-5/biosynthesis ; Mice ; Mice, Inbred C3H ; Plant Extracts/administration & dosage/immunology ; Research Support, Non-U.S. Gov't ; Spleen/cytology/drug effects/metabolism ; Stomach/drug effects/*immunology ; T-Lymphocytes/cytology/drug effects/metabolism ; Time Factors