RESUMEN
BACKGROUND: Autoimmune Polyglandular Syndrome type 1 (APS-1) is a rare recessive inherited disease, caused by AutoImmune Regulator (AIRE) gene mutations and characterized by three major manifestations: chronic mucocutaneous candidiasis (CMC), chronic hypoparathyroidism (CH) and Addison's disease (AD). METHODS: Autoimmune conditions and associated autoantibodies (Abs) were analyzed in 158 Italian patients (103 females and 55 males; F/M 1.9/1) at the onset and during a follow-up of 23.7 ± 15.1 years. AIRE mutations were determined. RESULTS: The prevalence of APS-1 was 2.6 cases/million (range 0.5-17 in different regions). At the onset 93% of patients presented with one or more components of the classical triad and 7% with other components. At the end of follow-up, 86.1% had CH, 77.2% AD, 74.7% CMC, 49.5% premature menopause, 29.7% autoimmune intestinal dysfunction, 27.8% autoimmune thyroid diseases, 25.9% autoimmune gastritis/pernicious anemia, 25.3% ectodermal dystrophy, 24% alopecia, 21.5% autoimmune hepatitis, 17% vitiligo, 13.3% cholelithiasis, 5.7% connective diseases, 4.4% asplenia, 2.5% celiac disease and 13.9% cancer. Overall, 991 diseases (6.3 diseases/patient) were found. Interferon-ω Abs (IFNωAbs) were positive in 91.1% of patients. Overall mortality was 14.6%. The AIRE mutation R139X was found in 21.3% of tested alleles, R257X in 11.8%, W78R in 11.4%, C322fsX372 in 8.8%, T16M in 6.2%, R203X in 4%, and A21V in 2.9%. Less frequent mutations were present in 12.9%, very rare in 9.6% while no mutations in 11% of the cases. CONCLUSIONS: In Italy, APS-1 is a rare disorder presenting with the three major manifestations and associated with different AIRE gene mutations. IFNωAbs are markers of APS-1 and other organ-specific autoantibodies are markers of clinical, subclinical or potential autoimmune conditions.
Asunto(s)
Enfermedad de Addison , Candidiasis Mucocutánea Crónica , Hipoparatiroidismo , Interferón Tipo I/inmunología , Poliendocrinopatías Autoinmunes , Factores de Transcripción/genética , Enfermedad de Addison/diagnóstico , Enfermedad de Addison/etiología , Adulto , Autoanticuerpos/sangre , Candidiasis Mucocutánea Crónica/diagnóstico , Candidiasis Mucocutánea Crónica/etiología , Femenino , Humanos , Hipoparatiroidismo/diagnóstico , Hipoparatiroidismo/etiología , Italia/epidemiología , Masculino , Mortalidad , Mutación , Poliendocrinopatías Autoinmunes/diagnóstico , Poliendocrinopatías Autoinmunes/genética , Poliendocrinopatías Autoinmunes/mortalidad , Poliendocrinopatías Autoinmunes/fisiopatología , Prevalencia , Proteína AIRERESUMEN
Many airway diseases in children, notably bronchiolitis, cystic fibrosis (CF), non-CF bronchiectasis including primary ciliary dyskinesia, pneumonia, and severe asthma are associated with retention of airway secretions. Medications to improve secretions clearance, the mucoactive medications, are employed to treat these diseases with varying degrees of success. This manuscript reviews evidence for the use of these medications and future directions of study.
Asunto(s)
Asma/tratamiento farmacológico , Bronquiectasia/tratamiento farmacológico , Bronquiolitis Viral/tratamiento farmacológico , Trastornos de la Motilidad Ciliar/tratamiento farmacológico , Fibrosis Quística/tratamiento farmacológico , Expectorantes/uso terapéutico , Fármacos del Sistema Respiratorio/uso terapéutico , Adolescente , Corticoesteroides/uso terapéutico , Niño , Preescolar , Antagonistas Colinérgicos/uso terapéutico , Desoxirribonucleasa I/uso terapéutico , Diuréticos Osmóticos/uso terapéutico , Bloqueadores del Canal de Sodio Epitelial/uso terapéutico , Humanos , Lactante , Macrólidos/uso terapéutico , Manitol , Proteínas Recombinantes/uso terapéutico , Solución Salina Hipertónica , Índice de Severidad de la EnfermedadRESUMEN
PURPOSE: Mitotane is the only chemotherapeutic agent available for the treatment of adrenocortical carcinoma (ACC), however, the anti-neoplastic efficacy is limited due to several side-effects in vivo. There is, therefore, a need of exploring for new anti-tumoral agents which can be used either alone or in combination with mitotane. The active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) acts as an anti-proliferative agent in human cancer by inhibiting the Wnt/beta-catenin pathway through the vitamin D receptor (VDR). The aim of this study was to study the effects of mitotane and 1α,25(OH)2D3, individually or in combination, in an in vitro model with H295R ACC cells, and to elucidate the molecular events behind their effects involving the Wnt/beta-catenin signaling. METHODS AND RESULTS: Multiple concentrations of mitotane and 1α,25(OH)2D3, individually or in combination, were tested on H295R cells for 24-96 h, and the effects analysed by MTT. A reduction in cell growth was observed in a dose/time-dependent manner for both mitotane and 1α,25(OH)2D3. In addition, a combination of clinically sub-therapeutic concentrations of mitotane with 1α,25(OH)2D3, had an additive anti-proliferative effect (Combination Index = 1.02). In a wound healing assay, individual treatments of both mitotane and 1α,25(OH)2D3 reduced the migration ability of H295R cells, with the effect further enhanced on combining both the agents. Western blotting and qRT-PCR analysis showed a modulation of the Wnt/beta-catenin and VDR signaling pathways. CONCLUSION: Our results show an additive effect of mitotane and 1α,25(OH)2D3 on the inhibition of H295R ACC cell growth and viability, and suggest that molecular mechanisms of their effects involve a functional link between VDR and Wnt/beta-catenin pathways.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/metabolismo , Corteza Suprarrenal/efectos de los fármacos , Carcinoma Corticosuprarrenal/metabolismo , Calcitriol/farmacología , Mitotano/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Corteza Suprarrenal/metabolismo , Línea Celular Tumoral , Humanos , beta Catenina/metabolismoRESUMEN
Osteopetrosis is a rare and heterogeneous genetic disorder characterized by dense bone mass that is a consequence of defective osteoclast function and/or development. Autosomal recessive osteopetrosis (ARO) is the most severe form and is often fatal within the first years of life; early hematopoietic stem cell transplant (HSCT) remains the only curative treatment for ARO. The majority of the ARO-causing mutations are located in the TCIRG1 gene. We report here the identification and characterization of an A to T transversion in the fourth base of the intron 2 donor splice site (c.117+4AâT) in TCIRG1, a mutation not previously seen in the Ashkenazi Jewish (AJ) population. Analysis of a random sample of individuals of AJ descent revealed a carrier frequency of approximately 1 in 350. Genotyping of five loci adjacent to the c.117+4AâT-containing TCIRG1 allele revealed that the presence of this mutation in the AJ population is due to a single founder. The identification of this mutation will enable population carrier testing and will facilitate the identification and treatment of individuals homozygous for this mutation.
Asunto(s)
Efecto Fundador , Osteopetrosis/genética , Mutación Puntual , Sitios de Empalme de ARN/genética , ATPasas de Translocación de Protón Vacuolares/genética , Análisis Mutacional de ADN , Frecuencia de los Genes , Humanos , Lactante , Intrones , Judíos/genéticaRESUMEN
BACKGROUND: IL-13, a helper T cell type 2 (Th2) cytokine, transforms cultured airway epithelial cells to goblet cells, and this is not inhibited by corticosteroids. IL-33 stimulates Th2 cytokines and is highly expressed in airways of persons with asthma. The effect of IL-33 on goblet cell differentiation and cytokine secretion has not been described. OBJECTIVE: We examined the effect of IL-33 on CXCL8/IL-8 secretion from goblet or normally differentiated human bronchial epithelial (NHBE) cells and signalling pathways associated with IL-33 activation in these cells. METHODS: Normal human bronchial epithelial cells were grown to goblet or normally differentiated ciliated cell phenotype at air-liquid interface in the presence or absence of IL-13. After 14 days, differentiated cells were exposed to IL-33 for 24 h. RESULTS: CXCL8/IL-8 secretion into the apical (air) side of the goblet cells was greater than from normally differentiated cells (P < 0.01), and IL-33 stimulated apical CXCL8/IL-8 release from goblet cells, but not from normally differentiated cells (P < 0.01). IL-33 increased ERK 1/2 phosphorylation in goblet cells (P < 0.05), and PD98059, a MAPK/ERK kinase inhibitor, attenuated IL-33-stimulated CXCL8/IL-8 secretion from goblet cells (P < 0.001). IL-13 induced ST2 mRNA (P < 0.02) and membrane-bound ST2 protein expression on the apical side surface of goblet cells compared with normally differentiated cells, and neutralization with anti-ST2R antibody attenuated IL-33-induced apical CXCL8/IL-8 secretion from goblet cells (P < 0.02). CONCLUSIONS AND CLINICAL RELEVANCE: Goblet cells secrete CXCL8/IL-8, and this is increased by IL-33 through ST2R-ERK pathway, suggesting a mechanism for enhanced airway inflammation in the asthmatic airway with goblet cell metaplasia.
Asunto(s)
Células Caliciformes/efectos de los fármacos , Células Caliciformes/metabolismo , Interleucina-8/biosíntesis , Interleucinas/farmacología , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Diferenciación Celular/efectos de los fármacos , Expresión Génica , Células Caliciformes/citología , Humanos , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-13/farmacología , Interleucina-33 , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Compuestos Orgánicos/farmacología , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Mucosa Respiratoria/efectos de los fármacosRESUMEN
We show that electrochemical formation of long probes with nanosharp tips can be controlled by choosing an appropriate thermodynamic pathway of metal to metal oxide and hydroxide transformation. Currently, convection-limited electropolishing (CLE) is extensively used. Nanosharp probes are produced by electrochemically etching a wire until it breaks into two pieces. This process is difficult to control because of the complexity of the associated hydrodynamic flows. We introduce transport-limited electropolishing (TLE), where the electrochemical reaction results in the formation of metal oxides and hydroxides which form a porous surface layer hindering the flow of electrolyte. The developed TLE method enables one to make long tapered needles. The taper can spread over more than 6 mm while the radius of tip curvature can be decreased down to 30 nm. These needles are strong and were successfully applied for piercing single smooth vascular muscle cells.
RESUMEN
BACKGROUND: Gastrointestinal stromal tumors (GISTs) and desmoid tumors (DTs) are two rare mesenchymal tumor. Anecdotal reports of individuals with both diseases led us to make the hypothesis that the association is a nonrandom event as the probability would be extremely low to observe such cases if they were independent events. PATIENTS AND METHODS: We evaluated the existence of patients with GIST and DT in a large multicenter cohort at 10 institutions in the United States, Australia and Europe. Data on gender, age at diagnosis, KIT, PDGFRA, CTNNB1 mutation status and follow-up time after diagnosis were collected. RESULTS: We identified 28 patients diagnosed with both tumors. DT was diagnosed after GIST in 75% of patients and concomitantly in 21%. In only one case (4%), GIST was diagnosed after DT. KIT or PDGFRA mutations were detected in 12 of 14 GIST, 9 in KIT exon 11, 2 in KIT exon 9 and 1 in PDGFRA. CONCLUSION: A statistical analysis of these 28 cases suggests a nonrandom association between GIST and DT. Further studies may be able to elucidate the underlying biology responsible for this association.
Asunto(s)
Fibromatosis Agresiva/complicaciones , Fibromatosis Agresiva/epidemiología , Tumores del Estroma Gastrointestinal/complicaciones , Tumores del Estroma Gastrointestinal/epidemiología , Adulto , Anciano , Anciano de 80 o más Años , Australia/epidemiología , Estudios de Cohortes , Europa (Continente)/epidemiología , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Estados Unidos/epidemiologíaRESUMEN
BACKGROUND: Glucocorticosteroids (GCS) are used to treat bronchial asthma, but are not uniformly effective, especially in severe asthma. IL-13 is a T helper type 2 cytokine implicated in the pathogenesis of asthma, and IL-13 induces mucus production and goblet cell hyperplasia in airway epithelial cells. The effect of GCS on IL-13-induced mucin production is not well characterized. OBJECTIVE: The aim of this study was to evaluate the effect of dexamethasone (Dex), a potent synthetic GCS, on IL-13-induced MUC5AC mucin expression and goblet cell proliferation in differentiated normal human bronchial epithelial cells (NHBECs). METHODS: NHBECs were cultured for 14 days at an air-liquid interface with IL-13, with or without Dex. MUC5AC protein secretion and mRNA expression was determined using ELISA and quantitative real-time PCR. IL-8 production was assayed using ELISA. Histochemical analysis was performed using H&E and periodic acid-Schiff stain, and MUC5AC immunostaining. RESULTS: Although Dex dose dependently inhibited IL-8 release induced by 5 ng/mL IL-13, Dex 0.001-1 µg/mL had no effect on IL-13 induced MUC5AC protein secretion or mRNA expression. Dex paradoxically increased MUC5AC induced by IL-13 at 0.5 and 1 ng/mL, but had no effect alone or with IL-13 at 0.1 ng/mL. Dex 0.001-1 µg/mL did not inhibit the differentiation of cells into goblet cells and MUC5AC-positive cells induced by IL-13. CONCLUSION AND CLINICAL RELEVANCE: Dex at therapeutic concentrations did not inhibit the effects of IL-13 on goblet cell differentiation, characteristic of severe asthma. Paradoxically, MUC5AC production was increased with lower dose IL-13 exposure. This may lead to airway mucus obstruction commonly seen in life-threatening asthma.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Dexametasona/farmacología , Células Caliciformes/citología , Células Caliciformes/efectos de los fármacos , Interleucina-13/farmacología , Mucina 5AC/biosíntesis , Mucosa Respiratoria/metabolismo , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Humanos , Interleucina-8/biosíntesis , Mucina 5AC/genética , ARN Mensajero/metabolismo , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/inmunología , Transcripción Genética/efectos de los fármacosRESUMEN
Mice immunized with hapten-autologous serum albumin conjugates (DNP-mouse serum albumin) were shown to contain immune B and T cells with specificity for the conjugate. Fractionation on antigen-coated Sepharose beads showed that B cells could be subdivided in two major groups: those reacting against the haptenic group (DNP) and those reactive against the new antigenic determinants (NADs) introduced into the protein carrier by the hapten coupling. It was shown previously that humoral antibodies formed against hapten-mouse serum albumin conjugates also were directed against these two groups of antigenic determinants and that the immune response to the NADs does not follow the genetic rules of high or low response against the hapten used. All together these findings support the distinct nature of the NADs over the haptenic groups, as recognized both at the humoral and cellular level. Absorption of mouse cells immune to hapten-autologous serum albumin conjugates on antigen-coated Sepharose beads using a variety of incubation conditions resulted in no specific retention of T cells. Therefore we had to resort to specificity studies of T cells in relation to T cell function. Relatively pure immune T cell suspensions were obtained using fractionation on anti-immunoglobulin-coated columns. DNP-MSA-specific T cells were shown to be very specific for the DNP-MSA conjugate with only one exception: they cross-reacted with antigenic determinants on DNP-rat serum albumin. As DNP-specific help was excluded in the present transfer system (as shown by the inability of cells from DNP-skin-painted mice and DNP-heterologous protein conjugate specific T cells [anti-immunoglobulin column purified] to help a DNP-MSA response), these results demonstrate the NAD specificity of the DNP-MSA-reactive T cells. The cross-reactivity pattern of DNP-MSA-specific T cells was similar to that found for humoral anti-NAD antibodies produced against the same immunogen. Whether B and T cells are activated by the same antigenic determinants is discussed.
Asunto(s)
Formación de Anticuerpos , Linfocitos B/inmunología , Proteínas Portadoras , Haptenos , Linfocitos T/inmunología , Animales , Especificidad de Anticuerpos , Suero Antilinfocítico , Bovinos/inmunología , Pruebas Inmunológicas de Citotoxicidad , Dinitrofenoles , Pruebas de Hemaglutinación , Reacción de Inmunoadherencia , Sueros Inmunes , Inmunización Pasiva , Lisina , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Ovalbúmina , Conejos/inmunología , Albúmina Sérica , Timo/inmunologíaRESUMEN
Immune lymph node cells from guinea pigs respond to soluble antigen in vitro by an increase in DNA synthesis. Optimal conditions for this proliferative response were studied in the present article. Under such conditions, immune cells showed increasing responses with increasing antigen concentration in vitro, the threshold dose of activation frequently being as low as 0.02 microg per culture. In contrast, normal lymph node cells (from FCA-stimulated animals) did only respond to antigen at very high doses (20 mg/culture), and immune cell dilution studies could be performed in normal cells without changing the kinetics of the antigen specific response of immune cells. Fractionation on anti-Ig columns indicated that purified, immune T lymphocytes were quite capable of proliferating in vitro upon antigen stimulation. However, our attempts to adsorb the proliferating cells onto chemically defined immunoadsorbants failed despite the fact that immune B cells (as measured by the rosette assay) were retained almost completely by such a procedure. Purified, immune T lymphocytes from guinea pigs immunized with different antigen concentrations in vivo and/or obtained at different times after immunization were tested for a differential sensitivity toward antigen-induced DNA synthesis in vitro. However, we were not able to demonstrate any regular increase in sensitivity to antigen in vitro, and if found, it seemed to be more dependent upon the number of antigen reactive cells in the population studied rather than upon differences in the average avidity of the receptors on the cells proliferating in vitro. The results in the present article are discussed in relation to current knowledge and hypotheses on T-lymphocyte receptors.
Asunto(s)
Sitios de Unión de Anticuerpos , Haptenos , Inmunidad Celular , Linfocitos T/inmunología , Animales , Formación de Anticuerpos , Especificidad de Anticuerpos , Bovinos/inmunología , Reacciones Cruzadas , Dinitrofenoles , Técnica del Anticuerpo Fluorescente , Vidrio , Cobayas , Pruebas de Inhibición de Hemaglutinación , Pruebas de Hemaglutinación , Reacción de Inmunoadherencia , Sueros Inmunes/aislamiento & purificación , Inmunización , Técnicas Inmunológicas , Ganglios Linfáticos/inmunología , Ovalbúmina , Conejos/inmunología , Ratas/inmunología , Albúmina Sérica , Albúmina Sérica Bovina , Linfocitos T/efectos de la radiación , Timidina/metabolismo , TritioRESUMEN
The present study describes the qualitative reactions of a xenogeneic anti-idiotype (Id) antiserum produced in a mouse-gamma-globulin-tolerant rabbit (5,936) against B6 anti-CBA IgG antibodies. The results showed that such an anti-Id antiserum reacts specifically against anti-H-2k antibodies and against H-2k alloantigen-activated T cells from the following pairs of congenic mice: B10 (H-2b) and B10.D2 (H-2d); and A.BY (H-2b) and A.SW (H-2s), but not against C3H.SW (H-2b) and C3H.OH (H-2o); and BALB/b (H-2b) and BALB/c (H-2d). CB 20 (BALB/c mice with the Ig-1b allotype) anti-CBA T blasts also express idiotypic determinants that react with rabbit 5,936 antiserum. Thus, positive reactions are obtained between rabbit 5,936 anti-Id antiserum and anti-H-2k IgG preparations and T blasts from mice carrying the Ig-1b or Ig-1e allotype, but not from mice carrying the Ig-1a allotype. These reactions are qualitatively independent of the H-2 genotype of the Id-producing mice. Such a finding strongly suggests that the Id-bearing receptor molecules on mouse T cells are coded for by genes that are associated with the Ig heavy-chain-linkage group and not to the mouse histocompatibility complex. Furthermore, the anti-Id antibodies studied react preferentially against anti-H-2k antibodies or T cells with specificity toward the IAk-region-associated serological specificities. Thus, genes associated with the Ig heavy-chain-linkage group seem to be structural genes for at least T-cell receptors with specificity for IA-region-coded membrane antigens.
Asunto(s)
Sitios de Unión de Anticuerpos , Linfocitos T/inmunología , Animales , Anticuerpos Antiidiotipos , Genes , Histocompatibilidad , Inmunización , Alotipos de Inmunoglobulinas , Inmunoglobulina G , Cadenas Pesadas de Inmunoglobulina , Ratones , Conejos , Bazo/citologíaRESUMEN
Spleen cells from mice immunized against ovalbumin (OA) or dinitrophenylated mouse serum albumin (DM) were found to be specifically cytotoxic in vitro towards target cells (chicken red blood cells) coated with these antigens. Inhibition of specific cytotoxicity was observed when free soluble antigen was added to the incubation mixtures. DM-immune cell cytotoxicity could be specifically and completely inhibited by DNP-lysine and was thus shown to be hapten specific. Complete and specific inhibition was also observed for OA-immune cell cytotoxicity using OA as inhibitor, but compared with the inhibition curves obtained with DNP-lysine, the OA cytotoxicity inhibition curves were shifted by a factor of about one hundred towards lower molar inhibitor concentrations. Very similar results were observed when the serum antibodies of DM- and OA-immune animals were analyzed by passive hemagglutination inhibition. With increasing time after immunization, both cytotoxicity inhibition curves and agglutination inhibition curves, shifted to lower antigen or hapten concentrations. Specific cytotoxicity against antigen-coated target cells was induced in nonimmune spleen cells (a) by serum from immune animals, and (b) by supernatants from in vitro immune cell cultures. In both instances, the factor which induced antigen-specific cytotoxic activity could be absorbed on anti-mouse Ig columns, thus demonstrating its immunoglobulin nature. The ability of target cell bound antibodies to induce cytotoxicity in nonimmune spleen cells was restricted to the 7S antibody class.
Asunto(s)
Reacciones Antígeno-Anticuerpo , Pruebas Inmunológicas de Citotoxicidad , Bazo/inmunología , Animales , Proteínas Portadoras , Células Cultivadas , Radioisótopos de Cromo , Epítopos , Eritrocitos/inmunología , Haptenos , Inmunoglobulina G , Ratones , Ratones Endogámicos CBA , Albúmina Sérica Bovina , Bazo/citologíaRESUMEN
Human blood mononuclear leukocytes stimulated with toxoplasma antigen, concanavalin A, mezerein plus lentil lectin, or staphylococcal enterotoxin A secreted a factor (macrophage-activating factor, or MAF) that enhanced the capacity of human macrophages to release H2O2 and to kill toxoplasmas. The same lymphoid supernatants contained IFN gamma but not IFN alpha or IFN beta. The MAF activity of six of seven unfractionated supernatants was completely eliminated by a monoclonal antibody that neutralizes IFN gamma, and MAF in the remaining supernatant was almost completely neutralized. Native IFN gamma partially purified by two independent protocols to specific activities of 1 X 10(6) and 10(7) U/mg protein was enriched in MAF activity at least as much as in antiviral activity. The capacity of macrophages to secrete H2O2 after incubation in partially purified native IFN gamma (mean peak stimulation, 8.8-fold) was greater than with unpurified lymphokines (3.8-fold) and sometimes equaled or exceeded the capacity of freshly harvested monocytes. The MAF activity of the partially purified native IFN gamma preparations was abolished by monoclonal anti-IFN gamma. Finally, IFN gamma of greater than 99% estimated purity was isolated (at Genentech, Inc.) from bacteria transformed with the cloned human gene for this lymphokine. Recombinant IFN gamma had potent MAF activity, stimulating the peroxide-releasing capacity of macrophages an average of 19.8-fold at peak response and enhancing their ability to kill toxoplasmas from 2.6 +/- 1.3% for untreated cells to 54 +/- 0.4% for treated cells. Attainment of 50% of the maximal elevation in peroxide-releasing capacity required a geometric mean concentration of 0.1 antiviral U/ml of recombinant IFN gamma, which is estimated to be approximately 6 picomolar for this preparation. Peroxide secretory capacity and toxoplasmacidal activity of macrophages peaked 2-4 d after exposure to IFN gamma. Peroxide-secretory capacity remained elevated during at least 6 d of continuous exposure, but the effect of IFN gamma was reversed within about 3 d of its removal. Activation was usually but not invariably accompanied by characteristic changes in cell morphology. Thus, IFN gamma activates human macrophage oxidative metabolism and antimicrobial activity, and appeared to be the only factor consistently capable of doing so in the diverse LK preparations tested.
Asunto(s)
Peróxido de Hidrógeno/sangre , Interferón gamma/fisiología , Linfocinas/fisiología , Activación de Macrófagos , Adulto , Anticuerpos Monoclonales/fisiología , Fenómenos Fisiológicos Sanguíneos , Concanavalina A/fisiología , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Macrófagos/metabolismo , Recombinación Genética , Toxoplasma/crecimiento & desarrolloRESUMEN
125I-labeled TNF(LuKII) (tumor necrosis factor) binds specifically to human and mouse cell lines sensitive to the cytotoxic effect of TNF, but not to cells made resistant to TNF. TNF-sensitive cells have cell surface receptors with a high affinity for TNF(LuKII). Mouse TNF competes with TNF(LuKII) for receptor binding. Scatchard analysis of the binding data yielded linear plots and suggests that TNF(LuKII) binds to homogeneous receptor sites. The number of TNF(LuKII) receptors on two TNF-sensitive cell lines is 200-300 per cell and the affinity constant of the receptor for TNF(LuKII) is approximately 1 X 10(-10) M.
Asunto(s)
Glicoproteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Células Cultivadas , Células HeLa/metabolismo , Humanos , Células L/metabolismo , Ratones , Receptores del Factor de Necrosis Tumoral , Especificidad de la Especie , Factor de Necrosis Tumoral alfaRESUMEN
TNF-resistant lines of L cells can be derived from TNF-sensitive populations by repeated exposure to TNF, and these resistant L cells, in contrast to sensitive L cells and other types of cells, lack demonstrable cell surface receptors for TNF. We have now found that TNF-resistant L cells produce a factor that is cytotoxic for L cells and has the following distinguishing characteristics of mouse TNF: it is a protein of 43 kD, composed of 16 kD subunits, that competes with TNF for receptor binding, induces hemorrhagic necrosis of the TNF-sensitive mouse sarcoma Meth A, has synergistic cytotoxic action with interferon, and its activity is neutralized by antibody to TNF. The two conclusions of this study are that cells selected for TNF resistance spontaneously produce a molecule resembling macrophage TNF, and that cells of nonhematopoietic origin are capable of producing TNF.
Asunto(s)
Glicoproteínas/biosíntesis , Inhibidores de Crecimiento/biosíntesis , Animales , Línea Celular , Resistencia a Medicamentos , Glicoproteínas/inmunología , Glicoproteínas/farmacología , Inhibidores de Crecimiento/inmunología , Inhibidores de Crecimiento/farmacología , Células L/efectos de los fármacos , Células L/metabolismo , Ratones , Conejos , Receptores de Superficie Celular/análisis , Receptores del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfaRESUMEN
A cDNA clone complementary to an interferon (IFN)-induced mRNA approximately 3 kb in length was identified and sequenced revealing homology with the endoplasmic reticular heat shock protein/ATPase gp96. Both IFN-alpha and -gamma transcriptionally upregulate expression of this gene. gp96 transcripts, protein, and ATPase activity are shown to be enhanced as a result of IFN treatment in two human cell lines and this effect requires de novo protein synthesis. gp96 molecules have recently been implicated in the presentation of endogenous antigens. A number of the key elements in this pathway, the transporter proteins, the major histocompatibility complex (MHC)-linked units of the proteasomes and the MHC class I molecules are known to be IFN inducible. Our results show that yet another molecule suggested to play an accessory role in the endogenous presentation pathway is IFN inducible. Further, our studies represent the first demonstration of modulation of expression of a heat shock protein by a cytokine and identify a new enzymatic activity upregulated in IFN-treated cells.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico/biosíntesis , Interferones/farmacología , Adenosina Trifosfatasas/biosíntesis , Secuencia de Bases , Línea Celular , ADN Complementario/aislamiento & purificación , Proteínas de Choque Térmico/genética , Humanos , Datos de Secuencia Molecular , Transcripción Genética , Regulación hacia ArribaRESUMEN
We investigated the effect of OKT3 antibody and interleukin 2 (IL-2) on Tac antigen expression and the proliferation of human peripheral blood mononuclear leukocytes. OKT3 monoclonal antibody at low, nonmitogenic concentrations (25 pg/ml) or IL-2 alone at optimal concentrations (20 U/ml) did not induce IL-2 receptor expression, as measured by Tac antibody or by T cell proliferation. However, costimulation with these concentrations of OKT3 antibody and IL-2 led to Tac antigen expression and T cell proliferation. These data suggest that the T cells are activated in two steps: OKT3 antibody at 25 pg/ml does not induce Tac antigen expression, but preactivates T cells to become responsive to IL-2. The addition of exogenous IL-2 then leads to expression of the IL-2 receptor, as recognized by Tac antibody, and to subsequent proliferation.
Asunto(s)
Antígenos de Superficie/análisis , Interleucina-2/fisiología , Linfocitos T/inmunología , Anticuerpos Monoclonales/fisiología , Antígenos de Superficie/inmunología , ADN/análisis , ADN/biosíntesis , Técnica del Anticuerpo Fluorescente , Humanos , Interferón gamma/fisiología , Interleucina-2/biosíntesis , Interleucina-2/metabolismo , Cinética , Activación de Linfocitos , Activación de Macrófagos , Mitomicina , Mitomicinas/farmacología , Biosíntesis de Proteínas , Linfocitos T/análisis , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
Cytokines affecting mononuclear phagocytes were screened for activation of human macrophages to secrete H2O2 and kill toxoplasmas. In contrast to recombinant interferon-gamma (rIFN gamma), the following factors, tested in partially or highly purified form and over a wide range of concentrations, did not augment these functions: native interferon-alpha (nIFN alpha), rIFN alpha A, rIFN alpha D, rIFN beta, colony stimulating factor (type 1) (CSF-1), CSF for granulocytes and macrophages (GM-CSF), pluripotent CSF (p-CSF), tumor necrosis factor (TNF), native interleukin 2 (nIL-2), and rIL-2. Partially purified migration inhibitory factor (MIF) enhanced H2O2-releasing capacity submaximally without inducing antitoxoplasma activity, and warrants further study.