Cellular immune response to ß2-glycoprotein-I valine/leucine247 phenotypes in Mexican patients with primary antiphospholipid syndrome.

Homozygote genotype V247 of the ß2-glycoprotein-I (ß2GP-I) gene has been associated with anti-ß2GP-I and thrombosis in patients with primary anti-phospholipid syndrome APS (PAPS). However, the cellular immune response to ß2GP-I247 has been little studied. OBJECTIVE: To evaluate the immune cellular proliferation in response to native and non-native ß2GP-I247 valine/leucine phenotype from Mexican patients with PAPS. METHODS: We studied 10 patients with PAPS and 10 healthy control subjects (HC). The polymorphism at position 247 of the ß2GP-I gene was determined by PCR-RFLP and the corresponding ß2GP-I protein was subsequently purified from normal human plasma by affinity chromatography. PBMC purified from patients and controls were stimulated with ß2GP-I under native and in non native (reduced) conditions. We also determined the anti-ß2GP-I production in vitro by B cell clones (EBV) generated in cocultures experiments. Differential Scanning Calorimetry (DSC) was studied to determine the structural differences between the ß2GP-I247 valine/leucine isoforms. Cytokine profile (IL-2, IL-4, IL-6, TNFα, INFγ) was evaluated in culture supernatants. RESULTS: PAPS and healthy control PBMCs had a higher proliferative response when stimulated with ß2GP-I under reduced cultures conditions compared to non-denatured conditions. PBMCs response from PAPS patients was higher. We observed more cell proliferation in response to ß2GP-I247 valine/leucine or valine isoforms in non-native conditions. In contrast, this response was not significant against ß2GP-I247 leucine. These findings were T CD4+-dependent. Similar results were obtained with B cell clones derived from PAPS patients, which showed more pronounced proliferation in non native conditions and higher against ß2GP-I247 valine. No differences were found in anti-ß2GP-I production, but high levels of IL-6 in vitro were identified. The structural analysis of both ß2GP-I247 isoforms by DSC showed a major conformational change due to a single mutation in the ß2GP-I variants. CONCLUSIONS: PAPS PBMCs had a higher cellular response against ß2GP-I247 in non-native culture conditions preferentially to the ß2GP-I247 valine phenotype. This effect is T CD4+ dependent and appears to be driven by tertiary structural changes adopted by ß2GP-I247 polymorphism.

Análisis de líquido sinovial / Synovial fluid analysis

En la actualidad el estudio del líquido sinovial (LS) es una herramienta que se utiliza con frecuencia en los laboratorios especializados y que permite establecer el diagnóstico de artropatías por cristales, apoya el diagnóstico de las artritis sépticas y ayuda a establecer otros diagnósticos reumatológicos como la monoartritis o los derrames articulares. El estudio completo del LS incluye los siguientes análisis: 1) macroscópico, 2) microscópico y 3) uso de tinciones específicas. Cada uno de los estudios proporciona información del estado de la articulación y ayuda a establecer el diagnóstico y tratamiento. Las características que se deben describir en el análisis macroscópico son: color, volumen y viscosidad. El estudio microscópico, confirma la existencia de un proceso inflamatorio, infeccioso y la presencia de cristales. El microscopio de luz polarizada es una herramienta fundamental para el estudio del LS y la diferenciación de los cristales, la cual no solo depende de la forma, sino también de la birrefringencia. Es importante mencionar que en el análisis microscópico los artefactos pueden confundir al observador inexperto. Una adecuada interpretación del análisis del LS requiere de la observación de por lo menos 2 observadores capacitados. La información que proporciona el análisis del LS al clínico da los elementos necesarios para establecer el diagnóstico del paciente así como el tratamiento del mismo. Las tinciones en el análisis del LS ayudan a la identificación de cristales no birrefringentes, como son los de hidroxiapatita de calcio. Actualmente, en el estudio del LS se están explorando nuevos campos que incluyen cuantificación de citocinas, quimiocinas e inmunoglobulinas y la caracterización de las estirpes celulares (AU)

At present, the study of the synovial fluid (SF) is a tool that is used frequently in specialized laboratories because it allows the establishment of diagnosis of crystal associated arthropathies, supports the diagnosis of septic arthritis and helps establish other rheumatologic diagnoses such as monoarthritis or joint effusion. The complete study of the SF includes the following analyses: 1. Macroscopic; 2. Microscopic; and 3. Specific stains. Each one provides information of the joint's state and helps in the establishment of diagnosis and treatment. The characteristics that must be described in the macroscopic analysis are: color, volume and viscosity. Microscopic analysis of the SF confirms the presence of an inflammatory or infectious processes and allows for the detection and identification of crystals. Polarized light microscope is a fundamental tool for the analysis of SF and for the identification of the crystals present in the samples, which not only depend on the form, but also of their birefringence. It is important to mention that in the microscopic analysis, artifacts can confuse the inexperienced observer. A suitable interpretation of the analysis of SF requires the observation by at least two experienced observers. The information that the analysis of SF provides to the clinicians gives them the necessary elements to establish the diagnosis and to decide on treatment. Specific stains in the analysis of SF help in the identification of non-birefringent crystals as those of calcium hydroxypatite. In SF analysis, new fields are being explored that include quantification of cytokines, chemokines, immunoglobulins and the characterization of cell lineages (AU)

[Synovial fluid analysis]. / Análisis de líquido sinovial.

At present, the study of the synovial fluid (SF) is a tool that is used frequently in specialized laboratories because it allows the establishment of diagnosis of crystal associated arthropathies, supports the diagnosis of septic arthritis and helps establish other rheumatologic diagnoses such as monoarthritis or joint effusion. The complete study of the SF includes the following analyses: 1. Macroscopic; 2. Microscopic; and 3. Specific stains. Each one provides information of the joint's state and helps in the establishment of diagnosis and treatment. The characteristics that must be described in the macroscopic analysis are: color, volume and viscosity. Microscopic analysis of the SF confirms the presence of an inflammatory or infectious processes and allows for the detection and identification of crystals. Polarized light microscope is a fundamental tool for the analysis of SF and for the identification of the crystals present in the samples, which not only depend on the form, but also of their birefringence. It is important to mention that in the microscopic analysis, artifacts can confuse the inexperienced observer. A suitable interpretation of the analysis of SF requires the observation by at least two experienced observers. The information that the analysis of SF provides to the clinicians gives them the necessary elements to establish the diagnosis and to decide on treatment. Specific stains in the analysis of SF help in the identification of non-birefringent crystals as those of calcium hydroxypatite. In SF analysis, new fields are being explored that include quantification of cytokines, chemokines, immunoglobulins and the characterization of cell lineages.