Safety and Clinical Responses in Ankylosing Spondylitis after Three Months of Etanercept Therapy

We aimed to evaluate the safety and clinical responses in Korean ankylosing spondylitis (AS) patients after three months of etanercept therapy. AS patients satisfying the Modified New York Criteria were enrolled. They were assessed for safety and clinical responses at enrollment and after three months of etanercept therapy. A total of 124 patients completed the study. After three months, the rate of ASsessment in AS International Working Group 20% improvement (ASAS 20) response was 79.8%. The rates of ASAS 40 and ASAS 5/6 responses were 58.5 and 62.8%, respectively. Significant improvement of Korean version of Bath AS Disease Activity Index (KBASDAI) (p<0.0001), Bath AS Functional Activity Index (BASFI) (p<0.0001), and Bath AS Metrology Index (BASMI) (p=0.0009) were achieved after three months. Quality of life was also significantly improved after three months, as demonstrated by scores for SF-36 (p<0.0001) and EQ-5D (p<0.0001). Erythrocyte sedimentation rate and C-reactive protein were significantly decreased (p<0.0001, p<0.0001, respectively). None of the patients developed tuberculosis and there were no serious adverse event. AS patients with inadequate response to conventional therapy showed significant clinical improvement without serious adverse events after three months of etanercept therapy.

Primary culture of alveolar epithelial type II cells and its bionomic study / 华中科技大学学报（医学）（英德文版）

To establish a better method of primary culture for alveolar epithelial type II cells (AEC II) and to study its bionomics, alveolar epithelial type II cells were isolated by digestion with trypsin and collagenase, which were then purified by plated into culture flask coated with rat immunoglobulin G. The purified AEC II were identified by alkaline phosphatase staining, electron microscopy, immunocytochemical staining of pulmonary surfactant protein A (SPA). The SPA expression and transfection characteristics were compared with those of A549 cell line. The results showed that AEC II could be isolated by digestion with trysin and collagenase and purified by adhesive purification by using IgG, with a yield of about 2-3 x 10(7), and a purity of about 75%-84%. Cells could be quickly identified with AKP staining. AEC II were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC II, and AKP staining is simple in cell identification. AEC II can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.

Rabbit IgG antibodies against Phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom

Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 µg) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 µg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms

El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 µg de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 µg.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes

Activation of microglia in mice spinal cords following passive transfer of purified IgG from patients with amyotrophic lateral sclerosis

To determine whether IgG from amyotrophic lateral sclerosis [ALS] patients could cause activation of microglia, proliferation of astrocytes, and infiltration by lymphocytes within mice spinal cords. A group of 5 mice received injections of IgG purified from sera of patients with ALS. A control group of 5 mice received IgG from healthy humans, whilst a third group of 2 mice served as non-injected controls. One mouse served as a positive control and was injected with lipopolysaccharide, a known activator of microglia. Mice were culled after one week, for immunocytochemistry of spinal cord sections to localize the complement receptor CD11b on activated microglia, glial fibrillary acidic protein on astrocytes, and CD4 and CD8 receptors on lymphocytes. Histological examination was used to determine the presence of inflammatory reaction. This work was conducted at the Institute of Neurology, Queen Square London, United Kingdom, from January to July 2004. There was no significant difference in activation of microglia between mice injected with ALS IgG and mice injected with control IgG [p = 0.631], although mice injected with ALS IgG exhibited greater microglial activation than non-injected mice [p = 0.044]. Proliferation of astrocytes was not significantly different between the 3 groups. CD4 and CD8 lymphocytes were both absent in mice injected with ALS IgG, mice injected with control IgG and non-injected mice. Activation of microglia following passive transfer of IgG from ALS patients to mice represents a non-specific inflammatory response, rather than a primary mechanism for motor neuron degeneration

Activated protein C resistance and lupus anticoagulant activity induced by plasma and purified monospecific human IgG anti-β2-glycoprotein-I antibodies / Resistencia a la proteína C activada y actividad de anticoagulante lúpico inducidas por plasma y por anticuerpos purificados humanos del IgG anti β2-glicoproteína I

ABSTRACT Introduction. We investigated the activated protein C resistance (APCR) phenotype and the lupus anticoagulant (LA), activity induced by anti-β2-glycoprotein-I (anti-β2GP-I) antibodies. Patients and methods. We studied plasma and sera samples from 29 patients with persistently positive anti-β2GP-I: 22 with thrombosis (12 with primary APS, 10 with APS secondary to SLE) and seven without thrombosis (all with SLE); 25 healthy subjects were studied as controls. We detected anticardiolipin antibodies (ACA); IgG (and its subclasses) and IgM anti-β2GP-I, on irradiated and non-irradiated plates by ELISA. APCR was assessed by the activated partial thromboplastin time (APTT)-based assay and by the modified test. The FV Leiden mutation was studied by PCR. LA determination included screening and confirmatory dRVVT. Serum anti-β2GP-I were affinity purified on sepharose columns and their isotype, subclass, and reactivity against various antigens were studied by ELISA. Results. We found that titers of IgG anti-β2GP-I on irradiated plates were higher than on non-irradiated plates (p = 0.002), IgG2 was the predominant subclass. Fifteen patients (13 with thrombosis) had LA and 15 (also 13 with thrombosis) induced the APCR phenotype. Eleven (all with thrombosis) had both. Two patients were heterozygous for the Leiden mutation. Two purified antibodies, monospecific for β2GP-I, induced an in vitro APCR phenotype and LA activity. Conclusions. Our results seem to indicate that the inhibition of the APC anticoagulant function by IgG2 anti-β2GP-I with LA activity may be one of the responsible mechanisms of thrombophilia in patients with APS.

Introducción. Investigamos la resistencia a la proteína C activada (RPCA) y la actividad de anticoagulante lápico (AL), inducidas por anticuerpos anti-β2-glicoproteína-I (anti-β2GP-I). Pacientes y métodos. Estudiamos los plasmas y sueros persistentemente positivos para anti-β2GP-I de 29 pacientes: 22 tuvieron trombosis (12 con síndrome de antifosfolípidos (SAF) primario y 10 con SAF secundario a lupus erítematoso generalizado (LEG)) y siete sin trombosis (todos con LEG). Como controles estudiamos 25 sueros de personas clínicamente sanas. Detectamos anticuerpos anticardiolipina, anti-β2GP-I IgG (y sus subclases) e IgM por ELISA en placas irradiadas y no irradiadas. Evaluamos la RPCA por medio del tiempo parcial de tromboplastina activada y por la prueba modificada. Estudiamos la mutación FV de Leiden por PCR y el anticoagulante lápico con el método de dRVVT screening y confirmatorio. Después de purificar los anti-β2GP-I séricos con una columna de antígeno unido a sefarosa, analizamos por ELISA sus isotipos, subclases y reactividad contra β2GP-I y algunos fosfolípidos. Resultados. Los títulos de anti-β2GP-I IgG fueron más altos en placas irradiadas que en no irradiadas (p = 0.002), predominó la subclase IgG2. Quince plasmas (13 de pacientes con trombosis) tuvieron AL y 15 (13 también de pacientes con trombosis) indujeron el fenotipo de RPCA. Once plasmas (todos de pacientes con trombosis) indujeron ambas actividades. Dos pacientes fueron heterocigotos para la mutación de Leiden. Dos anticuerpos purificados monoespecíficos para β2GP-I indujeron el fenotipo de la RPCA y la actividad de AL in vitro. Conclusiones. Nuestros resultados sugieren que la RPCA, inducida por los anti-β2GP-I que concomitantemente tienen actividad de AL, puede tener implicaciones patogénicas en la trombofílía del SAF.

Evaluación de la autoinmunidad en ratones BALB/c inmunizados con una biblioteca genómica de expresión de Trypanosoma cruzi / Evaluation of autoimmunuty in Balb/c mice immunized with a genomic library of expression of Trypanosoma cruzi

Uno de los problemas de la seguridad para las vacunas de ADN es la inducción de fenómenos de autoinmunidad. Nosotros examinamos el efecto de la inmunización con ácidos nucleicos de Trypanosoma cruzi en la inducción de diferentes autoanticuerpos en ratones de Balb/c. Los animales fueron divididos en cinco grupos: los primeros cuatro recibieron diferentes esquemas: 25 µg de la biblioteca genómica de expresión (grupo L), 25 µg de antígenos solubles de T. cruzi (grupo T), 25 µg del plásmido pcDNA3 (grupo P), 25 µg de genómica ADN de T. cruzi (grupo G) y un grupo control de animales no inmunizados. Los anticuerpos antinucleares y anticuerpos contra músculo cardíaco fueron evaluados por immunofluorescencia indirecta y los anticuerpos anti ADN de doble, simple cadena y el anti IgG factor reumatoideo fueron determinados semanalmente por ELISA. La vacunación no provocó la inducción de anticuerpos anti ADN de doble o simple cadena, anticuerpos antinucleares ni contra músculo cardíaco. Se observó un aumento transitorio del Factor Reumatoideo IgG en los ratones inmunizados con la biblioteca genómica de expresión de T. cruzi. Nuestros hallazgos sugieren que la inducción de respuestas autoinmunes frente al ADN utilizado en la inmunización es poco probable

Estudo comparativo dos resultados obtidos pelos métodos de imunocromatografia e o ELISA para detecção de anticorpos IgM e IgG anti-vírus dengue / Comparative study of the results obtained by immunocromatography methods and ELISA IgM and IgG antibodies detection antidengue virus

O dengue constitui sério problema de saúde pública no mundo, especialmente na maioria dos países tropicais. No Brasil, o diagnóstico sorológico é realizado pelo ensaio imunoenzimático para captura de IgM (MAC-ELISA) e IgG (ELISA IgG) anti-dengue, entretanto, inúmeros kits estão disponíveis no mercado. Nesse estudo, o Kit Dengue - Teste Rápido em Tira (KD-TRT) é comparado com as técnicas padrões. Foram utilizadas 72 soros, incluindo soros de pacientes com infecção confirmada ou presuntiva por dengue, com infecção por febre amarela e rubéola, bem como, de pacientes com sorologia dengue negativa. O KD-TRT apresentou sensibilidade de 54,2 porcento e especificidade de 100 porcento mediante análise dos soros de pacientes com ou sem dengue. Quando incluídas as amostras de febre amarela e rubéola, o KD-TRT apresentou os índices de 54,2 porcento e 91,2 porcento de sensibilidade e especificidade, respectivamente. Os resultados da detecção de anticorpos IgG do KD-TRT foram correlacionados com os resultados do ELISA IgG. O ELISA IgG detectou 20 (38,5 porcento) reações primárias, 17 (32,7 porcento) reações secundárias e 15 (28,8 porcento) reações negativas, enquanto que o KD-TRT detectou 11 (21,2 porcento), 7 (13,5 porcento) e 34 (65,3 porcento), respectivamente. O KD-TRT apresentou menor sensibilidade e especificidade em relação ao MAC-ELISA e baixa correlação de resultados com ELISA IgG.

Negative inotropic effect of chagasic IGG mediated by nitric oxide

In this paper we analyse the interaction of IgG from T. cruzi infected patients with cardiac muscarinic acetycholine receptors (mAChRs). Human chagasic IgG, activating M2 mAChR simulated the agonist actions excerting negative inotropic effect and simulation of nitric oxide synthase (NOS). Inhibitos of phospholipase C, protein Kinase C, calcium/calmodulin, NOS and guanylate cyclase activities prevented the chagasis effects upon contractility and NOS activity.

Effect of growth factors on the expression of proto-oncogenes c-fos and c-myc in FRTL-5 cell line

This study was performed to prove the hypothesis that oncogene expressions would have the same patterns with those of cellular growth to growth factors in FRTL-5 cells. Ribonucleic acids of FRTL-5 were extracted at 15', 30', 60' and 120' after administration of growth factors to quiescent FRTL-5, and blotted to the nitrocellulose membrane. They were hybridized with radiolabelled c-fos, c-myc and beta-actin probes. Hybridized dot blots were autoradiographed and the amount of radioactivity was measured by densitometry. Densitometric readings were used as the indices of oncogene expressions. Expressions of c-fos and c-myc were more prominent in combined administrations of TSH (10 mU/ml) and IGF-I (100 ng/ml) or IgG of Graves' disease (Graves' IgG; 1 mg/ml) and IGF-I than in combined administration of TSH and Graves' IgG. IgG of primary myxedema suppressed oncogene expressions by TSH or Graves' IgG, but not by IGF-I. From the above results, it was suggested that expressions of c-fos and c-myc to growth factors would have similar patterns with those of cell growth to growth factors in FRTL-5, and the actions of TSH and Graves' IgG would be manifested through same signal transduction system, but IGF-I would be manifested by its own.

The modulatory effect of immunoglobulin G on the CD23 and HLA-DR expression and cytokine production in different groups of asthmatic patients.

The therapeutic effect and mechanism of action of immunoglobulin G (IgG) on bronchial asthma are not defined. Recently, it has been proposed that mononuclear cell (MNC) infiltration in the airway plays a role in the pathogenesis of asthma. In this study, we evaluated the effect of IgG on the cell receptor expression and cytokine production of MNC from two groups (young atopic and old non-atopic) of stable asthmatic patients. MNCs from both asthmatic patients and normal healthy individuals were obtained after Ficoll-Hypaque separation. Cells were cultured in serum free AIM-V medium, with or without phytohemagglutinin (PHA, 5 micrograms/ml) and IgG (100 micrograms/ml). After culture, MNCs were harvested and stained with monoclonal antibodies for HLA-DR (Ia), CD23 and CD3. MNC supernatants were collected for IL-2 and IL-4 measurement. The results showed an enhancing effect of IgG on young atopic MNC proliferation when stimulated with PHA. The production of IL-2 and IL-4 from MNCs were significantly higher in old non-atopic asthmatics after PHA stimulation. The CD23, but not HLA-DR, expression on CD3 positive T cells and cytokines (IL-2 and IL-4) production were increased by IgG when stimulated with PHA in young atopic asthmatics. To the contrary, the effect of IgG on PHA stimulated MNC proliferation, CD23 and HLA-DR expression on CD23 positive T cells in old non-atopic asthmatics were trivial. Only IL-4 production can be significantly inhibited by IgG. These results suggested that the therapeutic effect of IgG on asthmatics might be variable in different groups of asthmatics.(ABSTRACT TRUNCATED AT 250 WORDS)