RESUMEN
Mature nodose and petrosal ganglia neurons (placodally derived afferent neurons of the vagal and glossopharyngeal nerves) contain TrkA and TrkC, and transport specific neurotrophins [nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4)]. This study evaluated neurotrophin influences on the presence of neuropeptides and/or neurotransmitter enzymes in these visceral sensory neurons. NGF, NT-3 and NT-4 (10-100 ng/ml) were applied (5 days) to dissociated, enriched, cultures of mature nodose/petrosal ganglia neurons, and the neurons processed for tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neurofilament (NF-200) immunocytochemistry. Addition of NGF to nodose/petrosal ganglia neuron-enriched cultures significantly increased the number of TH-immunoreactive (ir) neurons, decreased the number of VIP-ir neurons in the cultures, and did not affect the numbers of CGRP-ir neurons. The addition of an NGF neutralizing antibody attenuated the effects of NGF on TH and VIP-ir neurons. NT-3 increased the number of VIP-ir neurons in the nodose/petrosal ganglia cultures and did not alter the numbers of TH-, or CGRP-ir neurons. The addition of an NT-3 neutralizing antibody attenuated the effects of NT-3 on VIP-ir neurons. NT-4 had no significant effects on the numbers of TH, VIP and CGRP-ir neurons. The absence of neurotrophin-induced changes in the numbers of NF-200-ir neurons in culture showed the lack of neurotrophin-mediated changes in survival of mature vagal afferent neurons. These data demonstrate that specific neurotrophins influence the numbers of neurons labeled for specific neurochemicals in nodose/petrosal ganglia cultures. These data, coupled with previous evidence for the presence of TrkA and TrkC mRNA and of the retrograde transport of NGF and NT-3, suggest important roles for NGF and NT-3 in the maintenance of transmitter phenotype of these mature visceral afferent neurons.
Asunto(s)
Nervio Glosofaríngeo/efectos de los fármacos , Factores de Crecimiento Nervioso/farmacología , Neuronas Aferentes/efectos de los fármacos , Neurotransmisores/metabolismo , Ganglio Nudoso/efectos de los fármacos , Factores de Edad , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Recuento de Células/estadística & datos numéricos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Nervio Glosofaríngeo/citología , Nervio Glosofaríngeo/metabolismo , Técnicas In Vitro , Masculino , Factor de Crecimiento Nervioso/farmacología , Factores de Crecimiento Nervioso/metabolismo , Proteínas de Neurofilamentos/metabolismo , Neuronas Aferentes/citología , Neuronas Aferentes/metabolismo , Neurotrofina 3/farmacología , Ganglio Nudoso/citología , Ganglio Nudoso/metabolismo , Ratas , Ratas Sprague-Dawley , Tirosina 3-Monooxigenasa/metabolismo , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Repeated exposure to bacterial endotoxin causes a diminished response by the host to further exposure. One important feature of this hyporesponsiveness is a reduced macrophage production of nitric oxide (NO) via the inducible nitric oxide synthase (iNOS) pathway. Using a murine macrophage model, we observed that hyporesponsiveness was accompanied by a decrease in the levels of NO release (measured as nitrite), iNOS protein and iNOS gene transcription. The expression of the putative lipopolysaccharide (LPS) receptor, CD14, was not altered. In vivo genomic footprinting showed that the same binding sites are occupied in the iNOS promoter and enhancer of desensitized macrophages and of LPS-responsive macrophages, yet the composition of NF-kappaB in the nuclei of these cells was found to be altered. The transcriptionally inactive homodimer p50-p50 represented the predominant binding activity in nuclei from LPS-pretreated cells before and after stimulation. Nuclei from cells which had not been pretreated but were stimulated contained more of the transcriptionally active p50-p65 heterodimer than their pretreated counterparts. Consistent with this, the cytosolic steady-state level of an inhibitor of NF-kappaB activity, I-kappaBalpha, was decreased in normal cells but not in pretreated cells. We propose that the presence of an overwhelming excess of transcriptionally inactive p50 homodimers on their kappaB sites in the iNOS control region in pretreated cells may block kappaB site binding by p50-p65, thereby reducing the activity of the protein complex governing iNOS transcription.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/inmunología , Lipopolisacáridos/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , FN-kappa B/inmunología , Óxido Nítrico Sintasa/inmunología , Animales , Línea Celular , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/genética , Ratones , FN-kappa B/genética , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo IIRESUMEN
Nitric oxide (NO) has been shown to inhibit the proliferation of lymphocytes. However, in tumour-bearing rats treated with the immunomodulator OM 163, the regressing nodules were heavily infiltrated by T lymphocytes, although they contained high levels of NO. We show here that NO, while inhibiting the proliferation of lymphocytes, increased their life-span, pointing to the ambivalence of this molecule in the course of tumour growth and regression.
Asunto(s)
Óxido Nítrico/farmacología , Linfocitos T/citología , Linfocitos T/inmunología , Adyuvantes Inmunológicos/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Carcinoma/inmunología , Carcinoma/terapia , Supervivencia Celular/efectos de los fármacos , Escherichia coli , Inmunoterapia , Activación de Linfocitos/efectos de los fármacos , Óxido Nítrico/biosíntesis , Neoplasias Peritoneales/inmunología , Neoplasias Peritoneales/terapia , RatasRESUMEN
A 4-week study was conducted to shed light on the question of whether compounds impairing immune homeostasis may escape the standard safety testing. Wistar rats were orally treated with cyclosporin A at dosages of 0 (control: olive oil), 1, 5 or 25 mg/kg/day. Ten rats/sex/group (study segment 1) were not immunized while six other rats/sex/group (study segment 2) were immunized 4 days before killing to perform a plaque forming cell (PFC) assay. All rats were subjected to routine safety evaluations (OECD guideline 407) and determination of IgM and IgG serum levels. Other immune parameters were evaluated using cells from spleen and mesenteric lymph nodes (segment 1). Effects on safety parameters were similar for immunized and non-immunized rats. A slight decrease of body weight gain (males, 25 mg/kg) accompanied slight clinical chemical and histomorphologic evidence of renal tubulotoxicity. Changes in safety parameters indicative of immune system alterations were: increased thymic corticomedullary ratio (> or = 5 mg/kg) and 25 mg/kg) minimal lymphopenia, low thymus weight, thymic cortical lymphocytolysis and low lymphoid cellularity of spleen and lymph nodes. They were associated with (males at > or = 1 mg/kg) dose-related decreases of T-cell receptor+ and CD4+ cells and increases of CD8+ cells, and decreased PFC (> or = 5 mg/kg) and lymphoproliferative responses to mitogens and alloantigens (25 mg/kg). There were no changes in natural killer activity. The conventional assay identified the drug as a potential immunomodulator. Specific immune assays (phenotyping, PFC) improved the threshold of detection. These results did not support the incorporation of specific immune tests in the standard 4-week study protocol.
Asunto(s)
Ciclosporina/toxicidad , Sistema Inmunológico/efectos de los fármacos , Inmunosupresores/toxicidad , Administración Oral , Animales , Formación de Anticuerpos/efectos de los fármacos , Especificidad de Anticuerpos , Antígenos CD4/efectos de los fármacos , Ciclosporina/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Inmunización , Inmunosupresores/administración & dosificación , Riñón/efectos de los fármacos , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Linfopenia/inducido químicamente , Masculino , Distribución Aleatoria , Ratas , Ratas Wistar , Receptores de Antígenos de Linfocitos T/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/metabolismo , Organismos Libres de Patógenos Específicos , Bazo/efectos de los fármacos , Bazo/metabolismo , Bazo/patología , Timo/citología , Timo/efectos de los fármacos , Timo/metabolismo , Timo/patología , Aumento de Peso/efectos de los fármacosRESUMEN
Although PROb colonic tumor cells are immunosuppressive, the immunomodulator OM 163 induced the disappearance of macroscopic peritoneal nodules in 50% of rats bearing a peritoneal carcinomatosis (p.c.) induced by PROb cells. When the p.c. developed, the number of T lymphocytes was low and the expression of interferon (IFN)-gamma mRNA in tumor nodules decreased very rapidly. The TGF-beta 1 secreted by PROb cells could be responsible for the immunosuppression, because the PROb cell supernatant inhibited IFN-gamma production and T lymphocyte proliferation. When the rats were treated with OM 163, an infiltration of T lymphocytes was observed in tumor nodules, as well as a high expression of the IFN-gamma mRNA. The antitumor efficiency of the immunomodulator OM 163 could be explained in part by a direct effect of OM 163 on T lymphocytes, because in vitro it stimulated the proliferation and the secretion of IFN-gamma of T lymphocytes. OM 163 could also act at the TGF-beta 1 level. Although OM 163 alone or in combination with IFN-gamma did not modify the TGF-beta 1 secretion by PROb cells in vitro, the expression of the TGF-beta 1 mRNA and the TGF-beta 1 protein content was decreased in vivo in treated tumor nodules.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antineoplásicos/farmacología , Regulación hacia Abajo/efectos de los fármacos , Linfocitos/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Animales , Relación Dosis-Respuesta a Droga , Escherichia coli , Expresión Génica/efectos de los fármacos , Terapia de Inmunosupresión , ARN Mensajero/metabolismo , RatasRESUMEN
In a model of colon cancer, spleen cells from tumor-bearing rats are neither cytotoxic nor proliferative in vitro in the presence of tumor cells. Interleukin-13 (IL-13) induced an in vitro cytolytic activity of spleen cells from tumor-bearing rats in response to the tumor they bore, but had no effect on spleen cells from normal rats. This cytotoxic response was dependent on both adherent and non- adherent cells, involving both an antigen-presenting activity that was enhanced by IL-13 and a cytolytic activity of lymphocytes. So, IL-13 reversed the tumor-induced immunosuppression.
Asunto(s)
Citotoxicidad Inmunológica/efectos de los fármacos , Interleucina-13/farmacología , Neoplasias Experimentales/inmunología , Bazo/efectos de los fármacos , Animales , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Ratas , Ratas Endogámicas , Bazo/inmunología , Células Tumorales CultivadasRESUMEN
NKR-P1 has been identified as a triggering structure selectively expressed on rat natural killer (NK) cells and adherent lymphokine-activated killer (A-LAK) cells. In vivo treatment with anti-NKR-P1 monoclonal antibody (mAb 3.2.3) was shown to induce complete inhibition of NK cytotoxicity and elimination of LAK cell precursors in Lewis and Fisher rat strains. We investigated the effects of mAb 3.2.3 in a colon tumor model in BDIX rats. Inoculation of animals with mAb 3.2.3 even at very high doses induced a strong but incomplete inhibition of NK cytotoxicity in nylon-wool-non-adherent spleen and peripheral blood cells. Generation of adherent A-LAK cells from their spleen precursors was also strongly by not fully inhibited. We also investigated the effect of treatment with mAb 3.2.3 on the tumorigenicity of the NK-sensitive REGb cell line. When subcutaneously inoculated in syngeneic animals, REGb cells induce tumors that first grow for 2 weeks, then spontaneously regress and disappear. In contrast with previous results using anti-asialoGM1, no significant difference in tumor growth was observed between rats treated with mAB 3.2.3 and control animals, even with a long-term treatment. In vitro, mAb 3.2.3 exhibited the same incomplete efficiency. Nylon-wool-non-adherent spleen cells treated with mAb 3.2.3 plus complement were completely free of 3.2.3(bright) cells, but retained a substantial NK activity and generated LAK cells after culture with IL-2. After an overnight incubation in standard medium of 3.2.3-depleted spleen cells, 3.2.3(bright) cells were partially recovered and the NK cytotoxic activity, as well as the generation of LAK cells, was significantly enhanced. These results suggest that a strong expression of NKR-P1 is not required for BDIX mononuclear cells to exhibit NK function and generate LAK cells under IL-2 activation.
Asunto(s)
Antígenos de Superficie/biosíntesis , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Células Asesinas Naturales/inmunología , Lectinas Tipo C , Animales , Anticuerpos Monoclonales/farmacología , Antígenos de Superficie/inmunología , Adhesión Celular/fisiología , División Celular/efectos de los fármacos , Neoplasias del Colon/terapia , Citocinas/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Inmunoterapia , Células Asesinas Activadas por Linfocinas/efectos de los fármacos , Células Asesinas Activadas por Linfocinas/inmunología , Células Asesinas Naturales/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Masculino , Ratones , Subfamilia B de Receptores Similares a Lectina de Células NK , Trasplante de Neoplasias , Ratas , Ratas Endogámicas , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Células Tumorales CultivadasRESUMEN
Colorectal adenocarcinomas are inherently resistant to anthracyclines and other topoisomerase-II inhibitors. Resistance to doxorubicin of colon cancer cells (Caco2) depends on 2 main mechanisms. The first is typical multi-drug resistance, characterized by the mdr1 gene and its product the P170 membrane glycoprotein. P170 effluxes anthracyclines out of cancer cells and is antagonized in vitro by verapamil. The second mechanism, which develops when cell-culture density increases, we have designated confluence-dependent resistance. Confluence-dependent resistance depends on the reduced topoisomerase II content of the G0/G1-phase cells which accumulate in the confluent population. We show here that short treatments of confluent Caco2 cells with slightly toxic concentrations of DNA-damaging agents (cisplatin, melphalan or mitomycin C) produced a transient accumulation of cells in S- and G2/M-phases of the cell cycle. Concomitantly with the increase in the S-phase population, the topoisomerase II cellular level and the sensitivity of cells to doxorubicin were greatly enhanced. Overcoming confluence-dependent resistance through S-phase accumulation and inhibition of multi-drug resistance by verapamil were fully additive, and a nearly complete reversal of confluent Caco2 cells' resistance to doxorubicin was obtained when both strategies were combined.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Resistencia a Múltiples Medicamentos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Adenocarcinoma/patología , Ciclo Celular , Cisplatino/farmacología , Neoplasias del Colon/patología , ADN-Topoisomerasas de Tipo II/biosíntesis , Doxorrubicina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Melfalán/farmacología , Mitomicinas/farmacología , Inhibidores de Topoisomerasa II , Células Tumorales CultivadasRESUMEN
The mechanisms of immunosuppression induced by colon cancer in rats were investigated at the systemic and tumor levels. During tumor growth (after i.p. injection of rat colon adenocarcinoma cells in syngeneic BD IX rats), Con A-induced proliferation of splenic mononuclear cells decreased and nitric oxide (NO) production by splenic macrophages increased concomitantly. Incubating splenic mononuclear cells with an inhibitor of NO synthase, NG-monomethyl-L-arginine, restored lymphocyte proliferation. A low level of inducible NO synthase mRNA was detectable in tumors by Northern blotting, with a weak increase during tumor growth. The NO concentration measured in the tumor nodules increased weakly parallel to the tumor growth. Five and six weeks after tumor cell injection, tumor-infiltrating lymphocytes from disaggregated tumors did not proliferate in the presence of Con A. Addition of NG-monomethyl-L-arginine inhibited the production of NO in tumor dissociations and enhanced tumor-infiltrating lymphocyte proliferation. Glyceryl trinitrate (a NO-releasing compound) totally inhibited the lymphocyte proliferation in vitro while it slightly reduced the tumor cell proliferation. T lymphocytes were therefore more sensitive to NO than were tumor cells. Culture medium from tumor cells induced NO production by splenic macrophages, although the factor involved has not yet been identified. Furthermore, tumor cells could also play a part in NO production by tumors because the tumor cells were induced to produce NO by IFN-gamma plus IL-1. These results strongly suggest the participation of NO in the tumor-induced immunosuppression in rats.
Asunto(s)
Tolerancia Inmunológica , Neoplasias Experimentales/inmunología , Óxido Nítrico/fisiología , Aminoácido Oxidorreductasas/biosíntesis , Animales , Células Cultivadas , Femenino , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Óxido Nítrico Sintasa , RatasRESUMEN
We have previously suggested that quinine and cinchonine could be good candidates for clinical circumvention of multidrug resistance (MDR) in hematological malignancies because of their tolerance and their retained efficacy in serum. In the present study, we have used the well-characterized multidrug resistant human leukemic cell line K562/ADM to compare the effect in vitro of quinine and cinchonine on doxorubicin, mitoxantrone, and vincristine uptake and cytotoxicity. In serum-free medium, quinine induced a dose-dependent increase of doxorubicin uptake reaching about 200% at 40 microM, while it had a slight and no effect on mitoxantrone and vincristine uptake respectively. In the same conditions, cinchonine induced a rapid and significant increase in the accumulation of the three drugs, reaching a plateau phase between 5 and 10 microM. Quinine and cinchonine induced both potentiation of doxorubicin, vincristine and mitoxantrone cytotoxicity in K562/ADM cells. However, quinine reached a plateau phase at 10 microM, while cinchonine had a maximal effect at 5 microM and was significantly more potent at low concentrations. When diluted in plasma, cinchonine was less bound to proteins than quinine. The free fraction of alkaloids was 37-55% for cinchonine and 20-30% for quinine. Cinchonine-induced enhancement of vincristine cellular accumulation was little modified by plasma proteins. When incubated in whole blood, the fraction of cinchonine trapped in red blood cells was rapidly and completely exchangeable with plasma. We conclude that cinchonine is a stronger inhibitor of MDR than quinine.