RESUMEN
Oligomannose-coated liposomes (OMLs) comprised of dipalmitoylphosphatidylcholine, cholesterol and Man3-DPPE at a molar ratio of 1:1:0.1 and particle diameters of about 1000 nm can induce liposome-encased antigen-specific strong Th1 immunity. In this study, we evaluated the effect of particle sizes of OMLs on induction of Th1 immune responses in mice. Spleen cells obtained from mice immunized with antigen-encapsulating OMLs with 1000- and 800-nm diameters secreted remarkably high levels of IFN-γ upon in vitro stimulation. In addition, sera of mice that received these OMLs had significantly higher titers of antigen-specific IgG2a than those of IgG1, which are commonly associated with Th1 responses. In contrast, treatment with antigen-encapsulating OMLs with 400- and 200-nm diameters failed to induce IFN-γ secretion from spleen cells, although these OMLs did elicit elevation of antigen-specific IgGs. In addition, the titers of serum antigen-specific IgG2a were the same as those of IgG1 in mice that received 400-nm OMLs. Resident peritoneal mononuclear phagocytes (MNPs) treated with OMLs of diameter ≥ 600 nm secreted IL-12, which is essential for induction of Th1 immune responses, while those treated with OMLs of ≤ 400 nm failed to produce this cytokine. However, 400-nm OMLs did induce enhanced expression of MHC class II and costimulatory molecules on MNPs, similarly to OMLs of ≥ 600 nm. Taken together, these results strongly indicate that OMLs of diameter ≥ 600 nm are required to induce Th1 immune responses against OML-encased antigens, although OMLs of diameter ≤ 400 nm can activate MNPs.
Asunto(s)
Liposomas/química , Liposomas/inmunología , Manosa/química , Manosa/inmunología , Células TH1/inmunología , 1,2-Dipalmitoilfosfatidilcolina/inmunología , Animales , Antígenos/inmunología , Antígeno B7-2/metabolismo , Citocalasina D/farmacología , Femenino , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/metabolismo , Sistema Inmunológico , Inmunoglobulina G/sangre , Interferón gamma/efectos de los fármacos , Interferón gamma/metabolismo , Subunidad p35 de la Interleucina-12/metabolismo , Ratones , Tamaño de la Partícula , Absorción Peritoneal/efectos de los fármacos , Fagocitos/efectos de los fármacos , Fagocitos/metabolismo , Fagocitosis/efectos de los fármacos , Bazo/efectos de los fármacos , Bazo/metabolismoRESUMEN
Staphylococcal enterotoxin type B (SEB) is associated with food poisoning. Current methods for the detection of biologically active SEB rely upon its ability to cause emesis when administered to live kittens or monkeys. This technique suffers from poor reproducibility and low sensitivity and is ethically disfavored over concerns for the welfare of laboratory animals. The data presented here show the first successful implementation of an alternative method to live animal testing that utilizes SEB super-antigenic activity to induce cytokine production for specific novel cell-based assays for quantifiable detection of active SEB. Rather than using or sacrificing live animals, we found that SEB can bind to the major histocompatibility complex (MHC) class II molecules on Raji B-cells. We presented this SEB-MHC class II complex to specific Vß5.3 regions of the human T-cell line HPB-ALL, which led to a dose-dependent secretion of IL-2 that is capable of being quantified and can further detect 10 pg/mL of SEB. This new assay is 100,000 times more sensitive than the ex vivo murine splenocyte method that achieved a detection limit of 1 µg/mL. The data presented here also demonstrate that SEB induced proliferation in a dose-dependent manner for cells obtained by three different selection methods: by splenocyte cells containing 22% of CD4+ T-cells, by CD4+ T-cells enriched to >90% purity by negative selection methods, and by CD4+ T-cells enriched to >95% purity by positive selection methods. The highly enriched and positively isolated CD4+ T-cells with the lowest concentration of antigen-presenting cells (APC) (below 5%) provided higher cell proliferation than the splenocyte cells containing the highest concentration of APC cells.
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Alternativas a las Pruebas en Animales/métodos , Pruebas Inmunológicas de Citotoxicidad/métodos , Enterotoxinas/farmacología , Leucemia de Células T , Animales , Línea Celular Tumoral , Enterotoxinas/análisis , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Humanos , Límite de Detección , Sensibilidad y EspecificidadRESUMEN
Toll-like receptor 4 (TLR4) activation contributes to vascular dysfunction in pathological conditions such as hypertension and diabetes, but the role of chronic TLR4 activation on renal autoregulatory behavior is unknown. We hypothesized that subclinical TLR4 stimulation with low-dose lipopolysaccharide (LPS) infusion increases TLR4 activation and blunts renal autoregulatory behavior. We assessed afferent arteriolar autoregulatory behavior in male Sprague-Dawley rats after prolonged LPS (0.1 mg·kg-1·day-1 sq) infusion via osmotic minipump for 8 or 14 days. Some rats also received daily cotreatment with either anti-TLR4 antibody (1 µg ip), competitive antagonist peptide (CAP; 3 mg/kg ip) or tempol (2 mmol/l, drinking water) throughout the 8-day LPS treatment period. Autoregulatory behavior was assessed using the in vitro blood-perfused juxtamedullary nephron preparation. Selected physiological measures, systolic blood pressure and baseline diameters were normal and similar across groups. Pressure-dependent vasoconstriction averaged 72 ± 2% of baseline in sham rats, indicating intact autoregulatory behavior. Eight-day LPS-treated rats exhibited significantly impaired pressure-mediated vasoconstriction (96 ± 1% of baseline), whereas it was preserved in rats that received anti-TLR4 antibody (75 ± 3%), CAP (84 ± 2%), or tempol (82 ± 2%). Using a 14-day LPS (0.1 mg·kg-1·day-1 sq) intervention protocol, CAP treatment started on day 7, where autoregulatory behavior is already impaired. Systolic blood pressures were normal across all treatment groups. Fourteen-day LPS treatment retained the autoregulatory impairment (95 ± 2% of baseline). CAP intervention starting on day 7 rescued pressure-mediated vasoconstriction with diameters decreasing to 85 ± 1% of baseline. These data demonstrate that chronic subclinical TLR4 activation impairs afferent arteriolar autoregulatory behavior through mechanisms involving reactive oxygen species and major histocompatibility complex class II activation.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Homeostasis/efectos de los fármacos , Lipopolisacáridos/toxicidad , Circulación Renal/efectos de los fármacos , Animales , Presión Sanguínea/efectos de los fármacos , Óxidos N-Cíclicos/farmacología , Masculino , Nefronas/efectos de los fármacos , Nefronas/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Marcadores de Spin , Receptor Toll-Like 4/antagonistas & inhibidores , Vasoconstricción/efectos de los fármacosRESUMEN
BACKGROUND & OBJECTIVES: Patients with autoimmune rheumatic diseases may be at an increased risk of infection due to disease and use of disease-modifying antirheumatic drug (DMARD) therapy. The present study was done to evaluate the immune response to influenza vaccination in patients with rheumatoid arthritis (RA). METHODS: Fifty one RA patients on stable methotrexate (MTX) therapy (≥15 mg/wk), 51 newly diagnosed DMARD-naïve RA patients and 45 healthy controls received a single dose of inactivated seasonal trivalent influenza vaccine. Blood samples were collected just prior to and four weeks after vaccination. Pre- and post-vaccination antibody titres against the three virus strains were measured by hemagglutination inhibition assay. The impact of age, gender, DMARD treatment and pre-vaccination seroprotection on response to the vaccine was assessed by binary logistic regression analysis for each of the virus strains. RESULTS: Pre-vaccination antibody titres were found to be high in the three study groups for all influenza strains, except for Yamagata strain, the titres for which were low in healthy controls. Trivalent influenza vaccination was found to be safe and stimulated a good antibody response in all study groups. On regression analysis, there was no association of age, gender or MTX therapy with vaccine response, except for Yamagata strain where healthy controls had higher positive immune response (P=0.008; odds ratio - 3.37, 95% confidence interval: 1.36-8.32). INTERPRETATION & CONCLUSIONS: Our results indicated that influenza vaccination was safe in RA patients with no detrimental effect on disease activity. MTX therapy at a dose ≥15 mg/wk did not affect the vaccine response. Presence of high pre-vaccination seroprotective antibody levels in the study population indicates the need for re-examination of recommended annual influenza vaccination in such subgroups of population.
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Artritis Reumatoide/tratamiento farmacológico , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Vacunas contra la Influenza/administración & dosificación , Gripe Humana/tratamiento farmacológico , Adulto , Anciano , Anticuerpos Antivirales/sangre , Antirreumáticos/administración & dosificación , Antirreumáticos/efectos adversos , Artritis Reumatoide/sangre , Artritis Reumatoide/epidemiología , Artritis Reumatoide/virología , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Vacunas contra la Influenza/efectos adversos , Gripe Humana/sangre , Gripe Humana/prevención & control , Gripe Humana/virología , Masculino , Metotrexato/administración & dosificación , Persona de Mediana EdadRESUMEN
Recent advances in immunotherapeutics have not yet changed the routine management of autoimmune type 1 diabetes. There is an opportunity to repurpose therapeutics used to treat other diseases to treat type 1 diabetes, especially when there is evidence for overlapping mechanisms. Janus kinase (JAK) 1/JAK2 inhibitors are in development or clinical use for indications including rheumatoid arthritis. There is good evidence for activation of the JAK1/JAK2 and signal transducer and activator of transcription (STAT) 1 pathway in human type 1 diabetes and in mouse models, especially in ß-cells. We tested the hypothesis that using these drugs to block the JAK-STAT pathway would prevent autoimmune diabetes. The JAK1/JAK2 inhibitor AZD1480 blocked the effect of cytokines on mouse and human ß-cells by inhibiting MHC class I upregulation. This prevented the direct interaction between CD8+ T cells and ß-cells, and reduced immune cell infiltration into islets. NOD mice treated with AZD1480 were protected from autoimmune diabetes, and diabetes was reversed in newly diagnosed NOD mice. This provides mechanistic groundwork for repurposing clinically approved JAK1/JAK2 inhibitors for type 1 diabetes.
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Glucemia/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Diabetes Mellitus Tipo 1/inmunología , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Western Blotting , Linfocitos T CD8-positivos/inmunología , Quimiocina CXCL10/inmunología , Citocinas/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunohistoquímica , Técnicas In Vitro , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/inmunología , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 2/antagonistas & inhibidores , Ratones , Ratones Endogámicos NOD , Regulación hacia ArribaRESUMEN
TLR2-dependent cellular signaling in Mycobacterium tuberculosis-infected macrophages causes apoptosis and inhibits class II major histocompatibility complex (MHC-II) molecules antigen processing, leading to evasion of surveillance. Mycobacterium tuberculosis (MTB) lipoproteins are an important class of Toll-like receptor (TLR) ligand, and identified as specific components that mediate these effects. In this study, we identified and characterized MTB lipoprotein Rv1016c (lpqT) as a cell wall associated-protein that was exposed on the cell surface and enhanced the survival of recombinants M. smegmatis_Rv1016c under stress conditions. We found that Rv1016c lipoprotein was a novel TLR2 ligand and able to induce macrophage apoptosis in a both dose- and time-dependent manner. Additionally, apoptosis induced by Rv1016c was reserved in THP-1 cells blocked with anti-TLR-2 Abs or in TLR2-/- mouse macrophages, indicating that Rv1016c-induced apoptosis is dependent on TLR2. Moreover, we demonstrated that Rv1016c lipoprotein inhibited IFN-γ-induced MHC-II expression and processing of soluble antigens in a TLR2 dependent manner. Class II transactivator (CIITA) regulates MHC II expression. In this context, Rv1016c lipoprotein diminished IFN-γ-induced expression of CIITA IV through TLR2 and MAPK Signaling. TLR2-dependent apoptosis and inhibition of MHC-II Ag processing induced by Rv1016c during mycobacteria infection may promote the release of residual bacilli from apoptotic cells and decrease recognition by CD4+ T cells. These mechanisms may allow intracellular MTB to evade immune surveillance and maintain chronic infection.
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Presentación de Antígeno/efectos de los fármacos , Apoptosis/efectos de los fármacos , Proteínas Bacterianas/farmacología , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Lipoproteínas/genética , Macrófagos/microbiología , Mycobacterium tuberculosis/metabolismo , Proteínas Recombinantes/farmacología , Receptor Toll-Like 2/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Pared Celular/química , ADN Bacteriano , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Genes Bacterianos/genética , Humanos , Interferón gamma/efectos de los fármacos , Lipoproteínas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium smegmatis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Proteínas Nucleares/efectos de los fármacos , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/fisiología , Proteínas Recombinantes/genética , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia , Receptor Toll-Like 2/genética , Receptores Toll-Like/clasificación , Receptores Toll-Like/metabolismo , Transactivadores/efectos de los fármacos , Tuberculosis/microbiologíaRESUMEN
OBJECTIVE: The mechanisms underlying bone damage in rheumatoid arthritis (RA) are incompletely understood. We recently identified the shared epitope (SE), an HLA-DRB1-coded 5-amino acid sequence motif carried by the majority of RA patients as a signal transduction ligand that interacts with cell surface calreticulin and accelerates osteoclast (OC)-mediated bone damage in collagen-induced arthritis (CIA). Given the role of the SE/calreticulin pathway in arthritis-associated bone damage, we sought to determine the therapeutic targetability of calreticulin. METHODS: A library of backbone-cyclized peptidomimetic compounds, all carrying an identical core DKCLA sequence, was synthesized. The ability of these compounds to inhibit SE-activated signaling and OC differentiation was tested in vitro. The effect on disease severity and OC-mediated bone damage was studied by weekly intraperitoneal administration of the compounds to DBA/1 mice with CIA. RESULTS: Two members of the peptidomimetics library were found to have SE-antagonistic effects and antiosteoclast differentiation effects at picomolar concentrations in vitro. The lead mimetic compound, designated HS(4-4)c Trp, potently ameliorated arthritis and bone damage in vivo when administered in picogram doses to mice with CIA. Another mimetic analog, designated HS(3-4)c Trp, was found to lack activity, both in vitro and in vivo. The differential activity of the 2 analogs depended on minor differences in their respective ring sizes and correlated with distinctive geometry when computationally docked to the SE binding site on calreticulin. CONCLUSION: These findings identify calreticulin as a novel therapeutic target in erosive arthritis and provide sound rationale and early structure/activity relationships for future drug design.
Asunto(s)
Artritis Experimental/metabolismo , Artritis Reumatoide/metabolismo , Huesos/efectos de los fármacos , Calreticulina/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Péptidos/farmacología , Animales , Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Huesos/metabolismo , Calreticulina/metabolismo , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Epítopos , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Técnicas In Vitro , Ligandos , Ratones , Simulación del Acoplamiento Molecular , Osteoclastos/metabolismo , Biblioteca de Péptidos , Transducción de Señal/efectos de los fármacosRESUMEN
INTRODUCTION: Matrix metalloproteinase (MMP)-3 is a member of the MMP family that degrades the extracellular matrix. Application of MMP-3 to injured pulp tissue induces angiogenesis and wound healing, but its anti-inflammatory effects are still unclear. Here, we evaluated the anti-inflammatory functions of MMP-3 in vitro and in vivo. METHODS: Nitric oxide and inflammatory mediator synthesis in macrophages activated by lipopolysaccharide (LPS) was measured in the presence or absence of MMP-3. The mouse Mmp3 (mMmp3) expression vector containing full length cDNA sequence of mMmp3 or cDNA sequence of mMmp3 missing the signal peptide and pro-peptide regions was transfected to RAW264, a mouse macrophage cell line, and NO synthesis and inflammatory mediator expression were evaluated. Pulpal inflammation was histologically and immunohistochemically evaluated in a rat model of incisor pulpitis induced by the application of LPS for 9 hours in the presence or absence of MMP-3. RESULTS: NO and pro-inflammatory mediator synthesis promoted by LPS was significantly down-regulated by MMP-3 in vitro. The full length of mMmp3 down-regulated the LPS-induced NO synthesis and chemical mediator mRNA expression, however the mMmp3 missing the signal peptide failed to block the NO synthesis induced by LPS. The numbers of major histocompatibility complex class II+ and CD68+ cells, which infiltrated into the rat incisor pulp tissues in response to the topical application of LPS, were significantly decreased by the application of MMP-3 in vivo. CONCLUSIONS: These results indicate that MMP-3 possesses anti-inflammatory functions, suggesting its potential utility as an anti-inflammatory agent for pulpal inflammation.
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Antiinflamatorios/farmacología , Regulación hacia Abajo/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Metaloproteinasa 3 de la Matriz/farmacología , Pulpitis/inmunología , Animales , Antígenos CD/efectos de los fármacos , Antígenos de Diferenciación Mielomonocítica/efectos de los fármacos , Línea Celular , Células Cultivadas , Quimiocina CCL2/análisis , Ciclooxigenasa 2/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Interleucina-17/análisis , Interleucina-1beta/efectos de los fármacos , Interleucina-6/análisis , Lipopolisacáridos/farmacología , Masculino , Ratones , Óxido Nítrico/análisis , Pulpitis/prevención & control , Ratas , Ratas WistarRESUMEN
BACKGROUND: Bortezomib has been used to reduce HLA antibody in patients either before transplantation or as treatment for antibody-mediated rejection (AMR). Reports on its efficacy show mixed results. The mechanism of action of this agent is via proteasome inhibition. The primary route of synthesis of HLA class I molecules is dependent on peptide generation by the proteasome, whereas that of class II is not. We observed a differential effect of bortezomib on class I versus class II antibody and hypothesized that this was related to a reduced expression of class I HLA antigens. METHODS: The effect of bortezomib on HLA antibody levels was evaluated in 13 patients who were desensitized for incompatible renal transplantation. We calculated the percent difference in HLA antibody level before and after bortezomib treatment and the impact of bortezomib on HLA expression in lymphocytes of healthy control subjects. RESULTS: On average, the level of HLA class I donor-specific antibody (DSA) decreased by 32%, whereas that of class II DSA increased by 29%. In vitro bortezomib treatment of lymphocytes resulted in a mean decrease of 23% in MHC class I expression on B lymphocytes and no change (+1.08%) in MHC class II expression (P=0.0003). The amount of intracellular class I molecules was reduced by a mean of 29% with bortezomib. CONCLUSION: These data indicate that bortezomib reduces HLA class I antibody more effectively than class II antibody. This difference may be due to the reduced expression of class I molecules resulting from treatment with this proteasome inhibitor.
Asunto(s)
Ácidos Borónicos/farmacología , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/efectos de los fármacos , Fallo Renal Crónico/inmunología , Inhibidores de Proteasoma/farmacología , Pirazinas/farmacología , Anticuerpos/inmunología , Apoptosis , Bortezomib , Membrana Celular/metabolismo , Femenino , Rechazo de Injerto , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Fallo Renal Crónico/cirugía , Trasplante de Riñón , Linfocitos/inmunología , Masculino , Estudios RetrospectivosRESUMEN
Fish oil-enriched long chain n-3 polyunsaturated fatty acids disrupt the molecular organization of T-cell proteins in the immunological synapse. The impact of fish oil derived n-3 fatty acids on antigen-presenting cells, particularly at the animal level, is unknown. We previously demonstrated B-cells isolated from mice fed with fish oil-suppressed naïve CD4(+) T-cell activation. Therefore, here we determined the mechanistic effects of fish oil on murine B-cell major histocompatibility complex (MHC) class II molecular distribution using a combination of total internal reflection fluorescence, Förster resonance energy transfer and confocal imaging. Fish oil had no impact on presynaptic B-cell MHC II clustering. Upon conjugation with transgenic T-cells, fish-oil suppressed MHC II accumulation at the immunological synapse. As a consequence, T-cell protein kinase C theta (PKCθ) recruitment to the synapse was also diminished. The effects were independent of changes in B-T cell adhesion, as measured with microscopy, flow cytometry and static cell adhesion assays with select immune ligands. Given that fish oil can reorganize the membrane by lowering membrane cholesterol levels, we then compared the results with fish oil to cholesterol depletion using methyl-B-cyclodextrin (MßCD). MßCD treatment of B-cells suppressed MHC II and T-cell PKCθ recruitment to the immunological synapse, similar to fish oil. Overall, the results reveal commonality in the mechanism by which fish oil manipulates protein lateral organization of B-cells compared to T-cells. Furthermore, the data establish MHC class II lateral organization on the B-cell side of the immunological synapse as a novel molecular target of fish oil.
Asunto(s)
Linfocitos B/inmunología , Aceites de Pescado/farmacología , Antígenos de Histocompatibilidad Clase II/inmunología , Sinapsis Inmunológicas/fisiología , Animales , Colesterol/deficiencia , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/metabolismo , Isoenzimas/metabolismo , Masculino , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa C/metabolismo , Proteína Quinasa C-theta , beta-Ciclodextrinas/farmacologíaRESUMEN
BACKGROUND: The P140 phosphopeptide issued from the spliceosomal U1-70K small nuclear ribonucleoprotein protein displays protective properties in MRL/lpr lupus-prone mice. It binds both major histocompatibility class II (MHCII) and HSC70/Hsp73 molecules. P140 peptide increases MRL/lpr peripheral blood lymphocyte apoptosis and decreases autoepitope recognition by T cells. OBJECTIVE: To explore further the mode of action of P140 peptide on HSC70+ antigen-presenting cells. METHODS: P140 biodistribution was monitored in real time using an imaging system and by fluorescence and electron microscopy. Fluorescence activated cell sorting and Western blotting experiments were used to evaluate the P140 effects on autophagic flux markers. RESULTS: P140 fluorescence accumulated especially in the lungs and spleen. P140 peptide reduced the number of peripheral and splenic T and B cells without affecting these cells in normal mice. Remaining MRL/lpr B cells responded normally to mitogens. P140 peptide decreased the expression levels of HSC70/Hsp73 chaperone and stable MHCII dimers, which are both increased in MRL/lpr splenic B cells. It impaired refolding properties of chaperone HSC70. In MRL/lpr B cells, it increased the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulated lysosomal degradation during autophagic flux. CONCLUSION: The study results suggest that after P140 peptide binding to HSC70, the endogenous (auto)antigen processing might be greatly affected in MRL/lpr antigen-presenting B cells, leading to the observed decrease of autoreactive T-cell priming and signalling via a mechanism involving a lysosomal degradation pathway. This unexpected mechanism might explain the beneficial effect of P140 peptide in treated MRL/lpr mice.
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Autofagia/efectos de los fármacos , Proteínas del Choque Térmico HSC70/antagonistas & inhibidores , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Lupus Eritematoso Sistémico/patología , Fragmentos de Péptidos/farmacología , Animales , Presentación de Antígeno/efectos de los fármacos , Linfocitos B/patología , Proteínas del Choque Térmico HSC70/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/inmunología , Ratones , Ratones Endogámicos MRL lpr , Fragmentos de Péptidos/farmacocinética , Fragmentos de Péptidos/uso terapéutico , Transducción de Señal/efectos de los fármacos , Distribución TisularRESUMEN
OBJECTIVE: To evaluate the efficacy of two topically applied immunomodulative agents through the detection of lymphocyte subsets using monoclonal antibodies against CD4, CD8 and MHC II. METHODS: Fifty patients from the Departments of Medical Biochemistry, Dermatology and Pathology at Cairo University with different degrees of alopecia areata (AA) were included in this study. They were classified into two groups each of 25 patients. Each patient was treated with the immunomodulative agent on one side of the scalp and the other side was left as a control. Biopsies were taken from all patients at the beginning of treatment and at the end of the study. Tissue specimens were prepared for histologic and immunophenotypic analysis. The main outcome measures were the uses of diphencyprone (DPCP) and topical tacrolimus as two topical immunotherapeutic modalities in the treatment of AA. RESULTS: A clinical response of 68% was achieved in group A (treated with DPCP) while group B (treated with 0.1% tacrolimus) showed an insignificant clinical response. Decreased expression of CD4 and increased expression of CD8 and MHC II was detected in the post-treated areas compared with pretreated areas in cases treated with DCPC. In tacrolimus-treated cases, there was a decrease in CD4 and MHC II, with no change in CD8 between the pre- and post-treated areas. CONCLUSION: DCPC is one of the most accepted therapeutic modalities in the treatment of AA, with a favourable prognosis among patchy hair loss. MHC II expression was the one correlating with clinical response. Tacrolimus, though beneficial in other dermatoses, could not be considered effective in the treatment of AA.
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Alopecia Areata/tratamiento farmacológico , Alopecia Areata/inmunología , Ciclopropanos/uso terapéutico , Inmunosupresores/uso terapéutico , Tacrolimus/uso terapéutico , Administración Tópica , Adolescente , Adulto , Alopecia Areata/metabolismo , Anticuerpos Monoclonales , Biomarcadores , Antígenos CD4/efectos de los fármacos , Antígenos CD4/metabolismo , Antígenos CD8/efectos de los fármacos , Antígenos CD8/metabolismo , Niño , Ciclopropanos/inmunología , Egipto , Femenino , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunohistoquímica , Masculino , Dermatosis del Cuero Cabelludo/tratamiento farmacológico , Dermatosis del Cuero Cabelludo/inmunología , Dermatosis del Cuero Cabelludo/metabolismo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/metabolismo , Tacrolimus/inmunología , Resultado del Tratamiento , Adulto JovenRESUMEN
Recent studies provided evidence for a significant role of cathepsin S during extracellular remodeling in atherosclerosis. In this study, we investigated the effect of a specific cathepsin S inhibitor on atherosclerotic plaque progression in the brachiocephalic artery. Male and female Apoe-/- mice on a cholate-containing high-fat diet containing or lacking a specific cathepsin S inhibitor were evaluated for the remodeling of atherosclerotic lesions. The in vivo efficacy of the cathepsin S inhibitor was demonstrated by the inhibition of invariant chain processing in spleen. After 8 weeks of diet, brachiocephalic arteries were analyzed for plaque size, collagen, macrophage, and smooth muscle cell content, for elastic lamina breaks, and the number of buried fibrous caps. The size of atherosclerotic plaques in inhibitor-treated mice was reduced by 36% in male and 68% in female mice, and they showed significantly smaller numbers in elastin lamina breaks (60% less in males; 75% less in females), plaque macrophages (47% less in males; 40% less in females), and buried fibrous caps (50% less in males; 86% less in females). In conclusion, the inhibition of cathepsin S showed a strong atheroprotective activity, demonstrating the potential benefits of a small molecule anti-cathepsin therapy.
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Apolipoproteínas E/genética , Aterosclerosis/tratamiento farmacológico , Catepsinas/antagonistas & inhibidores , Animales , Antígenos de Diferenciación de Linfocitos B/efectos de los fármacos , Antígenos de Diferenciación de Linfocitos B/metabolismo , Aterosclerosis/genética , Tronco Braquiocefálico/patología , Catepsinas/metabolismo , Dieta Aterogénica , Femenino , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Factores Sexuales , Bazo/efectos de los fármacos , Bazo/patologíaRESUMEN
Peptides derived from exogenous proteins are presented by both MHC class I and II. Despite extensive study, the features of the endocytic pathway that mediate cross-presentation of exogenous antigens on MHC class I are not entirely understood and difficult to generalize to all proteins. Here, we used dendritic cells and macrophages to examine MHC class I and II presentation of hen egg-white lysozyme (HEL) in different forms, soluble and liposome encapsulated. Soluble HEL or HEL targeted to a late endosomal compartment only allowed for MHC class II presentation, in a process that was blocked by chloroquine and a cathepsin S (CatS) inhibitor; brefeldin A (BFA) also blocked presentation, indicating a requirement for nascent MHC class II. In contrast, liposome-encapsulated HEL targeted to early endosomes entered the MHC class I and II presentation pathways. Cross-presentation of HEL in early endosomal liposomes had several unique features: it was markedly increased by BFA and by blockade of the proteasome or CatS activity, it occurred independently of the transporter associated with antigen processing but required an MHC class I surface-stabilizing peptide, and it was inhibited by chloroquine. Remarkably, chloroquine facilitated MHC class I cross-presentation of soluble HEL and HEL in late endosomal liposomes. Altogether, MHC class I and II presentation of HEL occurred through pathways having distinct molecular and proteolytic requirements. Moreover, MHC class I sampled antigenic peptides from various points along the endocytic route.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase I/genética , Animales , Brefeldino A/farmacología , Pollos , Cloroquina/farmacología , Células Dendríticas/inmunología , Endocitosis/inmunología , Antígenos de Histocompatibilidad Clase I/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Humanos , Liposomas/química , Macrófagos/inmunología , Ratones , Ratones Endogámicos NOD/inmunología , Muramidasa/química , Muramidasa/genéticaRESUMEN
BACKGROUND: In the initial phase after cardiac transplantation, mononuclear cells infiltrate the graft initiating a relevant impulse for rejection. 3-Deazaadenosin (c3Ado), an analog of adenosine, has demonstrated in vitro anti-inflammatory properties. Furthermore, in vivo studies on arteriosclerosis development and septic myocardial dysfunction c3Ado revealed reduced cellular infiltration. In addition ischemia and reperfusion injury could be diminished in a pulmonary animal model. The aim of our study was to investigate the properties of c3Ado to reduce adhesion molecule expression and cellular infiltration in a fully allogeneic cardiac transplant model. METHODS AND RESULTS: Lewis rats were challenged with Wistar-Furth cardiac allografts. Untreated grafts were rejected within 7 days (group 1). In group 2, animals received 2 x 5 mg c3Ado SC per day. Grafts were harvested on days 1, 3, and 6 after transplantation for further examination (n = 4 per group and time point). Immunohistochemical examination revealed significant reduction of graft-infiltrating MHC II positive cells, T-cell receptor positive cells (R73), as well as ED1-positive monocytes and macrophages (P < .01) at days 3 and 6 after transplantation. Adhesion molecule (ICAM-1, VCAM-1) expression on days 1 and 3 after transplantation was almost completely diminished in c3Ado-treated grafts. CONCLUSION: Thus, c3Ado is able to reduce graft infiltration by preventing leukocyte evasion through the suppression of adhesion molecule expression. This may be a novel strategy to protect transplanted organs from early damage after transplantation and extend organ survival after transplantation.
Asunto(s)
Antiinflamatorios/uso terapéutico , Rechazo de Injerto/prevención & control , Trasplante de Corazón/inmunología , Leucocitos/fisiología , Tubercidina/uso terapéutico , Animales , Modelos Animales de Enfermedad , Trasplante de Corazón/patología , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/efectos de los fármacos , Masculino , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas WF , Trasplante Homólogo/inmunología , Trasplante Homólogo/patología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Lactoferrin, an 80-kDa iron-binding protein with immune modulating properties, is a unique adjuvant component able to enhance efficacy of the existing Mycobacterium bovis Bacillus Calmette Guerin (BCG) vaccine to protect against murine model of tuberculosis. Although identified as having effects on macrophage presentation events, lactoferrin's capability to modulate dendritic cells (DCs) function when loaded with BCG antigens has not been previously recognized. In this study, the potential of lactoferrin to modulate surface expression of MHC II, CD80, CD86 and CD40 from bone marrow-derived dendritic cells (BMDCs) was examined. Generally, lactoferrin decreased pro-inflammatory cytokines [tumor necrosis factor (TNF)-alpha, IL-6 and IL-12p40] and chemokines [macrophage inflammatory protein (MIP)-1alpha and MIP-2] and increased regulatory cytokine, transforming growth factor-beta1 and a T-cell chemotatic factor, monocyte chemotactic protein-1, from uninfected or BCG-infected BMDCs. Culturing BCG-infected BMDCs with lactoferrin also enhanced their ability to respond to IFN-gamma activation through up-regulation of maturation markers: MHC I, MHC II and the ratio of CD86:CD80 surface expression. Furthermore, lactoferrin-exposed BCG-infected DCs increased stimulation of BCG-specific CD3(+)CD4(+) splenocytes, as defined by increasing IFN-gamma production. Finally, BCG-/lactoferrin-vaccinated mice possessed an increased pool of BCG antigen-specific IFN-gamma producing CD3(+)CD4(+)CD62L(-) splenocytes. These studies suggest a mechanism in which lactoferrin may exert adjuvant activity by enhancing DC function to promote generation of antigen-specific T cells.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Vacuna BCG/inmunología , Células Dendríticas/inmunología , Lactoferrina/farmacología , Tuberculosis/prevención & control , Animales , Antígeno B7-1/efectos de los fármacos , Antígeno B7-1/inmunología , Antígeno B7-1/metabolismo , Antígeno B7-2/efectos de los fármacos , Antígeno B7-2/inmunología , Antígeno B7-2/metabolismo , Vacuna BCG/administración & dosificación , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Antígenos CD40/inmunología , Antígenos CD40/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Citocinas/efectos de los fármacos , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Ratones , Ratones Endogámicos C57BL , Mycobacterium bovis/inmunologíaAsunto(s)
Folículo Piloso/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Péptidos/farmacología , Proopiomelanocortina/farmacología , Hormona Adrenocorticotrópica/farmacología , Células Cultivadas , Folículo Piloso/inmunología , Antígenos de Histocompatibilidad Clase I/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Interferón gamma/farmacología , alfa-MSH/farmacología , betaendorfina/farmacologíaRESUMEN
Systemic lupus erythematosus (SLE) is characterized in its early stages by the expansion of autoreactive T cells that trigger B-cell activation with subsequent multi-organ injury. Dendritic cells (DCs) in lupus were found to display an aberrant phenotype with higher expression of the maturation markers major histocompatibility complex (MHC) class II, CD80 and CD86, as well as higher production of proinflammatory cytokines including interleukin-12 (IL-12), resulting in an increased ability to activate T cells. A peptide (hCDR1) based on the complementarity determining region-1 of an anti-DNA antibody ameliorated SLE in both induced and spontaneous lupus models by downregulating T-cell functions. Our objectives were to determine whether DCs play a role in promoting the beneficial effects of hCDR1. We showed here that treatment with hCDR1 lowered the expression levels of MHC class II, CD80 and CD86 on DCs. The latter effect was associated with downregulation of messenger RNA expression and secretion of IL-12, a cytokine that upregulated T-cell proliferation and interferon-gamma (IFN-gamma) secretion. Moreover, DCs derived from hCDR1-treated mice downregulated proliferation and IFN-gamma secretion by T cells from untreated mice. Upregulation of transforming growth factor-beta (TGF-beta) secretion by T cells, following treatment with hCDR1, resulted in downregulation of IFN-gamma production and contributed to the phenotypic changes and magnitude of IL-12 secretion by DCs. The ameliorating effects of hCDR1 are therefore mediated at least partially by the upregulated secretion of TGF-beta by T cells that contribute to the induction of DCs with immature phenotype and suppressed functions. The resulting DCs further downregulate autoreactive T-cell functions.
Asunto(s)
Autoantígenos/uso terapéutico , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Proteínas del Tejido Nervioso/uso terapéutico , Animales , Autoantígenos/farmacología , Antígeno B7-1/efectos de los fármacos , Antígeno B7-1/inmunología , Antígeno B7-2/efectos de los fármacos , Antígeno B7-2/inmunología , Células Dendríticas/efectos de los fármacos , Regulación hacia Abajo , Femenino , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Humanos , Interferón gamma/agonistas , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-12/antagonistas & inhibidores , Interleucina-12/inmunología , Interleucina-12/farmacología , Ratones , Ratones Endogámicos BALB C , Proteínas del Tejido Nervioso/farmacología , Péptidos/farmacología , Péptidos/uso terapéutico , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia ArribaRESUMEN
Equine dendritic cells (eqDC) can be generated from peripheral blood monocytes by propagation in GM-CSF and IL-4. Despite similarities with the generation of human DC, we found significant improvements for eqDC generation and functional influences on eqDC maturation. The fractionation of peripheral blood mononuclear cells (PBMC) by two subsequent gradients at densities of 1.090 and 1.077 as well as an adherence step in AIM V((R)) medium on dishes coated with extracellular matrix components (Primaria) improved the purity and yield of DC. After 3 days, eqDC cultures with GM-CSF alone developed into three subsets of (i) MHC II(neg) cells, (ii) MHC II(low) immature, endocytic cells and (iii) MHC II(high) spontaneously mature, non-endocytic DC. The immature DC fraction of the GM-CSF cultures matured, as detected by MHC II up-regulation, upon LPS exposure overnight. DC cultures in GM-CSF plus IL-4 resulted in higher cell yields, a loss of the immature MHC II(low) population but increased mature MHC II(high) DC, suggesting maturation. However, the MHC II(high) DC fraction was still endocytically active and did not lose their endocytic function after LPS treatment. They marginally up-regulated MHC II expression but this did not result in an enhanced stimulation of an allogeneic mixed lymphocyte reaction. However, LPS treatment clearly induced mRNA for IL-12p35 and p40, which was not observed by addition of IL-4 alone. Together our data indicate that IL-4 and LPS induce two different maturation programs. IL-4 induces a semi-maturation where the cells are still endocytic, which can be further matured to secrete cytokines in a second step by LPS.