Effect of the Traditional Korean Immunomodulating Formulation,Gamguntang (GGT), on Experimental Thyroiditis Model

The crude herbal formulation, Gamgungtang (GGT), is an immunomodulator showing marked down-regulation of several experimental autoimmune diseases. In this study, its effect on different experimental models of thyroid disease was investigated. Although very effective at preventing thyroid infiltrates in mice immunized with mouse deglycosylated thyroglobulin and complete Freund's adjuvant and in spontaneous models of thyroiditis, it completely failed to modify experimental autoimmune thyroiditis (EAT) induced in mice immunized with mouse thyroglobulin and lipopolysaccharide. There was no significant shift in the observed isotypes of anti-mouse thyroglobulin antibodies and only anti-mouse thyroglobulin antibodies in the spontaneous model were completely down-modulated by the GGT. One surprising fact to emerge was that GGT-treated donor mice, although protected from thyroid lesions themselves, were still able to transfer EAT showing that they must have been effectively primed while being treated with GGT. It is possible that the drug down modulated EAT by interfering with the trafficking of primed effector cells.     

实验性自身免疫性甲状腺炎小鼠细胞免疫状态研究

作者检测了实验性自身免疫性甲状腺炎(EAT)小鼠的细胞免疫状态,结果表明:EAT小鼠脾细胞Thy-1.2和Lyt-2阳性T细胞数显著下降并伴随L3T4/Lyt-2阳性T细胞数比值升高;甲状腺球蛋白刺激的淋巴细胞增殖明显增强,但NK细胞活性无显著变化;脾细胞产生TNF和IL-1的水平明显高于正常小鼠。提示T细胞免疫功能紊乱和细胞因子的大量释放可能是引起此病的重要因素。     

Effect of the traditional Korean immunomodulating formulation, Gamguntang (GGT), on experimental thyroiditis model

The crude herbal formulation, Gamgungtang (GGT), is an immunomodulator showing marked down-regulation of several experimental autoimmune diseases. In this study, its effect on different experimental models of thyroid disease was investigated. Although very effective at preventing thyroid infiltrates in mice immunized with mouse deglycosylated thyroglobulin and complete Freund's adjuvant and in spontaneous models of thyroiditis, it completely failed to modify experimental autoimmune thyroiditis (EAT) induced in mice immunized with mouse thyroglobulin and lipopolysaccharide. There was no significant shift in the observed isotypes of anti-mouse thyroglobulin antibodies and only anti-mouse thyroglobulin antibodies in the spontaneous model were completely down-modulated by the GGT. One surprising fact to emerge was that GGT-treated donor mice, although protected from thyroid lesions themselves, were still able to transfer EAT showing that they must have been effectively primed while being treated with GGT. It is possible that the drug down modulated EAT by interfering with the trafficking of primed effector cells.     

Effects of Anti-I-A and Anti-I-E Monoclonal Antibodies on the Induction and Expression of Experimental Autoimmune Thyroiditis in Mice

Susceptibility to experimental autoimrnune thyroiditis (EAT) in mice is linked to the I-A subregion of the major histocompatibility complex (MHC). The present study was undertaken to assess the effectiveness of anti-I-A^k monoclonal antibody (MAb) 10–2.16 in preventing or arresting the development of EAT. Spleen cells from CBA/J or (CBA/J x Balb/c) Fl mice given 10–2.16 prior to sensitization with mouse thyro-globulin (MTg) and adjuvant could not transfer EAT to normal recipients, and cells from these mice did not proliferate in vitro to MTg. Donor CBA/J mice given 10–2.16 before immunization and recipients of cells from such mice produced little MTg-specific lgGl or IgG2b antibody but did produce nearly as much IgG2a as controls. The effects of in vivo treatment with 10–2. 16 appear to be due to elimination of Ia + cells rather than to modulation of Ia or induction of suppressor T cells. When 10–2.16 was added to in vitro cultures it also prevented the proliferation and activation of sensitized CBA/J or Fl effector cell precursors. Other mAb specific for MHC class II gene products, but not associated with disease susceptibility, expressed by CBA/J (I-E^k) or FI (I-A^d) mice (14-4-4S or MK-D6 respectively), also prevented in vivo sensitization, but did not block in vitro activation. Anti-I-A^k was also effective in preventing EAT if multiple injections of mAb were given to recipients of sensitized EAT effector cells. These studies indicate that Ia + cells are required for initial sensitization and activation of EAT effector cells and also contribute to the development of severe histopathologic lesions in the thyroid during the final effector stage of EAT.     

Induction of a cytotoxic T cell response by co-injection of a T helper peptide and a cytotoxic T lymphocyte peptide in incomplete Freund's adjuvant (IFA): Further enhancement by pre-injection of IFA alone

We have previously demonstrated that it is possible to induce a consistent and strong cytolytic T lymphocyte (CTL) response to synthetic peptides, corresponding to poorly immunogenic malaria CTL epitopes, by co-injecting them with peptides representing defined T helper (Th) epitopes in incomplete Freund's adjuvant (IFA). In this study we have tested different immunization protocols to improve further the elicitation of the CTL response. We show that the CTL response to a mixture of Th + CTL peptides administered in IFA was further enhanced by a previous injection of the Th epitope peptide in IFA. Moreover, we found that the response could be significantly augmented by a pre-injection of IFA alone. This enhancement was observed only if the Th epitope was also present in the second injection. The number of lymph node cells recovered was 2–3-fold higher in mice pre-injected with IFA, but the increase in specific CTL activity, expressed as lytic units per animal, by pre-injection of IFA was at least 10–20-fold. Thus, pre-injection of IFA clearly increases the magnitude of a subsequent CTL response.     

非洲爪蟾TGF-β5和小鼠TRP-2联合免疫诱导抗原特异性CTLs

目的 研究非洲爪蟾转化生长因子一β5(aFGF-β5)基因免疫是否能增强肿瘤治疗性DNA疫苗诱导抗原特异性细胞毒性T淋巴细胞的能力。方法 构建编码小鼠肿瘤抗原酪氨酸酶相关蛋白2(mTRP-2)的真核表达质粒，与aTGF-β5真核表达质粒联合免疫C56BL/6小鼠，采用标准^51Cr释放试验检测抗原特异性细胞毒性T淋巴细胞的诱导。结果 aTGF-β5和mTRP-2联合免疫可在体外诱导H-2K^b限制的mTRP-2aa180-188特异性细胞毒性T淋巴细胞产生。结论 aTGF-β5免疫能有效提高肿瘤治疗性DNA疫苗诱导特异性细胞毒性T淋巴细胞的能力。     

Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway

We have explored the role of an activation-induced T cell molecule, 4-1BB (CDw137), in the amplification of tumor immunity by retrovirus-mediated transduction of the 4-1BB ligand (4-1BBL) into tumor cells. Mice inoculated with P815 tumor cells expressing 4-1BBL developed a strong cytotoxic T lymphocyte (CTL) response and long-term immunity against wild-type tumor. The optimal effect of 4-1BBL in CTL stimulation required B7-CD28 interaction since blockade of this interaction by antibodies down-regulated the expression of 4-1BB on T cells and decreased CTL activity. Furthermore, co-expression of 4-1BBL and B7-1 in the poorly immunogenic AG104A sarcoma enhanced the induction of effector CTL and the rejection of the wild-type tumor while neither 4-1BBL nor B7-1 single transfectants were effective, suggesting a synergistic effect between the 4-1BB and the CD28 co-stimulatory pathways. Our results underscore the importance of the 4-1BB T cell stimulation pathway in the amplification of an antitumor immune response.     

Identification of a shared epitope recognized by melanoma-specific, HLA-A3-restricted cytotoxic T lymphocytes

We previously established a melanoma-reactive cytotoxic T lymphocyte (CTL) line that recognizes multiple epitopes in the context of HLA-A3. To increase the number of peptides available for use in a vaccine for the treatment of melanoma, we identified one of these epitopes, SQNFPGSQK, through a combination of epitope reconstitution experiments and mass spectrometry. The SQNFPGSQK peptide was also found to be associated with HLA-A3 on an additional melanoma tumor line, thus indicating that the peptide is not unique to the melanoma tumor line from which it was isolated and thus, unlikely to arise through a mutational event. Although the protein origin of SQNFPGSQK has yet to be established, the shared nature of this epitope and the fact that it elicits a natural immune response indicates that it warrants further study to determine its usefulness as a vaccine component for the treatment of melanoma. The peptide may also be useful as a research tool for evaluating spontaneous anti-tumor immune responses in patients with melanoma.     

Inhibition of tumor growth by peptide specific cytotoxic T lymphocytes in a three-dimensional collagen matrix

Tumor associated, MHC I restricted antigenic peptides have been identified in both human and mouse tumors. Cytotoxic T lymphocytes (CTL) which recognize these tumor associated antigenic peptides are potential anti-cancer effectors. The anti-tumor activity of CTL is usually measured in vitro by the ⁵¹Cr release assay and in mice by tumor growth inhibition which is the most direct assessment of anti-tumor effect. In clinical studies, an in vivo tumor growth inhibition assay is not an option and an in vitro assay which corroborates with in vivo tumor growth is needed to assess the long-term outcome of CTL activity. Here, a three-dimensional (3-D) collagen gel assay was developed to measure in vitro the inhibition of mouse mammary tumor growth by anti-tumor CTL. BALB/c mouse CTL were induced with peptide E474 SFAVATTAL which was expressed by mouse mammary tumor cells D2F2. To measure D2F2 tumor growth inhibition in vitro, a mixture of tumor cells and anti-E474 CTL in a 1 μl cell bolus was embedded in the collagen gel. Complete eradication of tumor growth was observed at E:T ratio of or greater than 1:1. rIL-2 supplementation was necessary to achieve long-term tumor growth inhibition. Even spontaneous D2 tumor explant could be grown in the collagen gel and addition of anti-E474 to this culture reduced tumor growth. This assay system provides a realistic and sensitive alternative to the in vivo tumor growth inhibition assay and allows easy adaptation to test additional therapeutic reagents.     

Validation of a 1D patient-specific model of the arterial hemodynamics in bypassed lower-limbs: Simulations against in vivo measurements

The validation of a coupled 1D–0D model of the lower-limb arterial hemodynamics is presented. This study focuses on pathological subjects (6 patients, 72.7 ± 11.1 years) suffering from atherosclerosis who underwent a femoro-popliteal bypass surgery. The 1D model comprises four vessels from the upper-leg, peripheral networks are modeled with three-element windkessels and in vivo velocity is prescribed at the inlet.The model is patient-specific: its parameters reflect the physiological condition of the subjects. In vivo data are acquired invasively during bypass surgery using B-mode ultrasonography and catheter.Simulations from the model compare well with measured velocity (u) and pressure (p) waveforms: average relative root-mean-square error between numerical and experimental waveforms are limited to ?_p = 9.6%, ?_u = 16.0%. The model is able to reproduce the intensity and shape of waveforms observed in different clinical cases. This work also details the introduction of blood leakages along the pathological arterial network, and the sensitivity of the model to its parameters.This study constitutes a first validation of a patient-specific numerical model of a pathological arterial network. It presents an efficient tool for engineers and clinicians to help them improve their understanding of the hemodynamics in diseased arteries.     

Induction and functional characterization of β 2-microglobulin (β2-μ)-free HLA class I heavy chains expressed by β2-μ-deficient human FO-1 melanoma cells

The frequent loss of β2-microglobulin (β₂-μ) in malignant cells has stimulated interest in the functional characteristics of β₂-μ-free HLA class I heavy chains, since this information contributes to assess the impact of β₂-μ abnormalities on the interaction of malignant cells with immune cells. Therefore, the present study has investigated the ability of β₂-μ-free HLA class I heavy chains to modulate NK cell-mediated lysis of melanoma cells and to present melanoma-associated antigen (MAA)-derived peptides to HLA class I-restricted, MAA-specific cytotoxic T lymphocytes (CTL). β₂-μ-free HLA class I heavy chains were induced on B₂m null FO-1 cells by sequential incubation with IFN-α for 48 h at 37 °C and for 24 h at 26 °C. Transfection of cells with a wild-type H-2L^d gene (FO-1L^d) enhanced the induction of β₂-μ-free HLA class I heavy chains under such experimental conditions. β₂-μ-free HLA class I heavy chains expressed on the cell membrane did not protect the B₂m null FO-1 cells from NK cell-mediated lysis. Furthermore, FO-1 cells which express β₂-μ-free HLA-A2 heavy chains following transfection with a wild-type HLA-A2 gene were not lysed by HLA-A2-restricted, MAA-specific CTL lines and clones. These results indicate that association with β₂-μ is required for interaction of HLA class I molecules with NK inhibitory receptors and for peptide presentation to CTL.     

Induction of cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza virus infection in mice by inoculation of apathogenic H5N1 influenza virus particles inactivated with formalin

We investigated whether a vaccine derived from an apathogenic reassortant type A H5N1 influenza strain could induce immune responses in vivo that mediated protection from highly pathogenic avian influenza virus infection in mice. After two subcutaneous immunizations with formalin-inactivated H5N1 whole virus particles (whole particle vaccine), significant killing specific for cells presenting a nucleoprotein peptide from the vaccine strain of the virus was observed. Similar vaccination with viruses treated with ether and formalin, which are commonly used for humans as ether-split vaccines, induced little or no cytotoxic T-cell response. Furthermore, whole particle vaccines of the apathogenic H5N1 strain were more effective than ether-split vaccines at inducing antibody production able to neutralize a highly pathogenic H5N1 strain. Finally, whole particle vaccines of H5N1 protected mice against infection by an H5N1 highly pathogenic avian influenza virus more effectively than did ether-split vaccines. These results suggest that formalin-inactivated virus particles of apathogenic strains are effective for induction of both cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza viruses in vivo, resulting in protection from infection by a highly pathogenic H5N1 virus.     

Anisakis simplex allergy: a murine model of anaphylaxis induced by parasitic proteins displays a mixed Th1/Th2 pattern

The study of the singular hypersensitivity reactions to Anisakis simplex (A.s) proteins, may help us to undestand many of the unknown immune interactions between helmiths infections and allergy. We have developed a murine model of allergy to A. simplex, that mimics human A. simplex allergy to study the specific aspects of anaphylaxis induced by parasites. Male C3H/HeJ mice were intraperitoneally sensitized to A. simplex. Mice were then intravenous or orally challenged with A. simplex. Antigen-specific immunoglobulins, polyclonal IgE, anaphylactic symptoms, plasma histamine levels and cytokine profiles were determined. Comparative IgE immunoblot analyses were also performed. Specific IgE, IgG(1) and IgG(2a) were detected in sensitized mice since week 3. Polyclonal IgE raised and peaked with different kinetics. Intravenous A. simplex challenge produced anaphylaxis in mice, accompanied by plasma histamine release. Oral A. simplex challenge in similarly sensitized mice did not caused symptoms nor histamine release. Numerous A. simplex allergens were recognized by sensitized mouse sera, some of them similar to human serum. The A. simplex stimulated splenocytes released IL-10, IFN-gamma, IL-4, IL-13 and IL-5. We describe a new animal model of anaphylaxis. It exhibits characteristics of type I hypersensitivity reactions to Anisakis simplex similar to those observed in allergic humans. Different responses to i.v. or oral A. simplex challenges emerged, which did not reflect a window tolerization period. The cytokine profile developed (mixed Th(1)/Th(2) pattern) differed from the observed in classical models of anaphylaxis or allergy to food antigens. This model may permit to investigate the peculiar allergic reactions to parasitic proteins.     

Induction of a colitogenic phenotype in Th1-like cells depends on interleukin-23 receptor signaling

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Perpetual Proliferation of LYT-1 Cells Requires Repetitive Signals for IL-2 Receptor Induction by Antigen-presenting Cells

T cell lines with specificity for bovine insulin and ovalbumin were maintained by serial stimulation with antigen presented on irradiated syngeneic spleen cells, alternating 3 days later with subculture in IL-2 containing medium (CM). When the cultures were repetitively split in CM, with concomitant dilution of antigen-presenting cells, a gradual loss of proliferative capacity of the cells in the presence of CM was observed. Absorption studies revealed a 20-fold reduction of IL-2 receptors on the surface of T blasts assayed 12 days after antigenic stimulation as compared with day 5 blasts. This decrement in the number of IL-2 acceptor sites reflected an actual decrease in cell surface density of IL-2 receptors. Restimulation of the T blasts with antigen and spleen cells induced both a substantial increase in IL-2 receptor density and responsiveness to CM. Furthermore, the permanent presence of antigen and spleen cells during splitting of the T blasts in CM prevented the loss of responsiveness to IL-2. As an interpretation we propose that the Lyt-1 cells studied here clear their IL-2 receptors from the cell surface after interaction with IL-2. Thus, each new round of replication of the daughter cells would be dependent on induction of IL-2 receptors by activating signals provided by antigen/la structures on accessory cells as well as possibly accessory cell products such as IL-1, rendering Lyt-1 cells sensitive to regulatory influences.     

T2 relaxation times of 13C metabolites in a rat hepatocellular carcinoma model measured in vivo using 13C‐MRS of hyperpolarized [1‐13C]pyruvate

A single‐voxel Carr‐Purcell‐Meibloom‐Gill sequence was developed to measure localized T₂ relaxation times of ¹³C‐labeled metabolites in vivo for the first time. Following hyperpolarized [1‐¹³C]pyruvate injections, pyruvate and its metabolic products, alanine and lactate, were observed in the liver of five rats with hepatocellular carcinoma and five healthy control rats. The T₂ relaxation times of alanine and lactate were both significantly longer in HCC tumors than in normal livers (p < 0.002). The HCC tumors also showed significantly higher alanine signal relative to the total ¹³C signal than normal livers (p < 0.006). The intra‐ and inter‐subject variations of the alanine T₂ relaxation time were 11% and 13%, respectively. The intra‐ and inter‐subject variations of the lactate T₂ relaxation time were 6% and 7%, respectively. The intra‐subject variability of alanine to total carbon ratio was 16% and the inter‐subject variability 28%. The intra‐subject variability of lactate to total carbon ratio was 14% and the inter‐subject variability 20%. The study results show that the signal level and relaxivity of [1‐¹³C]alanine may be promising biomarkers for HCC tumors. Its diagnostic values in HCC staging and treatment monitoring are yet to be explored. Copyright © 2010 John Wiley & Sons, Ltd.