Frequency of anergy in a group of patients with rheumatoid arthritis on immunosuppressive therapy / Frecuencia de anergia en un grupo de pacientes con artritis reumatoide y terapia inmunosupresora

ABSTRACT Background: Tuberculin is the globally accepted delayed cutaneous hypersensitivity test for the diagnosis of latent tuberculosis. The alteration of cellular immunity induced by disease-modifying drugs used in rheumatoid arthritis may give a false negative result, also known as cutaneous anergy. There are no studies that determine the frequency of anergy in patients with rheumatoid arthritis and on immunosuppressive therapy. Objective: To determine the frequency and possible factors associated with cutaneous anergy in a group of patients with rheumatoid arthritis and on immunosuppressive therapy. Methods: Cross-sectional analytical observational study including 100 patients with rheumatoid arthritis on immunosuppressive therapy. They were tested for delayed cutaneous hypersensitivity with tuberculin, and a control test with tetanus toxoid. The non-reactivity of both tests was defined as anergy. Results: The overall frequency of cutaneous anergy was 9% (n = 11). It occurred in 33% of men versus 6% of women. The mean age was 57 years, and 89% were over 50 years-old. Being female behaved as a protective variable for the generation of anergy, OR 0.795 [95% CI, 0.658 - 0.959, P<.05]. All patients with anergy were being treated with corticosteroids, 44% with methotrexate, and 33% with biological therapy. Treatment with moderate to high dose prednisone and biological therapy were independently associated as risk factors for presenting with anergy, OR 1.044 [95% CI, 1.008-1.080 P<.05] and OR 1.096 [95% CI, 1.016-1.182, P<.05], respectively. The overall positivity for tuberculin was 13%. Symptoms associated with disease activation were present in 38% of these. All cases (n= 1) of confirmed active tuberculosis were excluded. Conclusions: The high prevalence of cutaneous anergy in patients with RA in the present study, and the evidence presented here, supports the recommendation of a second diagnostic test (tuberculin booster or Interferon-Gamma Release Assays) for the diagnosis of latent TB in patients with RA on immunosuppressive therapy.

RESUMEN Antecedentes: La tuberculina es la prueba de hipersensibilidad cutánea tardía mundialmente aceptada para el diagnóstico de tuberculosis latente. La alteración de la inmunidad celular inducida por los fármacos modificadores de la enfermedad utilizados en la artritis reumatoide puede dar un resultado falso negativo, también conocido como anergia cutánea. No hay estudios que determinen la frecuencia de anergia en pacientes con artritis reumatoide y terapia inmunosupresora. Objetivo: Determinar la frecuencia y los posibles factores asociados con la anergia cutánea en un grupo de pacientes con artritis reumatoide y terapia inmunosupresora. Métodos: Estudio observacional analítico transversal que incluyó a 100 pacientes con artritis reumatoide con terapia inmunosupresora. Se les realizó una prueba de hipersensibilidad cutánea tardía con tuberculina y una prueba de control con toxoide tetánico. La no reactividad de ambas pruebas se definió como anergia. Resultados: La frecuencia general de anergia cutánea fue del 9% (n = 11). Ocurrió en el 33% de los hombres versus el 6% de las mujeres, la edad promedio fue de 57 anos y el 89% tenía más de 50 anos. El sexo femenino se comportó como una variable protectora para la generación de anergia (OR 0,795; IC 95%: 0,658-0,959; p < 0,05). Todos los pacientes con anergia usaron corticosteroides, el 44% fue tratado con metotrexato y el 33% con terapia biológica. El tratamiento con dosis de moderadas a altas de prednisona y terapia biológica se asoció de manera independiente como factor de riesgo para la presentación de anergia: OR 1,044 (IC 95%: 1,008-1,080; p < 0,05) y OR 1,096 (IC 95%: 1,016-1,182; p < 0,05), respectivamente. La positividad general para la tuberculina fue del 13%. Los síntomas asociados con la activación de la enfermedad estaban presentes en el 38% de ellos. Se excluyeron todos los casos de tuberculosis activa confirmada (n = 1). Conclusiones: La alta prevalencia de anergia cutánea en pacientes con artritis reumatoide en el presente estudio y la evidencia presentada respaldan la recomendación de una segunda prueba de diagnóstico (refuerzo de tuberculina o IGRA) para el diagnóstico de tuberculosis latente en pacientes con artritis reumatoide y terapia inmunosupresora.

Candin Test in Evaluation of Cellular Immunity in Chronic Kidney Disease Patients - A Cross Sectional Observational Study

Introduction: Chronic kidney disease (CKD) patients are moresusceptible to infection due to impaired immune competency.Age, nutritional deficiencies, uremic toxins, dialysis,metabolism of parathyroid hormones and immunosuppressivemedications contribute to immune dysregulation. Aims ofthe present study were to find out the use of Candin Test toearly reorganization of immunocompromised state of cellularimmunity in patients of CKD and to find out correlationbetween Candin Test results and various factors alteringimmune system in CKD patients.Material and methods: The Cross-sectional observationalstudy was conducted on eighty adult patients qualifying thediagnostic criteria as per KDIGO guideline. Another ageand sex matched eighty healthy volunteers were selectedas control group. Patients having diabetes, HIV positive,malignancy, pregnancy, who are on steroids or any otherimmunosuppressive therapy, and those belonging to extremesof ages i.e.<18 years and >65 years were not included in thestudy. A detailed history, clinical examination and relevantinvestigations (Serum urea, creatinine, FBS, HIV, iPTH andUSG abdomen) were done in each subject. Each subject alsounderwent Candin Test to assess the level of cellular immunity.Result: Out of 80 cases, 35% patients showed positiveinduration while control group (n=80) demonstrated 58.8%indurations that revealed significantly more induration positivein controls (p=0.0024). In stage 3 CKD patients, 55.5% casesshowed positive induration, however positive indurationdecreases 52% and 24% in stage 4 and stage 5 respectively.Induration was significantly more positive in stage 3 and stage4 in comparison to stage 5 (p=0.007). Longer duration of CKDshowed lesser number of positive induration response butdifference was statistically non-significant (p=0.0521). Therewas significantly more induration response in no hemodialysisgroup (p=0.032) in comparison to hemodialysis group. Nosignificant difference was observed between induration andage. Mean eGFR of the patients with positive induration was20.14±13.083 and those without induration was 12.87±7.968with p=0.003, which is significant i.e. cases with positiveinduration have higher eGFR values. Mean Serum iPTH is215.50±119.279 in patients with positive induration and312.88±286.601 in patients with no induration which is notsignificant (p=0.091).Conclusion: Our study emphasises that Candin Test maybe useful to predict anergy in patients of CKD. As the CKDpatients approaches towards ESRD, cellular immunityalso decreases. It has also been observed that Candin Testresponse was diminished in patients on maintenance HD,which explains that as the frequency of HD increases patient'simmunity decreases.

Mechanism of oral tolerance / 国际儿科学杂志

Oral tolerance is a state of no or low response to a specific oral antigen,but there are still normal immune responses to other antigens.Anaphylaxis may occur when oral tolerance is not established or destroyed.In recent years,the incidence of food allergy in Chinese children has increased,with an average of 5.83 ％ reported by parents.Therefore,it is of great significance to explore the mechanisms of oral tolerance.The mechanisms of oral tolerance include active suppression,bypass suppression,clonal anergy/deletion.Oral administration of low-dose antigen can induce regulatory T cells to secrete inhibitory cytokines and actively inhibit effector T cells.Oral administration of high-dose antigen can induce clonal anergy/deletion.Regulatory B cells,dendritic cells,various cytokines,gut microbiota and short-chain fatty acids also play an important role in oral tolerance.This review focuses on the mechanism and some influencing factors of oral tolerance.

Induction of CD4+ Regulatory and Polarized Effector/helper T Cells by Dendritic Cells

Dendritic cells (DCs) are considered to play major roles during the induction of T cell immune responses as well as the maintenance of T cell tolerance. Naive CD4+ T cells have been shown to respond with high plasticity to signals inducing their polarization into effector/helper or regulatory T cells. Data obtained from in vitro generated bone-marrow (BM)-derived DCs as well as genetic mouse models revealed an important but not exclusive role of DCs in shaping CD4+ T cell responses. Besides the specialization of some conventional DC subsets for the induction of polarized immunity, also the maturation stage, activation of specialized transcription factors and the cytokine production of DCs have major impact on CD4+ T cells. Since in vitro generated BM-DCs show a high diversity to shape CD4+ T cells and their high similarity to monocyte-derived DCs in vivo, this review reports data mainly on BM-DCs in this process and only touches the roles of transcription factors or of DC subsets, which have been discussed elsewhere. Here, recent findings on 1) the conversion of naive into anergic and further into Foxp3- regulatory T cells (Treg) by immature DCs, 2) the role of RelB in steady state migratory DCs (ssmDCs) for conversion of naive T cells into Foxp3+ Treg, 3) the DC maturation signature for polarized Th2 cell induction and 4) the DC source of IL-12 for Th1 induction are discussed.

Effect of Cytomegalovirus and Diabetes Mellitus on the Cytokine Profile of Cellular Immunity in Tuberculosis Patients.

Aims: This study aims to investigate the effect of cytomegalovirus (CMV) and diabetes mellitus (DM) on the cell-mediated immunity against TB represented by cytokine profile Study Design: Case control study. Place and Duration of Study: This study was carried out in Specialized Chest and Respiratory Center in Baghdad, Iraq and Department of Medical Microbiology-College of Medicine -Babylon university Hilla-Iraq, the period of study was October 2012 to January 2013. Methodology: This study was applied on 70 TB patients .It involved also 30 apparently healthy control. The patients consists of 43 males and 27 females with age range 8-76 years old, 29 of them were diabetic .Blood samples were collected from patients and controls to estimate the immune parameters interferon-gamma (IFN-γ) and interleukin - 2(IL-2 )as , and anti –CMV IgG antibodies by enzyme linked immunosorbent assay (ELISA). Results: The immune parameters showed that there is no significant difference in the mean serum concentration between the patients and control groups for IFN-γ and IL-2 (P=0.05), while there was a significant increase in the mean serum concentration of anti- CMV IgG between study groups (P≤0.001). The study also showed that there is a significant decrease in the mean serum concentration of IL-2 and IFN-γ between diabetic TB patients comparing with those nondiabetic TB patients where p values were 0.008 and 0.024 respectively. Conclusion: Both CMV and diabetes mellitus have a role in the suppression of cellular immunity in TB patients.

Tolerancia inmunológica, un recorrido en el tiempo: cómo discriminar entre lo propio y lo extraño / Immune tolerance, a walk through time: How does the immune system differentiate between self and foreign

Resumen Desde los primeros estudios en inmunología, ha sido evidente la necesidad de entender cómo, en condiciones normales, el sistema inmune tolera los antígenos propios y ataca algunos antígenos extraños que percibe como potencialmente peligrosos, y cómo, bajo ciertas circunstancias, la pérdida de la tolerancia desencadena enfermedades autoinmunes. Ha pasado más de medio siglo desde que Billingham, Medawar y Brent demostraron en un modelo experimental algunos eventos involucrados en el desarrollo de la tolerancia inmunológica. Desde entonces, los inmunólogos de trasplante han centrado sus esfuerzos en dilucidar los mecanismos que conllevan al mantenimiento de la tolerancia, con la esperanza de eludir las complicaciones de la inmunosupresión no específica y conseguir la prevención del rechazo crónico. Medawar (1953) argumentaba que durante el trasplante el sistema inmune del individuo se hacía tolerante al tejido trasplantado, manteniéndose la respuesta a otros antígenos. Estudios recientes han demostrado que la pérdida de la tolerancia al trasplante está asociada con una hiperrespuesta a los antígenos del tejido trasplantado, hecho que ha atormentado a los inmunólogos clínicos, quienes han encaminado sus esfuerzos a desarrollar sistemas de medición precisos que les permita evaluar qué tan tolerante podría ser un individuo al trasplante. Los intentos por inducir tolerancia en el individuo, se basan en la comprensión de los mecanismos básicos de tolerancia, cuyo conocimiento se ha desarrollado paralelamente con una mejor apreciación de la complejidad de la tolerancia inmune. En particular, se ha avanzado mucho en la comprensión del papel esencial de las células dendríticas tolerogénicas (CDT) y del mantenimiento de la tolerancia por células T reguladoras.

Abstract Since the first studies in immunology, there has been a clear need to understand how, under normal conditions, the immune system tolerates its own antigens and attacks some foreign antigens that it perceives as potentially dangerous and how, in certain circumstances, the loss of tolerance triggers autoimmune diseases. It has been over half a century since Billingham, Brent and Medawar demonstrated, in an experimental model, the mechanisms involved in the development of immunological tolerance. Since then transplant immunologists have intensively investigated the mechanisms involved in maintaining tolerance, in the hope of avoiding the complications of non-specific immunosuppression, as well as the prevention of chronic rejection. An important characteristic was observed by Medawar, who argued that during transplantation an individual's immune system is tolerant to transplanted tissue, maintaining the response to other antigens. Recent studies have shown that loss of tolerance to transplantation is associated with a hyper-response to antigens of the transplanted tissue; a problem that has plagued clinical immunologists, who have focused their efforts on developing accurate measurement systems to enable them to measure how an individual could be tolerant to transplant. Attempts to induce tolerance in the individual are based on understanding the basic mechanisms of tolerance, in which there has been significant progress. This growth in knowledge has been in parallel with a better appreciation of the complexity of immune tolerance. In particular, progress has been made in understanding the essential role of tolerogenic dendritic cells (CDS) and the maintenance of tolerance by regulatory T cells.

Linfocitos T reguladores: subpoblaciones, mecanismo de acción e importancia en el control de la autoinmunidad / T regulatory lymphocytes: subpopulations, mechanism of action and importance in the control of autoimmunity

La regulación inmunológica constituye tanto un mecanismo importante para el mantenimiento de la homeostasis del sistema inmune como para el establecimiento de la tolerancia hacia antígenos propios evitando el desarrollo de enfermedades autoinmunitarias. Así mismo, juega un papel relevante en el mantenimiento de la tolerancia periférica mediante el control de una pequeña población de células T circulantes denominadas células T reguladoras (Treg), las cuáles parecen haber migrado del timo durante estadios relativamente tardíos¹. El término "células T reguladoras" se refiere a células que activan o suprimen la función de otras células. Aparentemente, controlan el desarrollo de enfermedades autoinmunitarias (lupus, tiroiditis, diabetes tipo I y enfermedad inflamatoria intestinal entre otras) el rechazo de injertos, y pueden jugar un papel crítico en el control del asma y la alergia.

Immune regulation is both an important mechanism for maintaining immune system homeostasis and for the establishment of tolerance towards self antigens in order to prevent the development of autoimmune diseases. It also plays an important role in maintaining peripheral tolerance by controlling a small population of circulating T cells, called regulatory T cells (Treg), which seems to have migrated from the thymus during relatively late stages¹. The term "regulatory T cells" refers to cells that activate or suppress the function of other cells. Apparently, controlling the development of autoimmune diseases (For instance, lupus, thyroiditis, type I diabetes and inflammatory bowel disease among others), graft rejection and may play a critical role in asthma and allergy.

Valoración de las pruebas diagnósticas para tuberculosis bovina en un rebaño bovino ubicado en zona de alta incidencia del estado Zulia Venezuela / Valuation of diagnostic tests for bovine tuberculosis in cattle herd located in a high incidence area of Zulia state Venezuela

Las pruebas de tuberculina son las de uso generalizado para el diagnóstico y el control de la tuberculosis (TBC) en el hombre y en los animales. Se caracteriza por una compleja mezcla de antígenos de mycobacterias capaces de inducir reacciones de hipersensibilidad en animales infectados, incluso con mycobacterias diferentes al Mycobacterium bovis, por efectos de reactividad cruzada. La preparación del derivado protéico purificado (PPD), es similar a la de la tuberculina, a diferencia de la concentración de proteínas, las cuales se separan por precipitación con agentes químicos y no por calor, aumentando su especificidad. Los primeros resultados obtenidos con las pruebas serológicas para el diagnóstico de tuberculosis bovina muestran que existe una gran reactividad antigénica cruzada entre las especies de mycobacterias, por lo que se requiere de antígenos más específicos. Se implementó un ELISA-TBC para la detección de anticuerpos anti M. bovis. El ensayo inmunoenzimático para el IFN-y bovino cuando se utilizó conjuntamente con el sistema de cultivo de sangre completa resultó en un ensayo in vitro rápido y sensible para detectar la reactividad de la inmunidad mediada por células al M. bovis en el ganado infectado. A partir de estas pruebas se compararon los resultados obtenidos para establecer la sensibilidad y especificidad utilizando como prueba oro, los datos obtenidos en el cultivo bacteriológico y la reacción en cadena de la polimerasa (PCR). Los animales reaccionantes a la tuberculina incluyeron animales positivos a PPD-B y PPD-A, así como animales negativos a cultivo bacteriológico y PCR. Los PPD-B positivos, no son en su totalidad, los mismos reaccionantes al IFN-y o al ELISA-TBC. Aún cuando su sensibilidad es baja, muestra mayor especificidad y concordancia que el resto de las pruebas utilizadas.

The tuberculin tests are widely used for diagnosis and control of tuberculosis (TB) in humans and animals. It is characterized by a complex mixture of mycobacteria antigens able to induce hypersensitivity reactions even in animals infected with mycobacteria other than M. bovis, for purposes of cross-reactivity. The preparation of purified protein derivative (PPD) is similar to the tuberculin, unlike the concentration of proteins which are separated by precipitation with chemical agents and not by increasing its specific heat. The first results obtained with the serological tests for diagnosis of bovine tuberculosis show that there is a great antigenic cross-reactivity between mycobacterias species so it requires more specific antigens. It implemented a cattle IFN-g test and ELISA-TBC to detect anti M. bovis activity. The immunoassay test for IFN-g used in conjunction with the cropping system of whole blood resulted in an essay in vitro rapid and sensitive to detect the reactivity of the cell-mediated immunity to M. bovis in livestock infected. Comparative test of the tuberculina, test of Gamma Interferon (INF-y) and a test ELISA-TBC, soon was taken to slaughter house to take linfoides weave samples and nodules, to which the test of chain reaction of Polimerasa was applied to them, to bacteriological culture and (PCR), for the identification from the pathogen. From these tests the patterns of immune response settled down and the obtained results of the different tests were compared to establish sensitivity and specificity using with t gold standard, the data collected in culture and PCR. The results were analyzed using the statistical method of analysis of variance for nonparametric tests. The PPD B-positive, are not the same reacting to IFN-y or at ELISA-TBC. Although its sensitivity is low, it shows greater specificity and consistency as the rest of the tests used.

N-acetyl-L-Cysteine Inhibits Functional Activation of Mouse Bone Marrow-Derived Dendritic Cells / 대한해부학회지

N-acetyl-L-cysteine (NAC) is a thiol-containing compound and acts as a precursor for glutathione (GSH). It behaves as an antioxidant in mammalian cells and also exerts anti-inflammatory effects. NAC is also known to affect several immune cells including eosinophils, B cells, T cells, and dendritic cells (DC) in many aspects. Even though it has been reported that NAC inhibits DC activation and shifts the immune response to Th2, these studies exhibit some contradictory results in detail and do not give any information with respect to the induction of regulatory T cells. In this study, we re-analyzed the effects of NAC on DC during their activation. We also evaluated whether it induced T cell anergy, Th1/Th2 shift, or regulatory T cells. NAC suppressed the elevation of intracellular reactive oxygen species during DC activation. In parallel, it down-regulated surface expression of CD40 and CD86, suppressed the decrease of phagocytic function, lowered the secretion of cytokines such as IL-6, IL-10, and IL-12. All these effects showed dose-dependency. Thus, it seems likely that NAC inhibited DC activation with regard to their phenotype and cytokine secretion. When we evaluated the T cell-stimulating capacity of these NAC-DC, T cell proliferation and secretion of both Th1 (IFN-gamma) and Th2 cytokine (IL-5) were decreased. This implies that the T cell-stimulating activity of NAC-DC decreased without any shift to Th1 or Th2 cytokine (IL-5). The secretion of IL-10 and TGF-beta in the supernatants were also decreased, which suggests that the decrease of T cell proliferation and cytokine secretion is due to the induction of T cell anergy, rather than regulatory T cells.

Frequency and Absolute Number of FoxP3+ Regulatory T Cells Correlate with Disease Progression of Chronic HIV-1 Infection / 中国病毒学·英文版

CD4+CD25+ Regulatory T cells (Treg) have been found to down-regulate immune activation in HIV-1 infection. However, whether the depletion of Treg benefits to the disease status of HIV infection remains undefined. To address this issue, we enumerated the Treg absolute counts and frequency in 75 antiviral-na(i)ve HIV-1-infected individuals in this study. It was found that HIV-infected patients displayed a significant decline in Treg absolute counts but a significant increase in Treg frequency. In addition, with disease progression indicated by CD4 T-cell absolute counts, circulating Treg frequency gradually increased; while Treg absolute counts were gradually decreased, suggesting that the alteration of Treg number closely correlated with disease progression in HIV infection.Functional analysis further showed that Treg efficiently inhibit both CD4 and CD8 T cell proliferation in vitro. Thus, our findings indicates that Treg actively participate in pathogenesis of chronic HIV infection,influencing the disease progression.

Maintenance of CD8+T-cell anergy by CD4+CD25+ regulatory T cells in chronic graft-versus-host disease

In a murine model of systemic lupus erythematosus (SLE)-like chronic graft-versus-host disease (cGVHD), donor CD8+T cells rapidly fall into anergy to host cells, while donor CD4+T cells hyperactivate B cells and break B-cell tolerance to self-Ags in the recipient mouse. The functional recovery of donor CD8+T cells can result in the conversion of cGVHD to acute GVHD (aGVHD), indicating that donor CD8+T-cell anergy is a restriction factor in the development of cGVHD. In this report, we present evidence that donor CD4+CD25+regulatory T cells (T(reg) cells) are critical in maintaining the donor CD8+T-cell anergy and thus suppressing the development of aGVHD in mice that are naturally prone to cGVHD. Our results provide a novel insight into the role of T(reg) cells in determining cGVHD versus aGVHD.

The Effects of Maturation Resistant Donor Dendritic Cells on Alloimmune Response in Mice / 대한신장학회잡지

BACKGROUND: Although dendritic cells (DCs) are the most influential antigen presenting cells maturation of DC is the critical control point toward either activation or regulation of immunity. We hypothesized that pretreatment with donor DCs, if which were maturation-resistant in vivo, could enhance engraftment by inducing inactivated state for allo- reactive T cell clones. METHODS: Immature DCs were prepared by 6- day culture of BM cells and we used paraformaldehyde for locking the DCs as immature phenotypes. We did in vitro and in vivo MLR to evaluate the effect of maturation resistant DCs on alloreactive T cells and we confirmed the effect of DCs in MHC full mismatched skin and islet transplantation model. RESULTS: Fixed DCs in immature state were resistant to maturation stimuli and weak stimulator for allo-reactive T cells (CB6F1-->C3H). In contrast, fixed DCs in mature state stimulated allogeneic T cell proliferation effectively. Splenocytes isolated from mice 2 weeks after maturation resistant DC injection could not be reactivated and maintained naive phenotype when cocultured with allogeneic splenocytes (BALB/c-->C57BL6). Consistent with this finding maturation resistant DC treatment suppressed MLR-driven T cell division (CB6F1-->C3H) as assessed by CFSE analysis. But, CD25+ T cells depletion by treatment with anti-CD25 prior to DCs transfer attenuated this regulatory effect of DCs. In a MHC mismatched transplantation model (CB6F1-->C3H), treatment with maturation-resistant DCs 2 weeks before operation, markedly prolonged skin and islet graft survival. But C3H mice pretreated with CB6F1 DCs rejected DBA1 (H-2q) skin graft within 14 days. CONCLUSION: These findings suggest the maintenance of immaturity of DCs is a key factor in modulating alloimmune responses through dendritic cells.

Immunotherapeutic Effects of CTLA4Ig Fusion Protein on Murine EAE and GVHD

BACKGROUND: CTLA4 (CD152), which is expressed on the surface of T cells following activation, has a much higher affinity for B7 molecules comparing to CD28, and is a negative regulator of T cell activation. In contrast to stimulating and agonistic capabilities of monoclonal antibodies specific to CTLA-4, CTLA4Ig fusion protein appears to act as CD28 antagonist and inhibits in vitro and in vivo T cell priming in variety of immunological conditions. We've set out to confirm whether inhibition of the CD28-B7 costimulatory response using a soluble form of human CTLA4Ig fusion protein would lead to persistent inhibition of alloreactive T cell activation. METHODS: We have used CHO-dhfr cell-line to produce CTLA4Ig fusion protein. After serum free culture of transfected cell line we purified this recombinant molecule by using protein A column. To confirm characterization of fusion protein, we carried out a series of Western blot, SDS-PAGE and silver staining analyses. We have also investigated the efficacy of CTLA4Ig in vitro such as mixed lymphocyte reaction (MLR) & cytotoxic T lymphocyte (CTL) response and in vivo such as experimental autoimmune encephalomyelitis (EAE), graft versus host disease (GVHD) and skin-graft whether this fusion protein could inhibit alloreactive T cell activation and lead to immunosuppression of activated T cell. RESULTS: In vitro assay, CTLA4Ig fusion protein inhibited immune response in T cell-specific manner: 1) Human CTLA4Ig inhibited allogeneic stimulation in murine MLR; 2) CTLA4Ig prevented the specific killing activity of CTL. In vivo assay, human CTLA4Ig revealed the capacities to induce alloantigen-specific hyporesponsiveness in mouse model: 1) GVHD was efficiently blocked by dose-dependent manner; 2) Clinical score of EAE was significantly decreased compared to nomal control; 3) The time of skin-graft rejection was not different between CTLA4Ig treated and control group. CONCLUSION: Human CTLA4Ig suppress the T cell-mediated immune response and efficiently inhibit the EAE, GVHD in mouse model. The mechanism of T cell suppression by human CTLA4Ig fusion protein may be originated from the suppression of activity of cytotoxic T cell. Human CTLA4Ig could not suppress the rejection in mouse skin-graft, this finding suggests that other mechanism except the suppression of cytotoxic T cell may exist on the suppression of graft rejection.

Inhibitory effects of CTLA4-Ig fusion protein on the proliferation of T cell and the antibody production of B cell / 천식및알레르기

BACKGROUND: Atopic asthma is characterized by activation of Th2-type T cells in the bronchial mucosa. Several reports have suggested an important role for costimulation through the CD28/CTLA4 (cytotoxic T lymphocyte-associated antigen 4)-B7 (CD80/CD86) pathway in allergen activation of T cells in animal models of allergen-induced asthma, because B7-CD28/ CTLA4 interaction can promote the differentiation and development of the Th2 lymphocyte subset. OBJECTIVE: In the present study, we intended to investigate a potential role of humanized CTLA4-Ig on the inhibition of T and B cell activation by blocking B7/CD28 interactions. METHOD: For this purpose we produced humanized CTLA4-Ig fusion protein by transfection to CHO cell and examined its inhibitory effects for activated T and B cell responses. We evaluated the inhibitory effect of MLR (mixed lymphocyte reaction) and con A-stimulated T cell proliferation. And we assayed wheather B cell was inhibited by stimulation of costimulatory signal in LPS-induced B cell response and PFC assay. RESULT: In vitro assay, humanized CTLA4-Ig fusion protein inhibited T cell-specific immune response in dose-dependent manner: CTLA4-Ig inhibited allogeneic stimulation in murine MLR, and the proliferation of T cell by the stimulation of Con A. But CTLA4-Ig did not inhibit directly the proliferative response of B cell by the stimulation of LPS. In addition, in vivo assay, CTLA4-Ig inhibited the production of antibody from B cell, which was presented by plaque-forming cell (PFC) assay. CONCLUSION: These findings suggest that humanized CTLA4-Ig is effective to inhibit the proliferation of activated T cell directly by blocking B7/CD28 costimulation. And humanized CTLA4-Ig influences antibody-producing capacity of B cell indirectly by regulating T cell.

CD28 and CTLA-4 expressions in the process of mice' T-cell inefficiency induced by staphylococcal enterotoxin A / 解放军医学杂志

Objective To explore the characteristics and regulations of CD28 and CTLA-4 expressions in the process of mice' T-cell inefficiency induced by staphylococcal enterotoxin A(SEA).Methods Twelve mice were averagely divided into three groups(4 each).The mice in group 1 received single injection of SEA,and the mice in group 2 and 3 received SEA injection twice and three times,respectively,with a 3 days interval.Mice were sacrificed at day 1,3,7 and 14 after the last injection,and then the splenic lymphocytes were isolated.The expressions of CD28 and CTLA-4 in cellular membrane and the intracellular expressions of CTLA-4 and IL-2 were detected with flow cytometry.Results In group 1,the CD28 expression in cellular membrane and the intracellular expression of IL-2 were significantly up-regulated,while the CTLA-4 expressions both in cellular membrane and intracellular expression were lower with no obvious changes.In group 2,the expressions of both CD28 and IL-2 were up-regulated slightly,the expressions of CTLA-4 increased significantly both in cellular membrane and intracellular expression,even more increase was the intracellular CTLA-4 expression.In group 3,the expressions of CD28 and CTAL-4 in cellular membrane and the intracellular CTLA-4 expression declined,and at day 7 the intracellular IL-2 expression was undetectable.Conclusions SEA may obviously promote the activation of naive T-cells when initially used to induce the splenic lymphocytes,while multiple stimulations(e.g.3 times) of SEA may lead T-cells to anergy.On the process of inefficiency,the declined IL-2 production may be closely related to the down-regulation of CD28 expression;the up-regulation of CTLA-4 expression may profit inducing inefficiency,but is not on the maintenance of inefficiency.

Oral Tolerance in Active Fatal Anaphylaxis / 대한면역학회지

We have investigated whether oral administration of ovalbumin (OVA) could prevent active systemic anaphylaxis to the antigen. Oral tolerance was induced by a single feecfing with 40 mg OVA before, but not after, sensitization characterized by diminished OVA-specific IgE and IgG responses. Feeding 15 mg OVA suppressed anaphylaxis and antibody responses to a lesser extent. Spleen cells from tolerant donors were incapable of transferring the tolerance to naive recipients. Pretreatment of cyclophosphamide (100 mg/kg) 2 days before OVA feeding did not restore the tolerance. Furthermore, in vitro cell mixing studies showed that the proliferation of spleen cells from OVA- sensitized donors was not inhibited by the addition of spleen cells from tolerant donors, arguing against the role of suppressor cells. Anergy was demonstrated by the ability to reverse the tolerant state after culturing tolerant cells in the presence of IL-2. These findings indicate that only a high-dose (40 mg) feeding OVA was found to be effective in inducing tolerance in this experimental system, and demonstrate anergy as the mechanism underlying oral tolerance to systemic anaphylaxis.

Anergy and Characteristics of Human T Cells Induced by Combination with B7-1 mAb and CsA / 中国肿瘤生物治疗杂志

To study the induced condition and characteristics of T cell anergy in vitro. Methods: Anergic Tcell was induced by combination of B7-1 mAb and cyclosporin A (CsA) in vitro, cytokine gene of anergic T cells was detected by RT-PCR. Results: T cell anergy was antigen-specific. The state of T cell anergy can be reversed by PHA, CD3 mAb and PMA plus A23187. IL-2 can prevent the induction of T cell anergy, but it can not reverse the state of un-responsiveness. IL-2 and IFN mRNA can not express in anergic T cells. In contrast, IL-4 and IL-10 mRNA were detectable. Conclusion: T cell anergy can be induced in vitro , cytokine profile of anergic T cells deviated to Th2-like phe-norype.

Expression of transcription factor AP-1 and NF-kappaB in anergic T cells / 中国免疫学杂志

Objective:To explore the relationship between transcription factor AP 1 and NF kappaB and IL 2 decrease in anergic T cells.Methods:Nuclear proteins of activated T cells and anergic T cells were extracted and then the expression of AP 1 and NF kappaB were determinated by gel electrophorotic mobility shift assay.Results:Compared to activated T cells,anergic T cells expressed defective AP 1 transcription factor;there were three forms NF kappaB complex in both activated T cells and agergic T cells,but NF kappaB transcription activity was higher in anergic T cells than that in activated T cells.Conclusion:It has been demonstrated that these alteration of transcription factors have been likely to be responsible for repression of IL 2 in anergic T cells.