RESUMEN
Systemic lupus erythematosus is a chronic inflammatory disease which involves multiple organs. Self-specific B and T cells play a main role in the pathogenesis of lupus and have been defined as a logical target for selective therapy. The protein annexin A1 (ANX A1) is a modulator of the immune system involving many cell types. An abnormal expression of ANX A1 was found on activated B and T cells during autoimmunity, suggesting its importance as a potential therapeutic target. We hypothesize that it may be possible to down-regulate the activity of autoreactive T and B cells from lupus patients in a humanized immunodeficient mouse model by treating them with an antibody against ANX A1. When cultured in the presence of anti-ANX A1, peripheral blood mononuclear cells (PBMC) from lupus patients showed a decreased number of immunoglobulin (Ig)G anti-dsDNA antibody-secreting plasma cells, decreased T cell proliferation and expression of activation markers and increased B and T cell apoptosis. We employed a humanized model of SLE by transferring PBMCs from lupus patients to immunodeficient non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. The humanized animals presented autoantibodies, proteinuria and immunoglobulin deposition in the renal glomeruli. Treatment of these NOD-SCID mice with an anti-ANX A1 antibody prevented appearance of anti-DNA antibodies and proteinuria, while the phosphate-buffered saline (PBS)-injected animals had high levels after the transfer. The treatment reduced the levels of autoantibodies to several autoantigens, lupus-associated cytokines and disease symptoms.
Asunto(s)
Anexina A1/inmunología , Anticuerpos/inmunología , Linfocitos B/inmunología , Modelos Animales de Enfermedad , Lupus Eritematoso Sistémico/inmunología , Linfocitos T/inmunología , Animales , Anexina A1/metabolismo , Anticuerpos/farmacología , Anticuerpos Antinucleares/inmunología , Anticuerpos Antinucleares/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Activación de Linfocitos/inmunología , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
The aim of this study was to provide proof-of-concept for quantitative and qualitative label-free detection of immune complexes through myeloid cells with imaging surface plasmon resonance. Surface plasmon resonance imaging was first applied to monitor the binding of human sera from healthy and rheumatoid arthritis (RA) patients to immobilized citrullinated RA-specific peptide antigens, histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Next, the binding of monocytoid cell line U937 to the resulting immune complexes on the sensor surface was monitored. As control, binding of U937 was monitored to immunoglobulin (Ig)G subclasses simultaneously. Cell response results were compared to results of cyclic citrullinated peptide 2 (CCP2) enzyme-linked immunosorbent assay (ELISA), clinical RA diagnosis and antigen-specific antibody distribution of the samples. Human IgG3 triggered the most pronounced response, followed by IgG1 and IgG4, while IgG2 did not result in U937 cell binding. Serum samples obtained from RA patients resulted in a significantly increased cell response to VCP2 compared to healthy controls. The strength of cell response towards VCP2 immune complexes showed significant correlation with levels of antigen-specific IgA, IgG and IgG3. Cellular responses on VCP2 immune complexes showed significant association with both CCP2-based serological positivity and European League Against Rheumatism (EULAR) criteria-based clinical RA diagnosis. Immunoglobulin-triggered binding of monocytoid cells can be monitored using a label-free multiplex technology. Because these binding events are presumably initiated by Fc receptors, the system provides a tool for biological detection of autoantibodies with diagnostic value, here exemplified by anti-citrullinated antibodies. This provides added information to antibody levels, as interaction with Fc-receptor-expressing cells is also affected by post-translational modification of the immunoglobulins.
Asunto(s)
Complejo Antígeno-Anticuerpo/análisis , Artritis Reumatoide/diagnóstico , Autoanticuerpos/metabolismo , Fragmentos de Péptidos/sangre , Péptidos Cíclicos/sangre , Proteínas Virales/sangre , Complejo Antígeno-Anticuerpo/metabolismo , Artritis Reumatoide/sangre , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Autoanticuerpos/química , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Humanos , Proteínas Inmovilizadas/sangre , Proteínas Inmovilizadas/química , Sueros Inmunes/química , Inmunoglobulina G/sangre , Fragmentos de Péptidos/química , Péptidos Cíclicos/química , Unión Proteica , Resonancia por Plasmón de Superficie , Células U937 , Proteínas Virales/químicaRESUMEN
The aim of this study was to test for the presence of prohepcidin in cord blood, to gauge its alteration during the early postnatal period, and to look for a possible association with neonatal iron homeostasis. Cord blood and postnatal venous blood samples were taken from 20 healthy neonates. In both kinds of samples the presence prohepcidin could be detected. No association was found between cord blood and postnatal samples prohepcidin and iron homeostasis. However, an association is demonstrated between cord blood prohepcidin values and mean cell hemoglobin concentration (MCHC). Prohepcidin increased postnatally in half of the neonates, indicating the active synthesis of the molecule. Interestingly, neonates with detectable non-protein-bound iron levels in cord blood were presented with lower prohepcidin concentrations. Association between cord blood prohepcidin and MCHC may suggest a possible link between hepcidin and fetal iron homeostasis.
Asunto(s)
Adaptación Fisiológica , Péptidos Catiónicos Antimicrobianos/sangre , Sangre Fetal/química , Hemoglobinas/análisis , Hepcidinas , Homeostasis , Humanos , Recién Nacido , Hierro/metabolismo , Regulación hacia ArribaRESUMEN
The adjuvant effect of gamma-inulin, a strong activator of the alternative complement pathway, is well-known, but its exact mechanism is not revealed yet. Here, we show that macrophages, isolated from the peritoneal cavity of gamma-inulin-injected mice and used as antigen-presenting cells, enhance the proliferation of antigen-specific T-cells up to 2.5-fold when compared with macrophages of non-treated animals. This effect is abrogated by the presence of anti-C3 F(ab')2 fragments and by prior decomplementation of the donor animals with CVF. It is demonstrated that treatment of mice with the adjuvant results in deposition of C3-fragments onto the surface of peritoneal macrophages, as does in vitro incubation of the cells with gamma-inulin in the presence of fresh autologous serum. Prior incubation of macrophages with gamma-inulin plus serum in vitro enhances subsequent C3 production. Because it has been shown earlier that CR1/2 expressed on activated T-cells and interacting with covalently bound C3-fragments plays an important role in the augmentation of the adaptive response, our present results reveal a mechanism that contributes to the adjuvant effect of gamma-inulin and point to a further link between innate and adaptive immunity.
Asunto(s)
Presentación de Antígeno/inmunología , Complemento C3/inmunología , Inulina/inmunología , Macrófagos Peritoneales/inmunología , Animales , Activación de Linfocitos , Ratones , Linfocitos T/inmunologíaRESUMEN
Histamine is produced from histidine by histidine decarboxylase (HDC) in many cells including normal and malignant lymphocytes. We examined the expression of HDC and the effect of histamine receptor antagonists on the proliferation of a human T cell line, Jurkat and on antigen-driven proliferation of lymphocytes from ovalbumin-immunized mice. Our results demonstrate that HDC is inducible in Jurkat cells by anti-CD3. The H1 receptor antagonist triprolidine dose dependently inhibits proliferation of both Jurkat cells and ovalbumin-stimulated murine lymphocytes, while the H2 antagonist ranitidine was ineffective. Alpha-fluoro-methyl-histidine blocking HDC activity did not inhibit the T cell proliferation, suggesting an existing pool of histamine in T cells.
Asunto(s)
Antagonistas de los Receptores Histamínicos H1/farmacología , Células Jurkat/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ovalbúmina/inmunología , Linfocitos T/inmunología , Triprolidina/farmacología , Animales , Histamina/metabolismo , Histidina Descarboxilasa/metabolismo , Humanos , Células Jurkat/enzimología , Células Jurkat/patología , RatonesRESUMEN
C3-fragments generated upon complement activation play an important role in the formation and regulation of immune responses. Receptors interacting with various activation fragments of this versatile complement component are expressed on a wide variety of cell types, such as lymphocytes, macrophages, dendritic cells, follicular dendritic cells, granulocytes, erythrocytes, consequently C3-products may influence several biological functions at different sites of the body, where complement activation takes place. In the last decade, genes, protein structure and functions played by murine complement receptors CR1 and CR2 (mCR1/2) have been deciphered. In this review, we wish to relate these properties, and fit it into the context of events following in vivo complement activation. We separately address the roles played by murine mCR1/2 as BCR coreceptor and as BCR independent structure, and propose a mchanism for the utilization of antigen-C3d conjugates bound on B cells. Finally, we raise some of the questions that remain to be elucidated in order to get a more precise picture of the functions of mCR1/2.
Asunto(s)
Receptores de Complemento 3b/fisiología , Receptores de Complemento 3d/fisiología , Animales , Autoinmunidad , Activación de Complemento , Expresión Génica , Tejido Linfoide/inmunología , Ratones , Receptor Cross-Talk , Receptores de Antígenos de Linfocitos B/fisiología , Receptores de Complemento 3b/química , Receptores de Complemento 3b/genética , Receptores de Complemento 3d/química , Receptores de Complemento 3d/genéticaRESUMEN
In spite of the intensive search for the determination of the continuously widening physiological and pathological roles of different stress proteins, their ultrastructural localization at the electron microscopic (EM) level has hardly been examined. As it becomes increasingly evident that the function and physiological effectiveness of stress proteins are highly dependent on their spatial location and their associations with diverse regulator proteins, the demand for morphological studies which can identify their detailed distribution within the cells is evident. The reason for the practical lack of studies carried out at the EM level, lies in the shortage of reagents with suitable specificity and avidity necessary for this type of examination. To create such a reagent, a polyclonal antibody was raised using a recombinant truncated form of the inducible Hsp-72 protein. The antibody was extensively characterized, using different immunochemical methods to determine and verify its specificity, and then it was tried in ultrastructural examinations. Using the new antibody, it was possible to analyze the intracellular distribution of Hsp-72 with the immunogold technique. The localization of Hsp-72 was demonstrated directly at the ultrastructural level in the cytoplasm (especially at the cisterns of the RER), in the nucleus (mainly around the heterochromatic regions) and at both sides of the nuclear envelope close to the membrane pores. Apart from these localizations, Hsp-72 was found in several membrane bordered intracellular structures, which mainly belong to the endosomal-lysosomal system. We provide the first morphological verification of the appearance of Hsp-72 on the surface of the cells. Also novel is the indication, that the stress protein may recycle from the cell surface using a common route which includes coated pits and the endosomal system.
Asunto(s)
Proteínas de Choque Térmico/ultraestructura , Región Variable de Inmunoglobulina/inmunología , Proteínas de Neoplasias/ultraestructura , Células 3T3 , Animales , Femenino , Técnica del Anticuerpo Fluorescente , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/inmunología , Sueros Inmunes , Ratones , Microscopía Electrónica , Chaperonas Moleculares , Proteínas de Neoplasias/inmunología , Conejos , RatasRESUMEN
Defence against the reactive oxidants produced during aerobic metabolism is a complex process and is provided by a system of enzymes and antioxidant compounds capable of preventing excess radical production, neutralising free radicals and repairing the damage caused by them. Regulation of the antioxidant system must provide sufficient, properly located, antioxidant compounds and enzymes. Damage to this system has been proved to play a role in various disorders. Long-term complications of diabetes mellitus are supposed to be partially mediated by oxidative stress. The authors summarise experimental and clinical investigations in this field and analyse the possible importance of the changes in the antioxidant system in the development of diabetic vascular complications.
Asunto(s)
Diabetes Mellitus/enzimología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Animales , Catalasa/metabolismo , Angiopatías Diabéticas/etiología , Glutatión Peroxidasa/metabolismo , Humanos , Ratas , Superóxido Dismutasa/metabolismoRESUMEN
Single-chain variable fragment (scFv) antibodies are genetically engineered molecules comprising the variable regions responsible for specific binding. scFv that recognize certain surface molecules on professional antigen presenting cells could therefore be suitable for targeting Ag to these cells. We have produced an scFv that recognizes murine complement receptors 1 and 2 (CR1/CR2) and genetically fused it with different numbers of influenza hemagglutinin peptides which contain both B and T cell epitopes. The CR1/CR2 specific hybridoma 7G6 was used for RT-PCR to obtain the variable regions, which were then combined to create an scFv fragment. The influenza hemagglutinin intersubunit peptide HA317-41 (IP) was engineered to the N terminus of the scFv in one, two or three copies. The so obtained IP(1-3)7G6scFv still bound the complement receptors; the peptides in the construct were recognized by the peptide specific monoclonal IP2-11-1 on Western blots and ELISAs. The CR1/CR2 positive B lymphomas A20 and 2PK3 presented the peptide to an I-Ed restricted IP specific T cell hybridoma more efficiently when incubated with the IP(1)7G6 constructs as compared to the free peptide. The results suggest that scFv could work as targeting devices in subunit vaccines.
Asunto(s)
Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Virus de la Influenza A/inmunología , Receptores de Complemento 3b/inmunología , Receptores de Complemento 3d/inmunología , Secuencia de Aminoácidos , Animales , Epítopos de Linfocito B/genética , Epítopos de Linfocito T/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ratones , Datos de Secuencia Molecular , Ratas , Receptores de Complemento 3b/genética , Receptores de Complemento 3d/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
During an ongoing immune response, immune complexes, composed of Ag, complement factors, and Igs, are formed that can interact with complement receptors (CRs) and IgG Fc receptors (Fc gamma R). The role of CR1/2 and Fc gamma R in the regulation of the immune response was investigated using OVA that was chemically conjugated to whole IgG of the rat anti-mouse CR1/2 mAb 7G6. FACS analysis using the murine B cell lymphoma IIA1.6 confirmed that the 7G6-OVA conjugate recognized CR1/2. Incubating IIA1.6 cells with 7G6-OVA triggered tyrosine phosphorylation and Ag presentation to OVA-specific T cells in vitro. Immunizing mice with 7G6-OVA at a minimal dose of 1 microgram i.p. per mouse markedly enhanced the anti-OVA Ig response, which was primarily of the IgG1 isotype subclass. The 7G6-OVA did not enhance the anti-OVA response in CR1/2-deficient mice. OVA coupled to an isotype control Ab induced a considerably lower anti-OVA response compared with that induced by OVA alone, suggesting inhibition by interaction between the Fc part of the Ab and the inhibitory Fc gamma RIIb on B cells. This findings was supported by the observation that IIA1.6 cells which were incubated with 7G6-OVA lost the ability to present Ag upon transfection with Fc gamma RIIb. In sum, 7G6-conjugated OVA, resembling a natural immune complex, induces an enhanced anti-OVA immune response that involves at least CR1/2-mediated stimulation and that may be partially suppressed by Fc gamma RIIb.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos/inmunología , Antígenos/metabolismo , Inmunoglobulina G/biosíntesis , Receptores de Complemento 3b/inmunología , Receptores de Complemento 3d/inmunología , Receptores Fc/metabolismo , Animales , Presentación de Antígeno , Antígenos/administración & dosificación , Sitios de Unión de Anticuerpos , Femenino , Inmunoglobulina G/administración & dosificación , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Fosforilación , Receptores de Complemento 3b/metabolismo , Receptores de Complemento 3d/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
Murine cells of the B lymphoblastoid line A20 and concanavalin A-elicited peritoneal macrophages are shown to activate and fix C3 fragments covalently when incubated in fresh, autologous serum under conditions allowing the initiation of the alternative complement pathway. For the detection of cell-bound C3, cytofluorimetry was performed using FITC-labeled F(ab')2 fragments of anti-mouse C3. Cell-bound C3 fragments are not internalized or shed by the cells under culture conditions for at least two hours. When the antigen-presenting capacity of serum-treated cells was tested using various antigens and experimental systems, augmentation of the proliferation of antigen-specific T cells was found. This enhancing effect was particularly pronounced at suboptimal antigen doses. The elevation of T cell proliferation induced by C3-opsonized antigen-presenting cells (APC) could be abrogated by F(ab')2 fragments of goat anti-mouse C3, suggesting the involvement of C3 receptors expressed by T cells in the process. Using the 7G6 mAb recognizing murine CR1/CR2, the presence of these complement receptors on activated T cells is demonstrated by cytofluorimetry and immunoprecipitation, as well. These results point to the role of C3 bound to acceptor sites on APC in the facilitation of antigen presentation, providing a further link between innate and adaptive immunity.
Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Complemento C3/metabolismo , Epítopos de Linfocito T/inmunología , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Complemento C3/inmunología , Complemento C3/fisiología , Vía Alternativa del Complemento , Hibridomas/inmunología , Hibridomas/metabolismo , Sueros Inmunes/farmacología , Inmunidad Innata , Inmunosupresores/farmacología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Receptores de Complemento 3b/biosíntesis , Receptores de Complemento 3d/biosíntesisRESUMEN
The authors review the knowledge accumulated on animal models of diabetes during the last decade. The most commonly used diabetes models and the underlying disease processes are summarized, respectively. The best known diabetogenic substances, Streptozotocin and Alloxan are described, including their usage, dosage dosing intervals, and mechanism of action. The latest results are included in the article which discuss the possible role of free radical-induced injury of beta cells in Streptozotocin and Alloxan diabetes.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Aloxano/farmacología , Animales , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 1/inducido químicamente , Modelos Animales de Enfermedad , Radicales Libres/farmacología , Humanos , Ratones , Ratones Endogámicos NOD , Ratas , Ratas Endogámicas BB , Estreptozocina/farmacologíaRESUMEN
Male, weaned Wistar albino rats (n = 8) were treated by single dose of intravenous 50 mg/bodyweight-kg streptozotocin (STZ) injection. Changes in carbohydrate and lipid metabolism, and scavenger capacity of red blood cells and liver homogenates were evaluated and compared to the respective values of the control group (n = 9) after 3 weeks. HbAlc was significantly higher (p < 0.005) in the STZ treated group. Plasma triglyceride also showed a marked elevation compared to the control group (p < 0.05). Scavenger capacity in erythrocytes was significantly (p < 0.05) higher in treated animals while no change was observed in liver homogenates. No alteration was observed in the superoxide dismutase activity of treated animals, but catalase activity was weaker (p < 0.05). Thiobarbituric acid reactive substances were in higher concentration in plasma of STZ treated animals (p < 0.01) and were in comparable amount in homogenates. The results suggest that 3 weeks after STZ treatment of rats, alterations can be observed in the scavenger system and of the examined tissues changes are most prominent in erythrocytes.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Depuradores de Radicales Libres/análisis , Estreptozocina/administración & dosificación , Animales , Metabolismo de los Hidratos de Carbono , Modelos Animales de Enfermedad , Eritrocitos/metabolismo , Inyecciones Intravenosas , Metabolismo de los Lípidos , Masculino , Ratas , Ratas Wistar , Estreptozocina/farmacologíaRESUMEN
Diabetic patients develop arteriosclerosis at an early age. Their disease progresses more rapidly than that of nondiabetics, due to the underlying cause of arteriosclerosis of which the origin is still unknown. Much attention has been paid recently to the causative role of glycosilated lipoproteins, free radical reactions and hyperinsulinaemia--insulin resistance. Disturbances of the carbohydrate metabolism are accompanied by disorders in lipid metabolism and in the antioxidant system. While proteins undergo glycosilation, free radicals are being released from inflamed cells and, during the course of glycosilation with subsequent lipid peroxidation. Oxidation of lipids and proteins form the basis of pathological processes that might initiate the development of arteriosclerosis. There are attempts to influence the above processes by scavengers--e.g. vitamins, Ca-antagonists, angiotensin converting enzyme inhibitors and antilipaemic agents.