RESUMEN
BACKGROUND: Osteosarcoma (OS) is a primary bone malignancy that mostly affects young people, characterized by high metastatic potential, and a marked chemoresistance that is responsible for disease relapse in most patients. Therefore, it is necessary to identify novel molecules to setup targeted strategies to improve the clinical outcome. The enzyme nicotinamide N-methyltransferase (NNMT) catalyses the N-methylation of nicotinamide and other analogs, playing a crucial role in the biotransformation of drugs and xenobiotics. NNMT overexpression was reported in a wide variety of cancers, and several studies demonstrated that is able to promote cell proliferation, migration and resistance to chemotherapy. The aim of this study was to explore the potential involvement of NNMT in OS. METHODS: Immunohistochemical analyses have been performed to evaluate NNMT expression in selected OS and healthy bone tissue samples. Subsequently, OS cell lines have been transfected with vectors targeting NNMT mRNA (shRNAs) and the impact of this downregulation on migration, cell proliferation, and response to chemotherapeutic treatment was also analysed by wound healing, MTT, SRB and Trypan blue assays, respectively. RESULTS: Results showed that OS samples display a significantly higher NNMT expression compared with healthy tissue. Preliminary results suggest that NNMT silencing in OS cell lines is associated to a decrease of cell proliferation and migration, as well as to enhanced sensitivity to chemotherapy. Data obtained showed that NNMT may represent an interesting marker for OS detection and a promising target for effective anti-cancer therapy.
Asunto(s)
Neoplasias Óseas , Nicotinamida N-Metiltransferasa , Osteosarcoma , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Adulto Joven , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Neoplasias Óseas/tratamiento farmacológico , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Resistencia a Antineoplásicos/genética , Nicotinamida N-Metiltransferasa/metabolismo , Nicotinamida N-Metiltransferasa/genética , Osteosarcoma/genética , Osteosarcoma/patología , Osteosarcoma/metabolismo , Osteosarcoma/tratamiento farmacológico , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND: Aerobic exercise (AE) may slow age-related cognitive decline. However, such cognition-sparing effects are not uniform across cognitive domains and studies. Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation and is also emerging as a potential alternative to pharmaceutical therapies. Like AE, the effectiveness of tDCS is also inconsistent for reducing cognitive impairment in ageing. The unexplored possibility exists that pairing AE and tDCS could produce synergistic effects and reciprocally augment cognition-improving effects in older individuals with and without cognitive impairments. Previous research found such synergistic effects on cognition when cognitive training is paired with tDCS in older individuals with and without mild cognitive impairment (MCI) or dementia. AIM: The purpose of this systematic review with meta-analysis was to explore if pairing AE with tDCS could augment singular effects of AE and tDCS on global cognition (GC), working memory (WM) and executive function (EF) in older individuals with or without MCI and dementia. METHODS: Using a PRISMA-based systematic review, we compiled studies that examined the effects of AE alone, tDCS alone, and AE and tDCS combined on cognitive function in older individuals with and without mild cognitive impairment (MCI) or dementia. Using a PICOS approach, we systematically searched PubMed, Scopus and Web of Science searches up to December 2021, we focused on 'MoCA', 'MMSE', 'Mini-Cog' (measures) and 'cognition', 'cognitive function', 'cognitive', 'cognitive performance', 'executive function', 'executive process', 'attention', 'memory', 'memory performance' (outcome terms). We included only randomized controlled trials (RTC) in humans if available in English full text over the past 20 years, with participants' age over 60. We assessed the methodological quality of the included studies (RTC) by the Physiotherapy Evidence Database (PEDro) scale. RESULTS: Overall, 68 studies were included in the meta-analyses. AE (ES = 0.56 [95% CI: 0.28-0.83], p = 0.01) and tDCS (ES = 0.69 [95% CI: 0.12-1.26], p = 0.02) improved GC in all three groups of older adults combined (healthy, MCI, demented). In healthy population, AE improved GC (ES = 0.46 [95% CI: 0.22-0.69], p = 0.01) and EF (ES = 0.27 [95% CI: 0.05-0.49], p = 0.02). AE improved GC in older adults with MCI (ES = 0.76 [95% CI: 0.21-1.32], p = 0.01). tDCS improved GC (ES = 0.69 [90% CI: 0.12-1.26], p = 0.02), all three cognitive function (GC, WM and EF) combined in older adults with dementia (ES = 1.12 [95% CI: 0.04-2.19], p = 0.04) and improved cognitive function in older adults overall (ES = 0.69 [95% CI: 0.20-1,18], p = 0.01). CONCLUSION: Our systematic review with meta-analysis provided evidence that beyond the cardiovascular and fitness benefits of AE, pairing AE with tDCS may have the potential to slow symptom progression of cognitive decline in MCI and dementia. Future studies will examine the hypothesis of this present review that a potentiating effect would incrementally improve cognition with increasing severity of cognitive impairment.
Asunto(s)
Disfunción Cognitiva , Demencia , Estimulación Transcraneal de Corriente Directa , Anciano , Disfunción Cognitiva/terapia , Ejercicio Físico , Humanos , Preparaciones FarmacéuticasRESUMEN
Osimertinib is a "third-generation'' oral, irreversible, tyrosine kinase inhibitor. It is used in the treatment of non-small cellular lung carcinoma and spares wild-type EGFR. Due to its reactive nature, osimertinib is, in addition to oxidative routes, metabolized through GSH coupling and subsequent further metabolism of these conjugates. The extent of the non-oxidative metabolism of osimertinib is unknown, and methods to quantify this metabolic route have not been reported yet. To gain insight into this metabolic route, a sensitive bioanalytical assay was developed for osimertinib, the active desmethyl metabolite AZ5104, and the thio-metabolites osimertinibs glutathione, cysteinylglycine, and cysteine conjugates was developed. The ease of synthesis of these metabolites was a key-part in the development of this assay. This was done through simple one-step synthesis and subsequent LC-purification. The compounds were characterized by NMR and high-resolution mass spectrometry. Sample preparation was done by a simple protein crash with acetonitrile containing the stable isotopically labeled internal standards for osimertinib and the thio-metabolites, partial evaporation of solvents, and reconstitution in eluent, followed by UHPLC-MS/MS quantification. The assay was successfully validated in a 2-2000â¯nM calibration range for all compounds except the glutathione metabolite, where the LLOQ was set at 6â¯nM due to low accuracy at 2â¯nM. Limited stability was observed for osimertinib, AZ5104, and the glutathione metabolite. The clinical applicability of the assay was demonstrated in samples of patients treated with 80â¯mg osimertinib once daily, containing all investigated compounds at detectable and quantifiable levels.
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Acrilamidas/farmacocinética , Compuestos de Anilina/farmacocinética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Monitoreo de Drogas/métodos , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacocinética , Acrilamidas/administración & dosificación , Acrilamidas/sangre , Acrilamidas/metabolismo , Administración Oral , Anciano , Anciano de 80 o más Años , Compuestos de Anilina/administración & dosificación , Compuestos de Anilina/sangre , Compuestos de Anilina/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/genética , Cromatografía Líquida de Alta Presión/métodos , Dipéptidos/sangre , Dipéptidos/síntesis química , Dipéptidos/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Glutatión/sangre , Glutatión/síntesis química , Glutatión/metabolismo , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/genética , Persona de Mediana Edad , Mutación , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/sangre , Inhibidores de Proteínas Quinasas/metabolismo , Espectroscopía de Protones por Resonancia Magnética , Compuestos de Sulfhidrilo/sangre , Compuestos de Sulfhidrilo/síntesis química , Compuestos de Sulfhidrilo/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
Ibrutinib is a targeted covalent inhibitor frequently used for the treatment of various lymphomas. In addition to oxidative metabolism, it is metabolized through glutathione coupling. The quantitative insight into this kind of metabolism is scarce, and tools for quantitation are lacking. The non-oxidative metabolism could prove a more prominent role when oxidative metabolism is impaired. Also, in-vitro studies could over-estimate the effect of CYP450-inhibition. To gain quantitative insight into this relatively unknown biotransformation pathway of the drug we have developed a validated simple, fast and sensitive bio-analytical assay for ibrutinib, dihydrodiol-ibrutinib, and the glutathione, cysteinylglycine and cysteine conjugates of ibrutinib in human plasma. The method emphasizes on simplicity, the thiol-conjugates were synthesized by a simple one step synthesis, followed by LC-purification. Sample preparation was done by a simple protein crash with acetonitrile containing labeled internal standards, evaporation of solvents, and reconstitution in eluent. Finally, the compounds were quantified using UHPLC-MS/MS. The assay was successfully validated in a 0.5-500nM calibration range for all compounds, and also a lower range of 0.05-50â¯nM was demonstrated for ibrutinib to accommodate for even the lowest trough levels. This assay has a considerably higher sensitivity than previous published assays, with the previous lowest LLOQ being 1.14â¯nM. Both, ibrutinib, dihydrodiol-ibrutinib and the cysteine conjugate were deemed stable under refrigerated or frozen storage conditions. At room temperature, the glutathione conjugate showed rapid degradation into the cysteinylglycine conjugate in plasma. Finally, the applicability of the assay was demonstrated in patient samples.
Asunto(s)
Cromatografía Liquida/métodos , Glutatión/sangre , Naftalenos/sangre , Pirazoles/sangre , Pirazoles/metabolismo , Pirimidinas/sangre , Pirimidinas/metabolismo , Espectrometría de Masas en Tándem/métodos , Adenina/análogos & derivados , Anciano , Estabilidad de Medicamentos , Glutatión/metabolismo , Humanos , Modelos Lineales , Masculino , Naftalenos/metabolismo , Piperidinas , Pirazoles/farmacocinética , Pirimidinas/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Tumor necrosis factor is released in the circulation and aqueous humor during endotoxin-induced uveitis, and induces acute uveitis when injected intraocularly in rats. To elucidate the role of tumor necrosis factor in the development of endotoxin-induced uveitis we analysed the effect of neutralizing anti-tumor necrosis factor antibodies and of pentoxifylline, a drug that inhibits tumor necrosis factor synthesis. Lewis rats were treated with: (a) a single intracardial injection of polyclonal rabbit anti-murine tumor necrosis factor antiserum prior to foot pad injection of 200 micrograms lipopolysaccharide; (b) an intraperitoneal injection of 10 mg pentoxifylline 1 hr before, at the time of, and 3 hr after foot pad injection of lipopolysaccharide; or (c) an intravitreal injection of 20 to 500 micrograms pentoxifylline together with 1 microgram lipopolysaccharide. The ocular inflammation was examined by slit-lamp and evaluated for the presence of hyperemia, flare, miosis, infiltrating cells or hypopyon. Levels of tumor necrosis factor in serum and aqueous samples were determined using a bioassay. Systemic treatment with either anti-tumor necrosis factor antibodies or pentoxifylline resulted in a significant inhibition, 90 and 70% respectively, of serum tumor necrosis factor activity at 3 to 4 hr after lipopolysaccharide injection. Systemic pentoxifylline treatment had no influence on the severity of uveitis. Anti-tumor necrosis factor antibody treatment, in contrast, caused an exacerbation of endotoxin-induced uveitis at t = 20 hr; mean uveitis score 3.9 vs. 1.4 in controls; P < 0.01. Intraocular administration of pentoxifylline together with lipopolysaccharide also had an aggravating effect on uveitis, that was associated with increased levels of intraocular tumor necrosis factor. The results show that inhibition of serum tumor necrosis factor activity does not block the development of endotoxin-induced uveitis. In fact, anti-tumor necrosis factor antibody treatment exacerbates the intraocular inflammation. These findings suggest that tumor necrosis factor may have other than proinflammatory properties in this uveitis model.
Asunto(s)
Anticuerpos/efectos adversos , Pentoxifilina/farmacología , Factor de Necrosis Tumoral alfa/inmunología , Uveítis/inducido químicamente , Animales , Anticuerpos/administración & dosificación , Humor Acuoso/química , Interleucina-6/análisis , Interleucina-6/sangre , Lipopolisacáridos , Masculino , Pentoxifilina/administración & dosificación , Pentoxifilina/efectos adversos , Ratas , Ratas Endogámicas Lew , Factor de Necrosis Tumoral alfa/análisis , Uveítis/sangre , Uveítis/prevención & controlRESUMEN
Lewis rats were injected with recombinant murine tumour necrosis factor-alpha either intravitreally (0.08-50 ng) or intracardially (1 microgram). The intraocular inflammatory response induced by tumour necrosis factor was examined by slit-lamp and protein extravasation into aqueous humor was determined. The phenotype of the inflammatory cells in the eye was analysed by immunohistochemistry. In addition, the kinetics of intraocular interleukin 6 production were determined. At 24 hr after intravitreal injection, a significant clinical uveitis was observed only in rats injected with 50 ng of tumour necrosis factor, when compared to saline-treated controls (P < 0.05). Maximal clinical uveitis and blood-aqueous barrier breakdown were already present at 4 hr after tumour necrosis factor injection. The uveitis was characterized by a massive cellular infiltrate in the anterior segment, consisting predominantly of polymorphonuclear cells and macrophages/monocytes, and to a lesser extent of T lymphocytes. Intraocular interleukin 6 mRNA expression and elevated levels of interleukin 6 in aqueous humor were detected 1 hr after tumor necrosis factor injection, reached a maximum at 3 to 4 hr after injection, and had declined again at 2 hr. Although intracardial injection of 1 microgram of tumour necrosis factor in Lewis rats induced a rise of circulating interleukin 6, it did not produce uveitis. The results obtained with intravitreally injected tumour necrosis factor indicate that intraocular TNF may play a pivotal role in the induction of uveitis in the rat. The transient intraocular production of interleukin 6 early during tumour necrosis factor-induced uveitis suggests that this cytokine may participate in the response induced by tumour necrosis factor.
Asunto(s)
Interleucina-6/biosíntesis , Factor de Necrosis Tumoral alfa/toxicidad , Uveítis Anterior/etiología , Animales , Humor Acuoso/metabolismo , Northern Blotting , Relación Dosis-Respuesta a Droga , Ojo/patología , Expresión Génica , Interleucina-6/genética , Cinética , Masculino , ARN Mensajero/genética , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/toxicidad , Factor de Necrosis Tumoral alfa/administración & dosificación , Uveítis Anterior/metabolismo , Uveítis Anterior/patologíaRESUMEN
PURPOSE: To determine the kinetics of tumor necrosis factor (TNF) and interleukin-6 (IL-6) in serum and aqueous humor of rats with different susceptibilities to endotoxin-induced uveitis (EIU), after footpad injection of lipopolysaccharide (LPS). METHODS: Samples were collected from EIU-susceptible Lewis rats and EIU-resistant Brown Norway (BN) rats for up to 72 hours after LPS injection. Specific bioassays were used to measure TNF and IL-6 activity. Northern blot analysis was used to assess intraocular IL-6 mRNA expression. RESULTS: High levels of TNF and IL-6 were detected in serum of both rat strains early after LPS injection. A second rise in serum TNF was observed at 18 to 20 hours in Lewis rats only. In aqueous humor of Lewis rats, high levels of TNF and IL-6 were observed early after LPS injection (2 to 8 hours) and concomitant with maximal uveitis (18 to 24 hours). Low levels of TNF and IL-6 were found in aqueous humor of BN rats. Ocular IL-6 mRNA was detected at the same time as IL-6 activity was measured in aqueous humor. CONCLUSIONS: The results of this study indicate that both TNF and IL-6 may play a role in the pathogenesis of EIU. The early release of TNF in aqueous humor during EIU suggests that this cytokine may serve as an initial mediator of intraocular inflammation. Furthermore, Northern blot analysis indicates that IL-6 is produced locally during EIU.
Asunto(s)
Interleucina-6/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Uveítis Anterior/metabolismo , Animales , Humor Acuoso/metabolismo , Toxinas Bacterianas , Northern Blotting , Línea Celular , Células Cultivadas , Endotoxinas , Enterotoxinas , Interleucina-6/genética , Cinética , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Salmonella , Uveítis Anterior/inducido químicamenteRESUMEN
Many studies have described the presence of circulating antibodies against corneal components in patients with corneal disease or uveitis, and in patients with skin or systemic disease with or without ocular involvement. The role of such antibodies in the underlying immunopathological process remains obscure. Here we describe the induction of autoantibodies against the rat cornea. Our attempts to induce corneal autoantibodies by various forms of keratitis and corneal trauma failed. However, circulating corneal autoantibodies could be detected by Western blotting after immunization of BN rats and Lewis rats with bovine corneal protein 54 (BCP 54). Rats immunized with rat corneal extracts (RaCE) or human serum albumin (HSA) as (auto) antigen did not develop corneal autoantibodies. During the study period (greater than 4 months), it was observed that the presence of circulating corneal autoantibodies did not elicit corneal inflammation. Severe keratitis did develop when BCP 54-immunized rats were challenged intracorneally with BCP 54, but the clinical signs were not significantly different from HSA-immunized rats after an intracorneal HSA challenge. Injection of corneal autoantibodies into the corneal stroma did not provoke keratitis. To the best of our knowledge this is the first study demonstrating corneal autoantibodies in rats without actual manipulation of the eye. This model may provide further insights in the role and significance of corneal autoantibodies in disease.
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Aldehído Deshidrogenasa , Autoanticuerpos/biosíntesis , Córnea/inmunología , Proteínas del Ojo/inmunología , Animales , Especificidad de Anticuerpos , Femenino , Inmunización , Masculino , Ratas , Ratas EndogámicasRESUMEN
The potential role of interleukin-6 (IL-6) was studied as an inflammatory mediator of endotoxin (or lipopolysaccharide [LPS])-induced uveitis (EIU) in the rat. In young Lewis rats, levels of intraocular IL-6, but not serum IL-6, correlated with the severity of uveitis and with aqueous humor protein levels in response to foot pad injections of LPS (P less than 0.001). Adult Lewis rats did not develop uveitis and had no intraocular IL-6, although IL-6 was released systemically. Resistance to EIU and absence of IL-6 levels in the aqueous humor, despite the ability to release serum IL-6, also were observed in brown Norway rats, irrespective of age and weight. Intravitreal injection of as little as 1 ng of human recombinant IL-6 induced uveitis in young Lewis rats. In adult Lewis rats, and in young animals made tolerant to LPS, intravitreal IL-6 still caused substantial leakage of plasma proteins into the anterior chamber but no influx of inflammatory cells. As early as 2 hr after intravitreal injection of IL-6, immunohistochemical analysis showed invasion of the iris, corneal stroma, and anterior chamber by polymorphonuclear leukocytes (PMN) and expression of major histocompatibility complex (MHC) class II antigen in the retina by large cells that were macrophage-marker ED2 negative. This was followed by massive PMN infiltration of the retinal layers and vitreous. The MHC class II antigen expression of ciliary and iris epithelium occurred at a later stage (greater than 8 hr).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Endotoxinas , Interleucina-6/inmunología , Salmonella , Uveítis Anterior/inmunología , Animales , Cámara Anterior/inmunología , Cámara Anterior/patología , Humor Acuoso/química , Humor Acuoso/inmunología , Proteínas del Ojo/análisis , Antígenos de Histocompatibilidad Clase II/inmunología , Técnicas para Inmunoenzimas , Inyecciones , Interleucina-6/análisis , Masculino , Neutrófilos/inmunología , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Proteínas Recombinantes , Retina/inmunología , Retina/patología , Uveítis Anterior/patologíaRESUMEN
The water-soluble fraction of bovine corneal epithelium was analysed by polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). Next to the principal soluble protein BCP 54, which has recently been identified as a corneal aldehyde dehydrogenase (ALDH), another abundant protein was observed, which we have denoted BCP 11/24, due to its estimated molecular weight of 11 kDa in SDS-PAGE and 24 kDa in high performance gel filtration under non-denaturing conditions. This protein was isolated and characterized by biochemical and immunochemical techniques. The isolation of BCP 11/24 was initially hampered by its tendency to bind non-covalently to BCP 54. BCP 11/24 behaves identically in reduced and unreduced SDS-PAGE and is probably not a glycoprotein. Isoelectric focusing indicated microheterogeneity of BCP 11/24, yielding bands with isoelectric points of 6.1, 5.9, 5.7 and 5.6. A rabbit antiserum directed against BCP 11/24, that did not recognize BCP 54, demonstrated that the distribution of BCP 11/24 in different ocular tissues as well as its light microscopic localization in corneal epithelium is strikingly similar to that of BCP 54. Together with its tendency to interact with BCP 54 in vitro, this suggests the possibility that BCP 11/24 is associated with BCP 54 in vivo, fulfilling a function which may be related to the activity of BCP 54 as a corneal ALDH. In contrast with BCP 54, however, BCP 11/24 was not detectable in corneal endothelium. The antiserum did not detect any immunologically related molecules in corneal epithelium extracts of sheep, human or rat origin, indicating that BCP 11/24 is probably not as highly conserved as BCP 54.
Asunto(s)
Aldehído Deshidrogenasa , Córnea/química , Proteínas del Ojo/análisis , Animales , Bovinos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Epitelio/química , Focalización Isoeléctrica , Peso MolecularRESUMEN
Several studies suggest a role for IL-6 in the pathogenesis of uveitis. Earlier we have shown that aqueous humour obtained from patients with uveitis contained raised levels of IL-6. In the study described here we investigated the IL-6 levels in vitreous fluid samples obtained from 75 uveitis patients with different uveitis entities. Vitreous samples from 14 patients with proliferative intraocular disorders (PID) and 29 eye bank eyes were used as controls. All the samples were tested in the IL-6 B9 bioassay as well as in a sensitive ELISA for IL-6. Raised IL-6 levels were detected in the vitreous fluid of uveitis patients as well as patients with PID, implicating IL-6 as a common inflammatory mediator. The highest mean level of IL-6 was found in the vitreous fluid of patients with acute retinal necrosis. The mean IL-6 levels measured by the ELISA were higher compared to the levels measured by the B9 bioassay. This may be caused by the presence of B9 bioassay inhibitory factors in the vitreous fluid of these patients.
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Oftalmopatías/inmunología , Interleucina-6/análisis , Uveítis/inmunología , Cuerpo Vítreo/inmunología , Bioensayo , Ensayo de Inmunoadsorción Enzimática , Bancos de Ojos , HumanosRESUMEN
The development of an onchocercal chorioretinopathy from the first detectable signs to a full blown oncho fundus is not fully understood. We investigated the intraocular humoral immune response against Onchocerca volvulus, human S-antigen, IRBP and crude retinal extract (using an ELISA) by examining paired aqueous humour and serum samples obtained from onchocerciasis patients (without [n = 10] and with ocular symptoms [n = 8]) and endemic controls [n = 14] from Sierra Leone (West Africa). A local intraocular anti-retinal IgG antibody production could not be demonstrated in onchocerciasis patients, whether they had ocular symptoms or not. A significantly higher level of O. volvulus antibodies and IgG was measured in the aqueous of onchocerciasis patients with ocular involvement, as compared to patients without ocular symptoms (Mann-Whitney ranksum test; p less than 0.001 and p less than 0.02 respectively). Since interleukin-6 (IL-6) plays an essential role in the differentiation of B cells into immunoglobulin producing plasma cells, we therefore measured this cytokine in paired aqueous and serum samples. Elevated IL-6 levels were found in the aqueous of two out of eight onchocerciasis patients tested. In view of these findings it seems improbable that retinal autoimmunity is a major factor in the pathogenesis of onchocercal chorioretinopathy. The high intraocular levels of antibodies against the parasite suggest a direct involvement of the parasite in the pathogenesis of onchocercal chorioretinopathy.
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Anticuerpos Antihelmínticos/análisis , Humor Acuoso/inmunología , Autoanticuerpos/análisis , Proteínas del Ojo/análisis , Onchocerca/inmunología , Oncocercosis Ocular/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Arrestina , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina G/análisis , Masculino , Persona de Mediana Edad , Retina/inmunologíaRESUMEN
In ophthalmo-immunological investigations only small samples of ocular tissues and fluid are available and assays which are feasible with very small volumes or cell numbers are mandatory. Indomethacin, which is known to augment the immune response both in vivo and in vitro was therefore tested for its effect on the monocyte migration inhibition (MIF) assay using low cell or antigen doses. The sensitivity of the MIF assay may be greatly increased by adding indomethaci during the first step of the assay. Titration of either the antigen dose, the mononuclear cells number or both per assay, resulted in a 10-50-fold increase in sensitivity of the assay, with a broad inter-individual variability. Increasing the sensitivity of the MIF assay with indomethacin has clear advantages with regard to the number of cells required but also confronts us with a new problem: activation of specific cells that circulate at very low frequencies in non-immunized individuals. The enhanced response could be reversed to some extent by adding prostaglandin E2 together with indomethacin to the first step of the assay. Moreover, adding leukotriene B4 to the first step of the assay had an enhancing effect over a limited concentration range. We conclude that in the presence of indomethacin, the MIF assay provides a highly sensitive technique for the demonstration of cellular immune responses in small samples of biological fluids containing very small numbers of antigen-specific lymphocytes.
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Inhibición de Migración Celular , Indometacina/farmacología , Monocitos/inmunología , Dinoprostona/farmacología , Humanos , Inmunización , Factores Inhibidores de la Migración de Leucocitos/biosíntesis , Leucotrieno B4/farmacología , Tuberculina/inmunologíaRESUMEN
The mechanisms underlying the induction of intraocular inflammation in the rat model of endotoxin-induced uveitis (EIU) and the subsequent development of tolerance after repeated endotoxin injections are poorly understood. Interleukin-6 (IL-6) was measured in the aqueous humor and serum of Lewis rats after single and repeated injections of endotoxin into the footpad. After a single injection, a rise in serum and aqueous-humor levels of IL-6 was seen after 2 and 16 hr, respectively. The highest aqueous-humor level of IL-6 was seen 20 hr postinjection and was tenfold that seen in the serum sample taken at the same time, suggesting intraocular synthesis of this cytokine. Four hours later the most active uveitis and the highest total aqueous-humor protein level were observed. Repeated injection of endotoxin still resulted in a moderate but significant systemic release of IL-6 but no detectable IL-6 in the aqueous humor and the absence of uveitis. Intravitreal injection of endotoxin-free human recombinant IL-6 (10-10(5) U) in rats resulted in uveitis, resembling the ocular response to endotoxin. There appeared to be a prozone effect regarding the total aqueous-humor protein concentration. The largest amount of aqueous-humor protein was seen in the eyes injected with 10(2) U of IL-6, but increasing concentrations of intravitreal IL-6 showed a corresponding decrease in protein levels. In the fellow saline-injected eyes, a clear consensual response was observed with regard to the extravasation of protein, although the uveitic grade in these eyes was low or zero. Repeated intravitreal injection of IL-6 resulted in ocular unresponsiveness in nine of 11 rats.(ABSTRACT TRUNCATED AT 250 WORDS)
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Interleucina-6/fisiología , Uveítis/inmunología , Animales , Humor Acuoso/inmunología , Toxinas Bacterianas , Endotoxinas , Enterotoxinas , Fondo de Ojo , Tolerancia Inmunológica , Interleucina-6/administración & dosificación , Masculino , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/administración & dosificación , Uveítis/inducido químicamenteRESUMEN
Interphotoreceptor retinoid binding protein (IRBP) is a 136,000 molecular weight photoreceptor cell protein capable of inducing an experimental autoimmune uveitis (EAU) in susceptible animal strains. The occurrence of serum antibodies against human (Hu) or bovine (Bo) IRBP was investigated in patients with uveitis and healthy controls. A sensitive ELISA detected anti-IRBP in approximately 50% of patients and controls, without apparent differences in the mean level, titre or avidity and irrespective of the origin of the antigen. Although the correlation (p less than 0.001) between anti-HuIRBP and anti-BoIRBP levels in uveitis sera suggested the presence of crossreacting antibodies, these sera also contained antibodies specific for either the human or the bovine antigen. The only difference between patients and controls was the greater ability of antibodies in uveitis sera (p less than 0.05) to recognize a synthetic peptide of HuIRBP, which induces severe EAU in rats. We conclude that autoantibodies to IRBP occur naturally in man and are not increased in patients with uveitis.
Asunto(s)
Autoanticuerpos/inmunología , Proteínas del Ojo/inmunología , Proteínas de Unión al Retinol/inmunología , Uveítis/inmunología , Secuencia de Aminoácidos , Animales , Bovinos , Reacciones Cruzadas/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/análisis , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/inmunología , Retina/inmunología , Toxoplasmosis Ocular/inmunologíaRESUMEN
The level of Interleukin-6 (IL-6) in the aqueous humor of 24 patients with 2 types of uveitis was measured with a specific bioassay using the murine hybridoma cell line B9. Sixteen patients had Fuchs' heterochromic cyclitis (FHC) and 8 had toxoplasma uveitis (TU). Sixty-three percent of each of the FHC and TU groups had raised levels of IL-6 in their aqueous (mean: 543 and 19,228 units/ml respectively). Thirteen control aqueous samples, obtained at surgery for senile cataract, showed IL-6 levels of less than 10 units/ml. Serum obtained at the same time as each aqueous humor sample also showed IL-6 levels of less than 10 units/ml, indicating that the raised levels of IL-6 found in the aqueous of uveitis patients did not result from serum leakage, but from local production. This is the first report on intraocular IL-6 levels, and indicates that IL-6 may play a role as an inflammatory mediator in uveitis.
Asunto(s)
Humor Acuoso/metabolismo , Interleucina-6/metabolismo , Iridociclitis/metabolismo , Uveítis/metabolismo , Adolescente , Adulto , Anciano , Bioensayo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Toxoplasmosis Ocular/complicaciones , Uveítis/etiología , Uveítis Posterior/metabolismoRESUMEN
Aqueous humor from 23 patients with Fuchs' heterochromic cyclitis (FHC) was analysed by a number of immunological methods. Intraocular IgG synthesis was found in 65% of patients and oligoclonal IgG bands, mainly of the IgG1 subclass, identified in 57%. There was a relative increase in IgG1 (P less than 0.01) as compared to patients with senile cataract. Local production of the cytokine Interleukin-6 was demonstrated in 63% of patients (P less than 0.01). Analysis of aqueous by HPLC and SDS-PAGE failed to reveal any abnormalities specific for FHC. These findings add further evidence to the theory of immune dysregulation in this condition.