RESUMEN
An open-label dose-escalation trial was performed to assess the safety and tolerability of high doses of coenzyme Q10 (CoQ10) in ALS. CoQ10, a cofactor in mitochondrial electron transfer, may improve the mitochondrial dysfunction in ALS. In this study, CoQ10 was safe and well tolerated in 31 subjects treated with doses as high as 3,000 mg/day for 8 months.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Sistema Nervioso Central/efectos de los fármacos , Degeneración Nerviosa/prevención & control , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/administración & dosificación , Ubiquinona/análogos & derivados , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/fisiopatología , Coenzimas , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos/fisiología , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Femenino , Depuradores de Radicales Libres/administración & dosificación , Depuradores de Radicales Libres/efectos adversos , Humanos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/metabolismo , Neuronas/metabolismo , Fármacos Neuroprotectores/efectos adversos , Fármacos Neuroprotectores/sangre , Ubiquinona/administración & dosificación , Ubiquinona/efectos adversos , Ubiquinona/sangreRESUMEN
In the spleens of mice infected intraperitoneally with the bacterium Listeria monocytogenes, both alphabeta and gammadelta T cells became rapidly activated, followed by a massive apoptotic death response predominantly within the gammadelta population. The death response involved two major splenic gammadelta T-cell subsets and was Fas/Fas ligand (Fas-L)-dependent. Among T cells isolated from the Listeria-infected spleen, Fas-L was almost exclusively expressed in gammadelta T cells. gammadelta T cells coexpressed Fas and Fas-L, suggesting activation-induced suicide as a mechanism of their death. In vivo treatment with an antibody specific for CD3epsilon induced activation, preferential Fas-L expression and apoptosis of gammadelta T cells, resembling the response pattern in listeriosis, whereas antibodies specific for T-cell receptor-beta (TCR-beta) or TCR-delta did not, suggesting that the complete response seen in listeriosis requires both gammadelta TCR engagement and additional stimuli. L. monocytogenes causes early nonspecific, Fas-independent lymphocyte death in heavily infected tissues. In contrast, the death response described here is selective, Fas-dependent and triggered at low local levels of bacteria, suggesting that it is controlled by interactions with other infection-activated host cells, and perhaps part of a regulatory circuit specifically curtailing gammadelta T cells.
Asunto(s)
Apoptosis , Listeriosis/inmunología , Glicoproteínas de Membrana/fisiología , Receptores de Antígenos de Linfocitos T gamma-delta/análisis , Linfocitos T/inmunología , Receptor fas/fisiología , Animales , Células Cultivadas , Proteína Ligando Fas , Cinética , Activación de Linfocitos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T/citologíaRESUMEN
Upon productive interaction of CD4 T cells with antigen-presenting cells (APCs), receptors and intracellular proteins translocate and form spatially segregated supramolecular activation clusters (SMACs). It is not known whether SMACs are required for CD8 T cell activation. CD8 T cells, unlike CD4 T cells, can be activated by a single peptide-MHC molecule, or by purified monovalent recombinant peptide-MHC molecules. We studied, by three-dimensional digital microscopy, cell conjugates of fresh ex vivo CD8 T cells (obtained from OT-1 mice, which are transgenic for T cell antigen receptor reactive with the complex of H-2K(b) and the ovalbumin octapeptide SIINFEKL) and peptide-pulsed APCs. Remarkably, even in T cell:APC conjugates that were formed in the presence of the lowest concentration of peptide that was sufficient to elicit T cell proliferation and IFN-gamma production; the theta isoform of protein kinase C was clustered in a central SMAC, and lymphocyte function-associated antigen 1 and talin were clustered in the peripheral SMAC. Conjugation of T cells to APCs that were pulsed with concentrations of peptide smaller than that required to activate T cells was greatly reduced, and SMACs were not formed at all. APCs expressing mutant H-2K(b) (Lys(227)) molecules that do not bind CD8 were unable to form stable conjugates with these T cells, even at high peptide concentrations. Thus, although CD8 and CD4 T cells may display different sensitivity to the concentration and oligomerization of surface receptors, SMACs are formed and seem to be required functionally in both cell types. However, unlike CD4 T cells, which can form SMACs without CD4, CD8 T cells from OT-1 transgenic mice depend on their coreceptor, CD8, for the proper formation of SMACs.
Asunto(s)
Células Presentadoras de Antígenos/fisiología , Antígenos CD8/fisiología , Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/fisiología , Animales , Antígenos CD4/fisiología , Citocinas/biosíntesis , Antígenos H-2/fisiología , Ratones , Ratones TransgénicosRESUMEN
The characterization of monoclonal antibodies raised against the foot-and-mouth disease virus isolates A22 Iraq/1964, Asia1 Shamir-Israel/1989, and SAT1 Zimbabwe/1989 with regard to neutralizing activity and sensitivity of their epitopes for treatment with trypsin, resulted in the identification of one non-neutralizing antibody in each panel that binds to a trypsin-sensitive epitope. Furthermore, each of these antibodies recognized 27 isolates of different provenance, representative of six serotypes. These antibodies are recommended for type-independent antigen detection by ELISA. The epitopes for these antibodies reside at the intertypically conserved N-terminus of capsid protein VP2. The two are specified by the lysines at positions two and three, but differ from each other as indicated by the variable heavy chain sequences of their antibodies.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Aphthovirus/aislamiento & purificación , Cápside/química , Cápside/metabolismo , Fiebre Aftosa/virología , Secuencia de Aminoácidos , Aphthovirus/clasificación , Aphthovirus/metabolismo , Cápside/genética , Proteínas de la Cápside , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Epítopos , Pruebas de Neutralización , Péptidos/química , Péptidos/inmunología , Tripsina/metabolismoRESUMEN
Previous findings suggest that during cognate T cell-B cell interactions, major histocompatability complex (MHC) class II molecules transduce signals, leading to Src-family kinase activation, Ca2+ mobilization, and proliferation. Here, we show that antigen stimulation of resting B cells induces MHC class II molecules to associate with Immunoglobulin (Ig)-alpha/Ig-beta (CD79a/CD79b) heterodimers, which function as signal transducers upon MHC class II aggregation by the T cell receptor (TCR). The B cell receptor (BCR) and MHC class II/Ig-alpha/Ig-beta are distinct complexes, yet class II-associated Ig-alpha/beta appears to be derived from BCR. Hence, Ig-alpha/beta are used in a sequential fashion for transduction of antigen and cognate T cell help signals.
Asunto(s)
Antígenos CD/metabolismo , Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Animales , Antígenos/inmunología , Linfocitos B/metabolismo , Antígenos CD79 , Células Cultivadas , Dimerización , Activación Enzimática , Antígenos de Histocompatibilidad Clase II/inmunología , Immunoblotting , Activación de Linfocitos , Ratones , Ratones Transgénicos , Fosforilación , Fosfotirosina/metabolismo , Pruebas de Precipitina , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transcripción GenéticaRESUMEN
The sequences of the antigenically relevant capsid proteins VP1-3 of 10 isolates obtained during an epizootic of serotype A foot-and-mouth disease virus in Iran, and collected within two and a half years, were found to be highly similar. However, each isolate differed by at least one amino acid from all others. This prompted us to analyze the immunological reactivity of the isolates. To this end, monoclonal antibodies (mAbs) against one isolate were generated and characterized with regard to neutralizing activity and reactivity with trypsinized virus. These mAbs as well as others raised against A22 virus were used for antigen profiling. This distinguished four antigenic conditions among the isolates and 16 reactivities among the mAbs. These findings, together with the observed sequence differences indicated the location of several epitopes. Many mAbs recognized the minor antigenic sites on VP2 and 3 and some the major site, the GH-loop of VP1. One epitope was composed of residues of the capsid proteins VP1 and 2.
Asunto(s)
Variación Antigénica/genética , Aphthovirus/inmunología , Enfermedades de los Bovinos/epidemiología , Fiebre Aftosa/epidemiología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Antígenos Virales/análisis , Antígenos Virales/química , Antígenos Virales/clasificación , Antígenos Virales/genética , Aphthovirus/clasificación , Aphthovirus/genética , Cápside/química , Cápside/genética , Bovinos , Enfermedades de los Bovinos/virología , Cartilla de ADN/química , ADN Viral/química , Brotes de Enfermedades/veterinaria , Fiebre Aftosa/virología , Irán/epidemiología , Datos de Secuencia Molecular , Pruebas de Neutralización/veterinaria , Mutación Puntual , ARN Viral/química , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The capsid protein encoding genes of five recent type Asia1 foot-and-mouth disease virus isolates, representative of three genotypes, were sequenced. The deduced amino acid sequences were aligned to each other and to two published sequences. The sequence differences suggested different antigenic properties of the isolates. One isolate was used to generate monoclonal antibodies (mAbs) which were analyzed for neutralizing activity and reactivity with trypsinized virus. Trypsin removes the major antigenic sites located at VP1. The five virus isolates formed three reaction patterns with the mAbs, irrespective of their genotype. Combination of all data allowed to suggest the location of the epitope of each antibody: the VP1 G-H and the VP2 B-C loop, the VP3 B-B knob, and the N-terminus of VP2, respectively, were involved.
Asunto(s)
Variación Antigénica , Aphthovirus/genética , Aphthovirus/inmunología , Cápside/genética , Variación Genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Aphthovirus/clasificación , Asia/epidemiología , Cápside/química , Bovinos , Fiebre Aftosa/virología , Datos de Secuencia Molecular , Pruebas de Neutralización , Alineación de Secuencia , Análisis de Secuencia de ADN , Tripsina/metabolismoRESUMEN
This report extends the knowledge on the epizootical situation of foot-and-mouth disease in Asia. RNA from six samples of type A and five of type O virus, isolated between 1987 and 1997 in Bangladesh, Iran, Malaysia and Turkey, was subjected to reverse transcription-dependent polymerase chain reactions that amplify large parts of the capsid protein VP1 encoding genome region. The amplification products were sequenced, and the sequences aligned to each other and to published sequences. This showed the type O isolates of 1987-1997 from Bangladesh to be of same genotype and closely related to isolates of 1988 and later from Saudi Arabia, 1990 from India, 1996 from Greece and Bulgaria, and 1997 from Iran. Among the analyzed type A isolates, those of 1992 and 1996 from Turkey were of same genotype and related to previously described isolates of 1987 from Iran and of 1992 from Saudi Arabia. The isolate of 1997 from Malaysia was found to be related to isolates from Thailand of 1993 and 1996. The isolates of 1987 from Bangladesh and 1997 from Iran, however, represent different so far not described genotypes. Monoclonal antibodies, raised against the vaccine production strains A22 Iraq, Asial Shamir, O1 Kaufbeuren and O1 Manisa, and the recent type A field isolates Saudi Arabia/92 and Albania/96, were used in an ELISA to compare the reaction patterns of many of the field isolates. The monoclonal antibodies were further characterized for virus-neutralizing activity and binding to trypsinized homologous virus. The failure of neutralizing antibodies in binding to trypsinized homologous as well as to heterologous virus suggested the epitopes to reside at the major antigenic component of the virus, which is the capsid protein VP1. Two non-neutralizing antibodies that bind to trypsin-sensitive epitopes cross-reacted, however, with heterologous virus. This indicates the existence of a trypsin-sensitive antigenic site outside of VP1. In summary, the results obtained by ELISA confirm the observed sequence differences, but indicate further sequence differences at minor antigenic sites that do not reside on VP1.
Asunto(s)
Aphthovirus/genética , ARN Viral/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Aphthovirus/inmunología , Aphthovirus/aislamiento & purificación , Asia , Secuencia de Bases , Cápside/genética , Cápside/inmunología , Proteínas de la Cápside , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
Lymphocyte circulation plays an important role in the generation of a specific immune response. Mature lymphocytes continuously circulate between blood and lymph, entering the lymphoid tissue via high endothelial venules. Trafficking across high endothelial venules of peripheral lymph nodes (PLN) depends on the expression of L-selectin. It has been shown that L-selectin is rapidly cleaved from the surface by a metalloproteinase after in vitro activation. Here, we show that ligation of CD4, without ligation of the T cell receptor for antigen, causes down-regulation of L-selectin on T helper cells. This down-regulation is caused by proteolytic cleavage by a metalloproteinase and is reversible by the addition of hydroxamic acid-based metalloproteinase inhibitors. We show that in vivo down-regulation of L-selectin in huCD4tg mice by mAb reduces the homing of lymphocytes to PLN in adoptive transfer experiments. Because CD4 is a coreceptor for HIV-1, the down-regulation of L-selectin induced by CD4 ligation could play a role in the pathogenesis of AIDS. We provide evidence that CD4 ligation by HIV-1 induces metalloproteinase-dependent L-selectin down-regulation. Reduced levels of L-selectin expression might contribute to immune deficiency in individuals infected with HIV by inhibiting T cell redistribution and decreasing the probability of an encounter between specific lymphocytes and viral antigens in PLN.
Asunto(s)
Antígenos CD4/metabolismo , Regulación hacia Abajo , Selectina L/metabolismo , Traslado Adoptivo , Animales , Anticuerpos Monoclonales , Antígenos CD4/inmunología , Técnicas de Cocultivo , VIH-1 , Humanos , Receptores de Hialuranos/biosíntesis , Células Jurkat , Selectina L/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Interleucina-2/biosíntesisRESUMEN
Activation of T cells by antigen-presenting cells (APCs) depends on the complex integration of signals that are delivered by multiple antigen receptors. Most receptor-proximal activation events in T cells were identified using multivalent anti-receptor antibodies, eliminating the need to use the more complex APCs. As the physiological membrane-associated ligands on the APC and the activating antibodies probably trigger the same biochemical pathways, it is unknown why the antibodies, even at saturating concentrations, fail to trigger some of the physiological T-cell responses. Here we study, at the level of the single cell, the responses of T cells to native ligands. We used a digital imaging system and analysed the three-dimensional distribution of receptors and intracellular proteins that cluster at the contacts between T cells and APCs during antigen-specific interactions. Surprisingly, instead of showing uniform oligomerization, these proteins clustered into segregated three-dimensional domains within the cell contacts. The antigen-specific formation of these new, spatially segregated supramolecular activation clusters may generate appropriate physiological responses and may explain the high sensitivity of the T cells to antigen.
Asunto(s)
Activación de Linfocitos , Agregación de Receptores , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Complejo CD3/inmunología , Complejo CD3/metabolismo , Línea Celular , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Ratones , Ratones Transgénicos , Péptidos/farmacología , Proteína Quinasa C/inmunología , Proteína Quinasa C/metabolismo , Receptores de Antígenos de Linfocitos T/agonistas , Talina/inmunología , Talina/metabolismoRESUMEN
A tumor cell line, named HS, was established from a bone metastasis of a patient with metastasizing paraganglioma. In vitro immunization of normal human peripheral blood mononuclear cells by coculturing with viable HS cells, followed by fusion with mouse myeloma cells, yielded a stable human/murine heterohybridoma producing the highly specific monoclonal anti-body KM-155. This MAB KM-155 is a member of the IgG3 subclass and shows no alpha GAL glycosylation that is specific for mouse but not for human antibodies. In pilot preclinical studies it could be demonstrated that MAB KM-155 is highly efficient in targeting a KM-155 antigen-expressing human tumor developing in nu/nu mice after xenografting. Moreover, the growth of KM-155 antigen-expressing human tumors in nu/nu mice was largely inhibited when the concentration of circulating MAB KM-155 was maintained at a high enough level by serial injections.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Inmunización Pasiva , Inmunoglobulina G/inmunología , Paraganglioma/secundario , Neoplasias de la Columna Vertebral/secundario , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antineoplásicos/aislamiento & purificación , Anticuerpos Antineoplásicos/uso terapéutico , Especificidad de Anticuerpos , Técnicas de Cocultivo , Reacciones Cruzadas , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Glicosilación , Humanos , Hibridomas/inmunología , Inmunoglobulina G/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Trasplante de Neoplasias , Paraganglioma/patología , Paraganglioma/terapia , Proyectos Piloto , Neoplasias de la Columna Vertebral/patología , Neoplasias de la Columna Vertebral/terapia , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Two highly metastatic human tumor cell lines, SLU-M1 SLU-M2, were established by in vivo selection in Balb/c-nu/nu mice of SLU-1 xenotransplants derived from an adenocarcinoma of the sigmoid colon. Metastatic spread was screened by transplantation of tissues from various organs of s.c.-tumor-bearing nu/nu mice. A monoclonal antibody, mab ME6H2, prepared against a membrane fraction of HT29 cells, also derived from an adenocarcinoma of the colon, showed high 125I-mab ME6H2 binding only to HT29 and SLU-1 cells, whereas hardly any binding was recorded for SLU-M1 and SLU-M2 cells. All cells of the HT29 and SLU-1 populations exhibited a positive immunofluoresence (IF) but only 1-5% of the SLU-M2 and 10-15% of the SLU-M1 subpopulation. A number of other tumor cell lines did not express the ME6H2 target antigen except for line MCF7, derived from an adenocarcinoma of the breast, which showed an IF positive reaction of 100% of the cells but only 25% of mab binding compared to HT29 and SLU-1 cells. The data indicate that expression of the ME6H2 target antigen is adenocarcinoma-specific and lack of expression is a marker for the metastatic potential of these cells. Mab ME6H2 was rapidly internalized upon binding to viable HT29 cells, resulting in an enhancement of cell growth in vitro and tumor growth in vivo. The mab ME6H2-defined target antigen was isolated from cell lysates by antibody affinity chromatography and was identified as a double band in SDS-PAGE with 31kD and 33kD molecular mass usually present in equal amounts.
Asunto(s)
Adenocarcinoma/patología , Biomarcadores de Tumor/análisis , Neoplasias del Colon/patología , Proteínas de la Membrana/análisis , Metástasis de la Neoplasia/patología , Adenocarcinoma/metabolismo , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , División Celular , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/patología , Neoplasias del Colon/metabolismo , Humanos , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Desnudos , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Monoclonal antibodies prepared against tyrosine phosphorylated epidermal growth factor receptor (EGFR) were tested for their effects on transmembrane signal transduction in A431 tumor cells. Monoclonal antibodies (mab) defined by SDS-sensitive epitopes, i.e., epitopes with conformational specificity, were most effective. Mab 5-125 reacting with a site of the extracellular EGFR domain blocked EGF-binding and cell proliferation in vitro, as well as tumor growth in vivo. However, this mab appeared not to be internalized upon binding to EGFR and did not trigger EGFR autophosphorylation. In contrast, mab 5-D43, also defined by an SDS-sensitive epitope and reacting with an extracellular EGFR site, did not block EGF binding but was readily internalized after binding to EGFR of untreated A431 cells. This mab induced EGFR tyrosine phosphorylation in cell lysates and tyrosine-specific autophosphorylation of insolubilized EGFR immune complexes. Cell growth in vitro was greatly stimulated in the presence of mab 5-D43. Since interaction of mab 5-D43 with EGFR induced most EGF-specific functions, although it did not bind to the EGF-specific site of EGFR, we have to assume that binding of mab 5-D43 to EGFR induced a conformational shift that activated the cytoplasmic EGFR kinase site. On the other hand, activation and/or accessibility of the EGFR kinase site could be blocked by mab 1-594, which is defined by an SDS-insensitive protein epitope of the cytoplasmic EGFR domain. Blocking of the EGFR kinase site by mab 1-594 also abolished EGF-induced tyrosine phosphorylation of endogenous cellular substrates with molecular masses of 145, 97, 85, 37, and 32 kDa, as well as of exogenous substrates such as GAT copolymer.
Asunto(s)
Receptores ErbB/fisiología , Transducción de Señal/fisiología , Animales , Anticuerpos Monoclonales , División Celular/fisiología , Citoplasma/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Células Tumorales CultivadasRESUMEN
Radiolabelled monoclonal antibodies (131I-MUC 8-22, 131I-MUC 2-63) were used for external scintigraphy of human glioma xenografts. To induce transplantation tumors. 5 x 10(6) cells (85HG-66) of an in vitro established human malignant astrocytoma (N66/85) were inoculated s.c. in BALB/c-nu/nu mice. The labelling of the immunoglobulins with 131iodine was carried out according to the iodogen method, the nude mice, bearing xenograft, received 30 m. 131I-labelled intact monoclonal immunoglobulins (200mCi: 7,4MBq) and the imaging was performed on days 4, 8 and 12 after the application. After 4 days, a clear tumor accumulation of iodinated MUC 2-63 antibodies recognizing surface determinants was visible. This enrichment of monoclonal antibodies (MAbs) led to a characteristic tumor presentation on day 8. Obviously, the MUC 2-63 antibodies remain in the tumor tissue for a long time, so that even on day 12 satisfactory tumor imaging is possible. On the other hand, neither with normal mouse IgG nor with MUC 8-22 antibodies - which react with intracellular structures - could a tumor localization be achieved. The result of the studies on the distribution of 131I-MUC 2-63 on day 19 was that the activity in the tumor tissue was about 4.4 times higher than in the blood and even more times higher than in solid organs.