RESUMEN
The location of the human TNF genes within the MHC complex has prompted much speculation about the role of TNF alleles in the etiology of MHC-associated autoimmune diseases. On sequencing the 5' regulatory region of the human TNFA gene a G (TNFA-308G) to A (TNFA-308A) transition polymorphism at position -308 was discovered. We have developed a simple PCR assay to facilitate the screening of the -308 polymorphism at the DNA level. In view of the possible linkage between the TNFA-308A allele and a certain MHC type, TNFA-308 genotypes in HLA-typed healthy individuals (n = 88) were determined. A statistically significant association between the TNFA-308A allele and HLA-DR3, DQB1*0201, DQA1*0501, A1, B8, and the NcoI 5.5-kb RFLP of the TNFB gene was observed. In addition, we determined the frequency of the TNFA-308A allele in patients with FS (n = 13), an HLA-DR4-associated disease. In this study, no association was found of Felty's syndrome with the TNFA-308A allele, indicating that this allele does not appear to be a susceptibility factor for FS.
Asunto(s)
Alelos , Síndrome de Felty/genética , Complejo Mayor de Histocompatibilidad/genética , Factor de Necrosis Tumoral alfa/genética , Secuencia de Bases , Humanos , Linfotoxina-alfa/genética , Datos de Secuencia MolecularRESUMEN
Our group recently developed oligonucleotide probes associated to the TA10 and 2B3 specificities (1). Unambiguous typings were observed in a panel of homozygous typing cells and a family, using 32P-end labeled probes and PCR-amplified DNA (DQB exon 2). To investigate whether these TA10 and 2B3 associated oligonucleotides could be used in routine HLA-typing we extended the study to a panel of healthy, unrelated individuals. When TA10 and 2B3 typings by oligonucleotides were compared with those obtained in routine serology a complete correlation was observed for TA10. A discrepancy was seen for 2B3 typing in material obtained from DQw5- (and possibly DQw4)-positive individuals which could be explained by the CYNAP (cytotoxicity-negative, absorption-positive) phenomenon, using the IIB3 monoclonal antibody in routine tissue typing. Our results suggest that HLA-DQB oligonucleotide typing for TA10 and 2B3 is an accurate and reliable method and can be used effectively in routine HLA typing.
Asunto(s)
Alelos , Antígenos HLA/genética , Antígenos HLA-DQ/genética , Sondas de Oligonucleótidos , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , Sondas de ADN de HLA , Epítopos , Cadenas beta de HLA-DQ , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
A characteristic of active cytomegalovirus (CMV) infection is its suppressive effect on in vitro assays of immune function. The expression of CD11b by the Cd4+ and Cd8+ lymphocytes allows the identification of subsets with distinct regulatory functions of pokeweed mitogen (PWM) induced B cell differentiation. In order to relate that result with our previous observation that CMV carriers have significantly increased numbers of CD4+, HNK1+ and CD8+, HNK1+ lymphocytes in their peripheral blood compared with non-carriers, we performed a three-colour flow cytometric analysis of the co-expression of Cd11b and HNK1 by CD4+ and CD8+ lymphocytes obtained from 27 CMV carriers and 42 non-carriers. The differences between CMV carriers and non-carriers were significant for the CD4+, HNK1+ lymphocytes (median [5th and 95th percentiles], 59 [18 and 123 versus 24/7 and 73 per mm3, respectively; P less than 0.001) and CD8+, HNK1+ lymphocytes (59 [18 259] versus 52 [23 and 139] per mm3; P less than 0.001), but not for the CD4+, CD11b+ lymphocytes (59 [18 and 135] versus 52 [17 and 104] per mm3) and the CD8+, CD11b+ lymphocytes (85 [34 and 293] versus 82 [21 and 248] per mm3). The CD4+, HNK1+ and CD8+, HNK1+ lymphocytes that were increased in CMV carriers compared with non-carriers included mostly CD11b-, but also CD11b+ lymphocytes. After sorting CD4+ and CD8+ lymphocytes for four CMV carriers into HNK1+ and HNK1- fractions, we analyzed their regulatory functions on PWM-driven B cell Helper function to PWM-driven B cell differentiation was exclusively associated with the CD4+, HNK1- lymphocytes; the CD4+, HNK1+ generally did not show helper or suppressor activity in this assay. Both CD8+, HNK1+ and CD8+, HNK1- lymphocytes showed suppressor activity. Thus, the NHK1 marker does not constitute a phenotypical correlate for suppressor cells of PWM-driven B-cell differentiation.
Asunto(s)
Linfocitos B/inmunología , Portador Sano/inmunología , Infecciones por Citomegalovirus/inmunología , Activación de Linfocitos , Linfocitos T/clasificación , Adolescente , Adulto , Antígenos de Diferenciación/análisis , Antígenos de Diferenciación de Linfocitos T/análisis , Antígenos CD57 , Antígenos CD8 , Femenino , Humanos , Antígeno de Macrófago-1 , Masculino , Glicoproteínas de Membrana/análisis , Mitógenos de Phytolacca americana , Linfocitos T/inmunologíaRESUMEN
The toxicity and therapeutic efficacy of the combination of recombinant interferon gamma (rIFN-gamma) and alpha (rIFN-alpha) was investigated in 15 patients with metastatic melanoma. Patients were treated with an escalating dose of rIFN-gamma and a fixed dose of rIFN-alpha administered s.c. 3 times a week. The maximum dose was well tolerated. The median survival time of the patients was 7 months; no clinical remissions were observed. In the majority of cases, expression of HLA class-I and -II antigens on the patients' peripheral blood lymphocytes and monocytes increased markedly during treatment. An increase in HLA-DR expression of peripheral blood T lymphocytes was correlated with a longer survival time. This suggests that activation of T lymphocytes may have a favourable influence on the course of metastatic disease. The in vitro anti-proliferative activity of IFNs on melanoma cell lines isolated from melanoma metastases during treatment of 3 patients was determined. In contrast to the lack of in vivo anti-tumour effect in patients, both rIFN-gamma and rIFN-alpha inhibited DNA synthesis of these melanoma cell lines in vitro, combined IFNs acting synergistically. Anti-proliferative activity observed in vitro occurred at IFN concentrations below the peak serum levels achieved in vivo.
Asunto(s)
Interferón Tipo I/administración & dosificación , Interferón gamma/administración & dosificación , Melanoma/terapia , División Celular/efectos de los fármacos , Separación Celular , Ensayos Clínicos como Asunto , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/análisis , Antígenos de Histocompatibilidad Clase II/análisis , Humanos , Inyecciones Subcutáneas , Interferón Tipo I/efectos adversos , Interferón Tipo I/farmacocinética , Interferón gamma/efectos adversos , Interferón gamma/farmacocinética , Leucocitos Mononucleares/inmunología , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/mortalidad , Metástasis de la Neoplasia , Proteínas Recombinantes , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
We studied the effects of herpes virus carrier status on peripheral blood T lymphocyte subsets in 334 healthy individuals. IgG-class antibodies against cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), and varicella-zoster virus (VZV) were used as markers for the carrier status of those viruses. CMV carrier status was associated with significant increases in the numbers of some T cell subsets, whereas the carrier status of EBV, HSV, and VZV had no significant effects. The 159 CMV-seropositive individuals had higher numbers of HNK1+ T cells than did the 175 CMV-seronegative individuals [mean (SD), 292 (196)/microL v 164 (89)/microL, respectively], including the CD4+HNK1+ T cells [38 (48)/microL v 9 (13)/microL, respectively] and the CD8+HNK1+ T cells [166 (146)/microL v 73 (54)/microL, respectively]. Morphological and cytochemical studies showed that the expression of HNK1 by the CD4+ and CD8+ T cells was associated with the occurrence of azurophilic cytoplasmatic granules and a loss of nonspecific esterase activity. The numbers of CD4+HNK1+ and CD8+HNK1+ T cells increased proportionally to the levels of the IgG-class CMV antibody titers. We suggest that the increased numbers of CD4+HNK1+ and CD8+HNK1+ granular T cells in CMV carriers reflect the persistent interaction between CMV and the immune system of its hosts.
Asunto(s)
Portador Sano/patología , Infecciones por Herpesviridae/transmisión , Linfocitos T/clasificación , Factores de Edad , Anticuerpos Antivirales/análisis , Femenino , Herpesviridae/inmunología , Humanos , Masculino , Factores Sexuales , Linfocitos T/patología , Linfocitos T/ultraestructuraRESUMEN
The effect of cytomegalovirus (CMV) infection on the repopulation of the peripheral blood with T-lymphocytes was studied in recipients of lymphocyte-depleted bone marrow transplants (BMT) who had hematologic and cytogenetic evidence of engraftment. Lymphocyte depletion was performed using counterflow centrifugation and resulted in a median depletion of 98.4% (range 94.4%-99.8%) of the T cells. Between 8 and 105 days after BMT, the T-cell repopulation was characterized by a relative preponderance of T cells lacking the CD3 marker and a slow repopulation of CD3+, CD4+, and CD8+ T cells. The CD8+ T cells repopulated at a faster rate in patients with CMV infection than in those not infected with CMV. At the end of the 9- to 12-month follow-up period, patients with CMV infection had normal numbers of CD4+ and CD8+ T cells but increased numbers of HNK1+ T cells. Those without CMV infection had subnormal numbers of CD4+ T cells, normal numbers of CD8+ T cells, and numbers of HNK1+ T cells that attained the upper limit of the normal range. Most of the HNK1+ T cells in both patient groups coexpressed the CD8 marker. We conclude that the occurrence of CMV infection in recipients of lymphocyte-depleted BMT is associated with an increase in the number of T cells coexpressing CD8 and HNK1, just as in recipients of nondepleted BMT.
Asunto(s)
Trasplante de Médula Ósea , Infecciones por Citomegalovirus/sangre , Linfocitos T/microbiología , Adolescente , Adulto , Citomegalovirus , Hematopoyesis , Humanos , Leucemia/terapia , Persona de Mediana EdadRESUMEN
The influence of the cytomegalovirus (CMV) carrier status on peripheral lymphocyte subsets was studied in 70 healthy individuals. IgG-class antibodies against CMV late antigen were used as markers for the presence of CMV in those individuals. The 39 CMV-seropositive individuals had significantly higher numbers of CD3+ (P = 0.009), CD8+ (P = 0.005) and HNK1+ (P = 0.002) cells than the 31 CMV-seronegative individuals. Two-colour immunofluorescence studies revealed that the HNK1+ cells coexpressing CD4 or high density CD8 were particularly increased in the number under the influence of CMV, but not the HNK1+ cells coexpressing CD16. Since HNK1 and CD16 are markers associated with natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC), we investigated the influence of the CMV carrier status on those functions. The NK and ADCC functions of peripheral blood mononuclear cells (PBMC), HNK1+ and HNK1- cells were correlated with the percentages of CD16+ cells among those cells. Although CMV-seropositive individuals had significantly less CD16+ cells among their HNK1+ cells than CMV-seronegative individuals (mean and s.d.: 39 and 19%, versus 58 and 11%, P = 0.003), the NK and ADCC functions of the HNK1+ cells were similar in both groups. Also, the CMV carrier status did not influence significantly those functions of PBMC and HNK1- cells. We conclude that the CMV carrier status, i.e. CMV-seropositivity, is associated with a significant increase in the numbers of HNK1+ lymphocytes coexpressing T cell markers. That situation may reflect the continuing interaction between CMV and the immune system of its host.
Asunto(s)
Portador Sano/inmunología , Infecciones por Citomegalovirus/inmunología , Inmunidad Innata , Linfocitos T/clasificación , Adulto , Anciano , Anticuerpos Antivirales/análisis , Citotoxicidad Celular Dependiente de Anticuerpos , Separación Celular , Citomegalovirus/inmunología , Citotoxicidad Inmunológica , Femenino , Citometría de Flujo , Humanos , Células Asesinas Naturales/inmunología , Masculino , Persona de Mediana EdadRESUMEN
T lymphocyte subset numbers in bone marrow grafts were correlated with the cytomegalovirus (CMV) antibody status of the donors and with the occurrence of acute graft-versus-host disease (GVHD) in their recipients. We studied whether or not the (previously reported) association between donor CMV antibodies and GVHD could be explained by CMV-related changes in T cell subsets in the marrow grafts. There were no significant correlations between any of the T cell subsets in the marrow grafts and the occurrence of grades II-IV GVHD. A particular subset of CD8+ T cells carrying the HNK1 marker was significantly increased in the marrow grafts of CMV-seropositive donors. Although recipients of marrow from CMV-seropositive donors received an average of five times more CD8+ HNK1+ T cells than those with CMV-seronegative donors, that situation was not associated significantly with the development of GVHD.
Asunto(s)
Trasplante de Médula Ósea , Infecciones por Citomegalovirus/inmunología , Enfermedad Injerto contra Huésped/microbiología , Linfocitos T/clasificación , Donantes de Tejidos , Enfermedad Aguda , Anticuerpos Antivirales/análisis , Antígenos de Diferenciación de Linfocitos T , Infecciones por Citomegalovirus/sangre , Enfermedad Injerto contra Huésped/inmunología , Humanos , Fenotipo , Pruebas SerológicasRESUMEN
The combination of the Propidium Iodide method and automatic reading and scoring with a microscope fluorimeter is an accurate, reproducible and stable procedure for HLA-DR, MB and MT typing.
Asunto(s)
Pruebas Inmunológicas de Citotoxicidad/métodos , Antígenos de Histocompatibilidad Clase II/inmunología , Prueba de Histocompatibilidad/métodos , Linfocitos/inmunología , Antígenos HLA-DR , Humanos , PropidioRESUMEN
Optimal conditions are defined for hybridoma formation between mouse spleen cells and mouse myeloma cells. The results of using different numbers of spleen cells in the fusion process is reported in 2 parts. Part I deals with the number of spleen cells in relation to hybridoma formation and antibody production. Part II treats of the purity of hybridoma clones and the loss of antibody production following fusion. Part I. Two series of experiments show that when cell fusion is performed properly the total number of antibody producing clones is greater than in non-standard conditions. The yield of hybridomas obtained with a ratio of mouse myeloma to mouse spleen cells of 1:10 did not differ from that reported by De Blas et al. (1981). The number of hybridomas formed seems to depend mainly on the number of mouse spleen cells available. The most satisfactory yield of monoclonal antibodies is obtained under conditions producing growth in approximately 100% of the wells. Part II. Two weeks after fusion a number of antibody producing clones were cultured in limiting dilution. Analysis of the hybridomas indicated that at least 40% of the antibody producing clones disappear during the first 3 weeks. Antibody producing hybridomas were as a rule not outgrown by non-antibody producing clones.
Asunto(s)
Fusión Celular , Separación Celular/métodos , Hibridomas/inmunología , Activación de Linfocitos , Animales , Células Productoras de Anticuerpos/inmunología , Recuento de Células , Células Clonales/inmunología , Humanos , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Mieloma Múltiple/inmunología , Bazo/citologíaRESUMEN
A new high quality young-calf serum, Hy-clone calf serum (HcCS), was tested for use in hybridoma culture. This calf serum alone had little growth promoting activity and was much inferior to fetal calf serum (FCS). Red cell lysate (RCL) used in combination with the young-calf serum showed very good growth promoting activity. Growth was increased about threefold over that in the presence of FCS. However, HcCS and RCL could not substitute for feeder cells when hybridomas were cultured as single cells under conditions of limiting dilution. It is thought likely that the potent growth promoting factor in red cell lysate is hemoglobin.
Asunto(s)
Linfocitos B/fisiología , Hibridomas/fisiología , Animales , División Celular , Fusión Celular , Medios de Cultivo , Sulfato de Dextran , Dextranos/farmacología , Eritrocitos/fisiología , Sustancias de Crecimiento/sangre , Lipopolisacáridos/farmacología , RatonesRESUMEN
Accelerated proliferation of hybridoma cells was observed in the presence of human umbilical cord serum (HUCS). This had very strong growth-promoting activity, even at a concentration of 2%. A comparison was made between HUCS and other B cell growth promoters, such as lipopolysaccharide (LPS) and dextran sulfate (DxS), macrophage supernatant, and human endothelial culture supernatant (HECS). The growth-promoting effect of HUCS was superior. Using a microcytotoxicity assay, we found no significant differences in the number of antibody producing clones with the various culture media, except for fetal calf serum.
Asunto(s)
Linfocitos B/inmunología , Fusión Celular , Sangre Fetal/fisiología , Sustancias de Crecimiento/farmacología , Hibridomas/inmunología , Animales , Línea Celular , Medios de Cultivo , Técnicas de Cultivo/métodos , Citotoxicidad Inmunológica , Replicación del ADN , Femenino , Humanos , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Plasmacitoma/inmunología , Embarazo , Cordón UmbilicalRESUMEN
A routine method has been developed and tested in the laboratory for the automatic reading of HLA typing and screening for antibodies with the microlymphocytotoxicity test. The assay is more sensitive than the NIH technique, is rapid, produces objective results, and can be easily linked up with existing manual procedures. Multipurpose reading machines are now commercially available.