RESUMEN
Severe acute respiratory syndrome coronavirus 2 infection and development of coronavirus disease 2019 presents a major health care challenge of global dimensions. Laboratory diagnostics of infected patients, and the assessment of immunity against severe acute respiratory syndrome coronavirus 2, presents a major cornerstone in handling the pandemic. Currently, there is an increase in demand for antibody testing and a large number of tests are already marketed or are in the late stage of development. However, the interpretation of test results depends on many variables and factors, including sensitivity, specificity, potential cross-reactivity and cross-protectivity, the diagnostic value of antibodies of different isotypes, and the use of antibody testing in identification of acutely ill patients or in epidemiological settings. In this article, the recently established COVID-19 Task Force of the German Society for Clinical Chemistry and Laboratory Medicine (DGKL) addresses these issues on the basis of currently available data sets in this rapidly moving field.
Asunto(s)
Anticuerpos Antivirales/sangre , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/diagnóstico , Pruebas Inmunológicas/métodos , Neumonía Viral/diagnóstico , Pruebas Serológicas/métodos , Betacoronavirus , COVID-19 , Prueba de COVID-19 , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/inmunología , Humanos , Pandemias , Neumonía Viral/sangre , Neumonía Viral/inmunología , SARS-CoV-2RESUMEN
In the murine model of pulmonary infection with Cryptococcus neoformans, IL-4 receptor α (IL-4Rα)-dependent polyfunctional T(h)2 cells induce disease progression associated with alternative activation of lung macrophages. To characterize the effector role of IL-4Rα-dependent alternatively activated macrophages (aaMph), we intra-nasally infected mice with genetically ablated IL-4Rα expression on macrophages (LysM(Cre)IL-4Rα(-/lox) mice) and IL-4Rα(-/lox) littermates. LysM(Cre)IL-4Rα(-/lox) mice were significantly more resistant to pulmonary cryptococcosis with higher survival rates and lower lung burden than non-deficient heterozygous littermates. Infected LysM(Cre)IL-4Rα(-/lox) mice had reduced but detectable numbers of aaMph expressing arginase-1, chitinase-like enzyme (YM1) and CD206. Similar pulmonary expression of inducible nitric oxide synthase was found in LysM(Cre)IL-4Rα(-/lox) and IL-4Rα(-/lox) control mice, but macrophages from LysM(Cre)IL-4Rα(-/lox) mice showed a higher potential to produce nitric oxide. In contrast to the differences in the macrophage phenotype, pulmonary T(h)2 responses were similar in infected LysM(Cre)IL-4Rα(-/lox) and IL-4Rα(-/lox) mice with each mouse strain harboring polyfunctional T(h)2 cells. Consistently, type 2 pulmonary allergic inflammation associated with eosinophil recruitment and epithelial mucus production was present in lungs of both LysM(Cre)IL-4Rα(-/lox) and IL-4Rα(-/lox) mice. Our results demonstrate that, despite residual IL-4Rα-independent alternative macrophage activation and ongoing T(h)2-dependent allergic inflammation, abrogation of IL-4Rα-dependent aaMph is sufficient to confer resistance in pulmonary cryptococcosis. This is even evident on a relatively resistant heterozygous IL-4Rα(+/-) background indicating a key contribution of macrophage IL-4Rα expression to susceptibility in allergic bronchopulmonary mycosis.
Asunto(s)
Criptococosis/inmunología , Enfermedades Pulmonares Fúngicas/inmunología , Macrófagos/inmunología , Receptores de Superficie Celular/inmunología , Células Th2/inmunología , Animales , Cryptococcus neoformans/inmunología , Cryptococcus neoformans/aislamiento & purificación , Modelos Animales de Enfermedad , Femenino , Macrófagos/citología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Receptores de Superficie Celular/deficienciaRESUMEN
BACKGROUND: Advanced liver disease predisposes to bacterial translocation and endotoxaemia which can contribute to elevated circulating levels of IL-10 and down-regulation of MHC class II on antigen-presenting cells. We sought to evaluate antigen-specific T-cell responses toward common viral antigens in order to investigate defects in cellular immunity in cirrhosis. METHODS: Peripheral blood was obtained from 22 cirrhotic patients with systemic inflammation, 13 cirrhotic patients without systemic inflammation and 14 healthy controls. C-reactive protein was used as an indicator for systemic inflammation using a cut-off of 10 mg/l. Intracellular Th1 cytokines were quantified after T cell-stimulation with the viral peptides EBNA1 and BZLF1 or the bacterial superantigen SEB by flow cytometry. Serum levels of lipopolysaccharide-binding protein (LBP) and IL-10 were quantified by ELISA. RESULTS: Compared to healthy controls, patients with cirrhosis had higher circulating levels of LBP and IL-10, an expansion of peripheral blood CD14+ monocytes with low HLA-DR expression and an increased fraction of CD25-positive CD4+ and CD8+ T cells. These findings were most pronounced in cirrhotic patients with systemic inflammation but fell short of reaching statistical significance when comparing against cirrhotic patients without systemic inflammation. In the former group TNF-α production in CD4+ and CD8+ T cells was reduced after stimulation with SEB, whereas there was no significant difference between the total cohort of cirrhotic patients and controls. After stimulation with the overlapping peptide pools for viral antigens EBNA1 and BZLF1, the number of responding T cells and the amount of TNF-α or IFN-γ production did not differ between the three pre-defined groups. However, cirrhotic patients with null-responses to EBV peptides had significantly higher serum IL-10 levels than responders to EBV peptides. Furthermore, TNF-α production in responding T cells was attenuated in patients with a high frequency of CD14+ HLA-DR- monocytes. CONCLUSION: Our data suggest that bacterial translocation, endotoxaemia, inflammation and T cell activation in cirrhosis are accompanied by an increase in circulating anti-inflammatory cytokines, reduced monocytic MHC class II expression and attenuated cytokine production in T cells. These changes are likely to contribute to altered adaptive immune responses during infection or after vaccination.
Asunto(s)
Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Antígenos HLA-DR/metabolismo , Interleucinas/sangre , Cirrosis Hepática/patología , Monocitos/inmunología , Proteínas de Fase Aguda , Adulto , Anciano , Anciano de 80 o más Años , Proteína C-Reactiva/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas Portadoras/sangre , Estudios de Casos y Controles , Citocinas/sangre , Femenino , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Cirrosis Hepática/inmunología , Cirrosis Hepática/metabolismo , Masculino , Glicoproteínas de Membrana/sangre , Persona de Mediana Edad , Monocitos/patologíaRESUMEN
Background: Chronic CMV infection drives the clonal expansion and accumulation of terminally differentiated, dysfunctional CMV-specific T-cells. CMV infection also appears to accelerate the differentiation of non-CMV-specific T-cells; however, the extent of this phenomenon is unclear. Methods: The distribution of CD4 and CD8 T-cells into four memory subsets determined by CD45RA and CCR7 expression was analyzed in 96 CMV-infected (CMV+) and 81 CMV-uninfected (CMV-) older individuals. In CMV+ individuals, the distribution of IFN-γ producing CMV-specific T-cells into the same subsets was analyzed following stimulation with 16 different CMV antigens using flowcytometry (intracellular cytokine staining). We used previously published results to extrapolate the relative size of the entire CMV-specific CD4 and CD8 T-cell response from the summated response to selected antigens. The T-cell memory subset distribution across all CMV antigen-induced responses (weighted mean) was then used to calculate memory subset proportions (in % of CD4 or CD8 T-cells) of CMV-specific and non-CMV-specific T-cells. These were compared to the corresponding proportions in CMV- individuals. Results: Only a minority (20%-30%) of CMV+ individuals displayed overall proportions of terminally differentiated T-cell memory subsets above an upper outlier boundary defined in CMV- individuals. The calculated proportions of these subsets among non-CMV-specific T-cells in CMV+ individuals also exceeded the corresponding proportions in CMV- people, suggesting that their differentiation could be CMV-driven. In CMV+ people showing overall subset distributions within the outlier limits, the memory subset distributions of non-CMV-specific T-cells were more like those in CMV- people. Logistic regression revealed that CMV infection, age, and sex all had significant effects on one or more of the non-CMV-specific CD4 or CD8 T-cell memory subsets in CMV+ individuals, with CMV infection showing the strongest effect overall. Surprisingly, except for the CD45RA-/CCR7- CD4 T-cell subset, we only found weak correlations between corresponding memory subset proportions among all T-cells and CMV-specific T-cells. Conclusion: Our analysis supports an effect of CMV infection on non-CMV-specific T-cells; however, it is limited to a minority of individuals and not closely related to the degree of memory subset differentiation of CMV-specific T-cells. We propose that unknown predisposing factors might determine to what extent CMV infection affects non-CMV-specific T-cell differentiation.
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Infecciones por Citomegalovirus , Citomegalovirus , Humanos , Receptores CCR7 , Subgrupos de Linfocitos T , Linfocitos T CD8-positivosRESUMEN
The absence of IFN-γ signaling leads to an increased inflammatory response in many murine models of autoimmune diseases induced by a CFA-assisted immunization schedule. We investigated the role of endogenous IFN-γ in arthritis induced by immunization with glucose-6-phosphate isomerase (G6PI) in CFA in DBA/1 mice. Surprisingly, and in contrast to our previous findings in collagen-induced arthritis (CIA), G6PI-induced arthritis was found to be reduced in IFN-γ receptor-deficient (IFN-γR KO) mice, demonstrating a proinflammatory role for IFN-γ in this model. Milder disease in IFN-γR KO mice was associated with less vigorous innate and adaptive immune responses early (day 9) after immunization: less proliferation of myeloid cells in the spleen, less osteoclast formation, less G6PI-reactive Th cells (as measured by ex vivo stimulation and flow cytometry and by in vivo skin reactivity to G6PI) and lower G6PI-specific immunoglobulin serum levels. Surprisingly, on day 21, despite continued milder disease in IFN-γR KO mice, their Th cell responses were no longer diminished but augmented as compared to wild-type mice, and their numbers of immature myeloid splenocytes were also more increased. These data reveal that IFN-γ signaling is critical for the induction of the early immune responses which trigger G6PI-induced arthritis. The strikingly different clinical consequences of absent IFN-γ signaling in G6PI-induced arthritis compared with the very similarly induced CIA emphasize that the role of a single cytokine in experimentally induced arthritis depends critically on the very nature of the inciting (auto)antigen and in particular on the kinetics of the disease manifestation elicited by the antigen.
Asunto(s)
Artritis Experimental/inmunología , Glucosa-6-Fosfato Isomerasa/inmunología , Interferón gamma/inmunología , Receptores de Interferón/inmunología , Animales , Artritis Experimental/genética , Artritis Experimental/patología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Femenino , Citometría de Flujo , Predisposición Genética a la Enfermedad , Glucosa-6-Fosfato Isomerasa/administración & dosificación , Inmunidad Celular/inmunología , Inmunidad Innata/inmunología , Inmunización , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Osteoclastos/inmunología , Osteoclastos/metabolismo , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptor de Interferón gammaRESUMEN
Reactive oxygen species (ROS) are important in the immune defense against invading pathogens, but they are also key molecules in the regulation of inflammatory reactions. Low levels of ROS production due to a polymorphism in the neutrophil cytosolic factor 1 (Ncf1) gene are associated with autoimmunity and arthritis severity in mouse models induced with adjuvant. We established an adjuvant-free arthritis model in which disease is induced by injection of the autoantigen collagen type II (CII) and depends on IL-5-producing T cells and eosinophils. In addition, the transgenic expression of mutated mouse CII allowed us to investigate an autoreactive immune response to an autologous Ag and by that natural tolerance mechanism. We show that a deficient ROS production, due to a spontaneous mutation in Ncf1, leads to increased autoantibody production and expansion of IL-33R-expressing T cells, impaired T cell tolerance toward tissue-specific CII, and severe arthritis in this unique model without disturbing adjuvant effects. These results demonstrate that the insufficient production of ROS promotes the breakdown of immune tolerance and development of autoimmune and adjuvant-free arthritis through an IL-5- and IL33R-dependent T cell activation pathway.
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Artritis Experimental/etiología , Interleucina-5/metabolismo , NADPH Oxidasas/genética , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina/metabolismo , Estallido Respiratorio/fisiología , Linfocitos T/patología , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/inmunología , Colágeno Tipo II/administración & dosificación , Colágeno Tipo II/efectos adversos , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Interleucinas , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , NADPH Oxidasas/fisiología , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T/inmunologíaRESUMEN
Regulatory T cells (Tregs) fulfill a central role in immune regulation. We reported previously that the integrin alphaEbeta7 discriminates distinct subsets of murine CD4+ regulatory T cells. Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. alphaE-CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues. In contrast, alphaE -positive subsets (CD25+ and CD25-) displayed an effector/memory phenotype expressing high levels of E/P-selectin-binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites. Accordingly, alphaE -expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Memoria Inmunológica/inmunología , Animales , Artritis Experimental/inmunología , Antígenos CD4/inmunología , Inmunofenotipificación , Inflamación/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Interleucina-2/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/inmunologíaRESUMEN
OBJECTIVES: Glucose-6-phosphate isomerase (G6PI)-induced arthritis is a spontaneously remitting experimental arthritis model. It was hypothesised that regulatory T cells (Tregs) are involved in remission and their role in G6PI-induced arthritis was investigated. METHODS: Tregs were depleted by injection of anti-CD25 before immunisation of DBA/1 mice with G6PI. The severity of arthritis was assessed clinically and histologically and the number and function of G6PI-specific T helper (Th) cells were determined by flow cytometry. Th cells and monocytes/macrophages were depleted using anti-CD4 or clodronate-containing liposomes. RESULTS: Injection of anti-CD25 depleted Tregs transiently. Normal numbers of Tregs were restored 5 weeks after G6PI immunisation. Whereas arthritis started to resolve in control mice 3 weeks after immunisation with G6PI, severe arthritis was still present in the anti-CD25-treated mice 12 weeks after immunisation. The most striking ex vivo correlate of non-remitting arthritis was a strong increase in G6PI-specific Th cells 3 days after G6PI immunisation. This difference between treated and control mice declined at later time points. Depletion of CD4 cells ameliorated arthritis in controls but not in anti-CD25-treated mice. In contrast, clodronate-containing liposomes were an effective treatment in both groups. CONCLUSIONS: Tregs control the transition from acute self-limiting to non-remitting destructive G6PI-induced arthritis already in the preclinical disease stage. Once established, non-remitting destructive arthritis is not controlled by restoration of normal Treg numbers. These findings question the rationale of therapeutic approaches augmenting Treg number or function in established arthritis.
Asunto(s)
Artritis Experimental/inmunología , Linfocitos T Reguladores/inmunología , Enfermedad Aguda , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/patología , Linfocitos T CD4-Positivos/inmunología , Enfermedad Crónica , Progresión de la Enfermedad , Glucosa-6-Fosfato Isomerasa/inmunología , Inmunización , Inmunoglobulina G/biosíntesis , Subunidad alfa del Receptor de Interleucina-2/inmunología , Ratones , Ratones Endogámicos DBARESUMEN
OBJECTIVES: Inducible costimulator (ICOS) and its ligand (ICOSL) regulate T and B cell responses. Glucose-6-phosphate isomerase (G6PI)-induced arthritis requires T and B lymphocytes. It was hypothesised that blocking ICOS/ICOSL interactions ameliorates G6PI-induced arthritis and reduces G6PI-specific B and T lymphocyte responses. METHODS: DBA/1 mice were injected with a blocking, non-depleting anti-ICOSL monoclonal antibodies (mAbs) during the induction or effector phase of G6PI-induced arthritis. G6PI-specific antibody responses were measured by ELISA. G6PI-specific T helper (Th) cell responses were assayed by polychromatic flow cytometry. RESULTS: Transient blockade of ICOS/ICOSL interactions profoundly reduced the severity of G6PI-induced arthritis. ELISA and proliferation assays showed no clear ex vivo correlates of protection. Polychromatic flow cytometry revealed two major findings: the absolute number of G6PI-specific Th cells was markedly diminished in secondary lymphatic organs from mice with blocked ICOS/ICOSL interactions. Within the pool of G6PI-specific Th cells the frequency of interleukin 17 (IL17), interferon gamma or tumour necrosis factor alpha producers or polyfunctional Th cells (expressing two or more of these cytokines) was higher in treated than in control mice. CONCLUSIONS: ICOS costimulation is not mandatory for the differentiation of Th1 or Th17 cells. Instead, the lack of ICOS costimulation results in reduced survival of G6PI-specific Th cells irrespective of their functional differentiation. This study demonstrates that a thorough examination of the quantity and the quality of antigen-specific immune responses is useful to determine ex vivo correlates of efficacy for immunomodulating treatments.
Asunto(s)
Anticuerpos Bloqueadores/uso terapéutico , Antígenos de Diferenciación de Linfocitos T/inmunología , Artritis Experimental/prevención & control , Artritis Reumatoide/prevención & control , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/prevención & control , Diferenciación Celular/inmunología , Citocinas/biosíntesis , Glucosa-6-Fosfato/inmunología , Inmunoglobulinas/biosíntesis , Ligando Coestimulador de Linfocitos T Inducibles , Proteína Coestimuladora de Linfocitos T Inducibles , Interleucina-17/análisis , Ligandos , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Ratones , Ratones Endogámicos DBA , Proteínas/inmunología , Células TH1/inmunologíaRESUMEN
EBV is a candidate trigger of rheumatoid arthritis (RA). We determined both EBV-specific T cell and B cell responses and cell-associated EBV DNA copies in patients with RA and demographically matched healthy virus carriers. Patients with RA showed increased and broadened IgG responses to lytic and latent EBV-encoded Ags and 7-fold higher levels of EBV copy numbers in circulating blood cells. Additionally, patients with RA exhibited substantial expansions of CD8(+) T cells specific for pooled EBV Ags expressed during both B cell transformation and productive viral replication and the frequency of CD8(+) T cells specific for these Ags correlated with cellular EBV copy numbers. In contrast, CD4(+) T cell responses to EBV and T cell responses to human CMV Ags were unchanged, altogether arguing against a defective control of latent EBV infection in RA. Our data show that the regulation of EBV infection is perturbed in RA and suggest that increased EBV-specific effector T cell and Ab responses are driven by an elevated EBV load in RA.
Asunto(s)
Artritis Reumatoide/inmunología , Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Subgrupos de Linfocitos T/inmunología , Antirreumáticos/farmacología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/virología , Linfocitos B/metabolismo , Linfocitos B/virología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Humanos , Inmunoglobulina G/inmunología , Memoria Inmunológica , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/virología , Carga ViralRESUMEN
BACKGROUND: Fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) are autonomously activated to maintain inflammation and joint destruction in co-transplantation models. To elucidate inducing mechanisms involved in this altered behavior, the arthritogenic potential of FLSs from murine antigen-induced arthritis (AIA) were investigated in a transfer model. METHODS: FLSs were isolated, expanded in vitro, and transferred into knee joint cavities of severe combined immunodeficient (SCID) mice. Their arthritogenic capacity was assessed by monitoring joint swelling and evaluation of histological parameters 70 to 100 days after transfer. RESULTS: FLSs from AIA mice were able to transfer arthritis into recipient SCID mice. FLS transfer induced a chronic arthritis with recruitment of inflammatory cells and marked cartilage destruction. Long-lasting inflammation was not required for imprinting of arthritogenicity in FLSs since cells isolated from acute arthritic joints were fully competent to transfer arthritis. We also observed arthritogenic potential in FLSs isolated from contralateral non-arthritic joints in our monoarticular arthritis model. CONCLUSIONS: We show that the transformation of FLSs into arthritogenic cells occurs early in arthritis development. This challenges current hypotheses on the role of these cells in arthritis pathogenesis and opens up the way for further mechanistic studies.
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Artritis Experimental/patología , Artritis Reumatoide/patología , Fibroblastos/patología , Articulación de la Rodilla/patología , Sinoviocitos/patología , Animales , Movimiento Celular , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Inflamación/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones SCIDRESUMEN
In the past few years it has been become increasingly clear that T cells capable of actively suppressing immune responses are thought to be in part responsible for the maintenance of peripheral self tolerance. In healthy rodents and humans, CD4(+) T cells constitutively expressing the interleukin (IL)-2 receptor alpha-chain (CD25) are able to exert such suppressive function in vitro and in vivo. Despite great efforts in our understanding of the biology of such immunoregulatory T cells, there are still certain points incompletely understood. Although some authors suggest that immunoregulatory cytokines such as IL-10 or transforming growth factor-beta are critical for the suppressive effect of these cells, this is controversial and the exact molecular nature and the targets of suppression are largely unknown. Thus far, until regulatory T cells can be used for diagnostic or therapeutic purposes many questions have to be answered. In this review we summarize the current knowledge on the function and properties of this T cell subset and discuss their potential role in human autoimmune or chronic inflammatory diseases.
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Inmunoterapia , Linfocitos T Reguladores/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/terapia , Citocinas/inmunología , Humanos , Inmunoterapia Adoptiva , Activación de Linfocitos , Neoplasias/inmunología , Neoplasias/terapia , Receptores de Interleucina-2/inmunologíaRESUMEN
PURPOSE: Near-infrared range fluorescence (NIRF) imaging is a potential tool to diagnose biologic processes in vivo. This applicability study sought to define whether imaging with fluorochrome Cy5.5 can identify arthritis in murine antigen-induced arthritis (AIA). MATERIALS AND METHODS: On day 7 of AIA (n = 9 mice), fluorescence intensities in inflamed and contralateral knee joints (the latter as internal control) were measured before and after intravenous injection of Cy5.5 (until 72 hours). Cy5.5 joint deposition was verified by confocal laser-scanning microscopy. Dye phagocytosis was evaluated in cultured macrophages (cell line PMJ2-R) by FACS analysis. Cy5.5 binding to serum protein was tested by NIRF scanning and gel electrophoresis. RESULTS: Between 2 and 72 hours, the arthritic knee joints showed significantly higher fluorescence intensities compared with contralateral joints. Microscopy confirmed Cy5.5 deposition in the synovial membrane. Cultured macrophages actively phagocytosed Cy5.5. Cy5.5 bound mainly to albumin as the main serum protein. CONCLUSION: NIRF imaging with Cy5.5 can identify arthritic joints in vivo, likely due to nonspecific deposition.
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Artritis Experimental/diagnóstico , Carbocianinas , Fluorometría , Articulación de la Rodilla/patología , Macrófagos , Espectroscopía Infrarroja Corta , Animales , Antígenos de Diferenciación , Carbocianinas/farmacocinética , Estudios de Factibilidad , Colorantes Fluorescentes/farmacocinética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía ConfocalRESUMEN
RATIONALE AND OBJECTIVES: The detection of arthritis by autofluorescence was investigated using an antigen-induced arthritis model. METHODS: For autofluorescence investigations of joints, a mobile fluorescence-detector was constructed consisting of a lens/mirror system attached to a conventional spectrofluorometer and optimized fiber optic cables reaching to and from the site of investigation. Autofluorescence measurements were performed at 7 arthritic and 7 healthy mice. Fifteen antigen-induced arthritis and 3 healthy mice were used for histologic examinations. RESULTS: In the exudative stage (day 1), a decrease of emission signal intensities for excitation wavelengths at 300 nm (emission, 355-365 nm) and 360 nm (emission, 475-485 nm) was observed. Signals increased on day 7 (maximum of cellular infiltration). Chronic inflammation (day 14 and 21) led to a decrease of signals again. CONCLUSION: Arthritis influences autofluorescence signals in vivo. The detected excitation/emission pairs can be assigned to collagen/elastin and NAD(P)H. Signal intensities of NAD(P)H differed significantly from controls at day 1 and 7.
Asunto(s)
Artritis Experimental/diagnóstico , Fluorescencia , Animales , Artritis Experimental/patología , Colágeno/análisis , Elastina/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , NAD/análisis , NADP/análisisRESUMEN
RATIONALE AND OBJECTIVES: Autofluorescence can be exploited to obtain spectroscopic information about tissues or organs in a noninvasive fashion. The knowledge of normal organ patterns is a prerequisite for subsequent characterization of pathological states, eg, inflammation or tumors. Therefore, the aim of this study was to investigate the autofluorescence properties of healthy organs in mice. MATERIALS AND METHODS: Organs from C57Bl/6 mice were removed in toto and stored in physiologic sodium chloride solution on ice (non-perfused specimens). Investigations were performed with a custom-made mobile fluorescence detector. Excitation-emission matrices (EEMs) were measured in selected organs (bladder, brain, kidney, liver, and spleen) (n = 5). Afterwards, single-emission spectra were obtained in selected organs (bladder, colon, brain, kidney, liver, and spleen) and peak fluorescence signal intensities were calculated (n = 9). RESULTS: EEMs showed that excitation at wavelengths from 300-310 nm (emission spectra in all samples of bladder and brain; probably caused by collagen/elastin) and from 350-360 nm (emission spectra in all samples with the exception of spleen; probably caused by NAD(P)H) seem to be best suited for autofluorescence measurements in organs. The single-emission spectra measurements were noticeably different in terms of occurrence (yes/no response) and intensity of fluorescence emission peaks in different organs. CONCLUSION: Combined autofluorescence measurements of collagen/elastin (for structural information) and NAD(P)H (for functional information) allow conclusions about the target organs. Therefore, autofluorescence measurements seem to be a diagnostic tool feasible for characterization of tissue.
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Encéfalo/fisiología , Colon/fisiología , Riñón/fisiología , Hígado/fisiología , Espectrometría de Fluorescencia , Bazo/fisiología , Vejiga Urinaria/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Characterization of the response of the host immune system is important in understanding the bidirectional interactions between the host and microbial pathogens. For research on the host site, flow cytometry has become one of the major tools in immunology. Advances in technology and reagents allow now the simultaneous assessment of multiple markers on a single cell level generating multidimensional data sets that require multivariate statistical analysis. We explored the explanatory power of the supervised machine learning method called "induction of decision trees" in flow cytometric data. In order to examine whether the production of a certain cytokine is depended on other cytokines, datasets from intracellular staining for six cytokines with complex patterns of co-expression were analyzed by induction of decision trees. After weighting the data according to their class probabilities, we created a total of 13,392 different decision trees for each given cytokine with different parameter settings. For a more realistic estimation of the decision trees' quality, we used stratified fivefold cross validation and chose the "best" tree according to a combination of different quality criteria. While some of the decision trees reflected previously known co-expression patterns, we found that the expression of some cytokines was not only dependent on the co-expression of others per se, but was also dependent on the intensity of expression. Thus, for the first time we successfully used induction of decision trees for the analysis of high dimensional flow cytometric data and demonstrated the feasibility of this method to reveal structural patterns in such data sets.
RESUMEN
The therapeutic benefit of B cell depletion in patients with rheumatoid arthritis has provided proof of concept that B cells are relevant for the pathogenesis of arthritis. It remains unknown which B cell effector functions contribute to the induction or chronification of arthritis. We studied the clinical and immunological effects of B cell depletion in glucose-6-phosphate isomerase-induced arthritis. We targeted CD22 to deplete B cells. Mice were depleted of B cells before or after immunization with glucose-6-phosphate isomerase (G6PI). The clinical and histological effects were studied. G6PI-specific antibody responses were measured by ELISA. G6PI-specific T helper (Th) cell responses were assayed by polychromatic flow cytometry. B cell depletion prior to G6PI-immunization prevented arthritis. B cell depletion after immunization ameliorated arthritis, whereas B cell depletion in arthritic mice was ineffective. Transfer of antibodies from arthritic mice into B cell depleted recipients did not reconstitute arthritis. B cell depleted mice harbored much fewer G6PI-specific Th cells than control animals. B cell depletion prevents but does not cure G6PI-induced arthritis. Arthritis prevention upon B cell depletion is associated with a drastic reduction in the number of G6PI-specific effector Th cells.
Asunto(s)
Artritis/inmunología , Linfocitos B/inmunología , Glucosa-6-Fosfato Isomerasa/inmunología , Animales , Formación de Anticuerpos/inmunología , Artritis/etiología , Artritis/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , RatonesRESUMEN
INTRODUCTION: CD4+ T cells express K(2P)5.1 (TWIK-related acid-sensitive potassium channel 2 (TASK2); KCNK5), a member of the two-pore domain potassium channel family, which has been shown to influence T cell effector functions. Recently, it was shown that K(2P)5.1 is upregulated upon (autoimmune) T cell stimulation. The aim of this study was to correlate expression levels of K(2P)5.1 on T cells from patients with rheumatoid arthritis (RA) to disease activity in these patients. METHODS: Expression levels of K(2P)5.1 were measured by RT-PCR in the peripheral blood of 58 patients with RA and correlated with disease activity parameters (C-reactive protein levels, erythrocyte sedimentation rates, disease activity score (DAS28) scores). Twenty patients undergoing therapy change were followed-up for six months. Additionally, synovial fluid and synovial biopsies were investigated for T lymphocytes expressing K(2P)5.1. RESULTS: K(2P)5.1 expression levels in CD4+ T cells show a strong correlation to DAS28 scores in RA patients. Similar correlations were found for serological inflammatory parameters (erythrocyte sedimentation rate, C-reactive protein). In addition, K(2P)5.1 expression levels of synovial fluid-derived T cells are higher compared to peripheral blood T cells. Prospective data in individual patients show a parallel behaviour of K(2P)5.1 expression to disease activity parameters during a longitudinal follow-up for six months. CONCLUSIONS: Disease activity in RA patients correlates strongly with K(2P)5.1 expression levels in CD4+ T lymphocytes in the peripheral blood in cross-sectional as well as in longitudinal observations. Further studies are needed to investigate the exact pathophysiological mechanisms and to evaluate the possible use of K(2P)5.1 as a potential biomarker for disease activity and differential diagnosis.
Asunto(s)
Artritis Reumatoide/metabolismo , Artritis Reumatoide/fisiopatología , Linfocitos T CD4-Positivos/metabolismo , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Artritis Reumatoide/inmunología , Western Blotting , Separación Celular , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
T-helper (Th) lymphocytes contribute to arthritis pathogenesis by helping B cells to produce antibodies, by producing cytokines that activate effector cells involved in the destruction of cartilage and bone, and by contributing to osteoclast differentiation. There are murine models of arthritis, most notably collagen- and proteoglycan-induced arthritis, in which arthritis depends on T-cell recognition of antigens that are expressed in the joints. In spite of this, we still do not know the antigens recognised by arthritogenic Th cells in humans. Moreover, current evidence for Th cells exerting arthritogenic effector functions within the joints is only indirect.
Asunto(s)
Artritis Experimental/fisiopatología , Artritis/fisiopatología , Linfocitos T Colaboradores-Inductores/fisiología , Animales , Antígenos/inmunología , Artritis/inmunología , Artritis Experimental/inmunología , Modelos Animales de Enfermedad , Clorhidrato de Fingolimod , Humanos , Articulaciones/inmunología , Articulaciones/fisiopatología , Lisofosfolípidos/agonistas , Ratones , Glicoles de Propileno/farmacología , Esfingosina/agonistas , Esfingosina/análogos & derivados , Esfingosina/farmacología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
INTRODUCTION: T-helper (Th) lymphocytes are critically required for the pathogenesis of glucose-6-phosphate isomerase (G6PI)-induced arthritis, but neither the G6PI epitopes recognized by arthritogenic T cells nor their pathogenic effector functions have been fully elucidated to date. We aimed at identifying arthritogenic G6PI peptides. METHODS: We used a library of overlapping peptides spanning the entire G6PI sequence to identify the epitopes recognized by G6PI-specific Th cells. Immunodominant peptides were then used to immunize mice. Arthritis development was evaluated clinically and histologically. The humoral and cellular immune responses upon peptide immunization were analyzed by ELISA and multiparameter flow cytometry, respectively. RESULTS: We identified six immunodominant T-cell epitopes in DBA/1 mice, of which three are arthritogenic. One of these peptides (G6PI469-483) is identical in man and mice. Immunization with this peptide induces arthritis, which is less severe and of shorter duration than arthritis induced by immunization with full-length G6PI. Upon immunization with either G6PI or peptide, the antigen-specific Th cells produce IL-17, RANKL, IFNgamma and TNFalpha. CONCLUSIONS: We identified immunodominant and arthritogenic epitopes of G6PI. Not all immunodominant peptides are arthritogenic. This is the first description of arthritis induced by immunization with a self-peptide in mice.