RESUMEN
Human and mouse respiratory tracts show anatomical and physiological differences, which will benefit from alternative experimental models for studying many respiratory diseases. Pig has been recognized as a valuable biomedical model, in particular for lung transplantation or pathologies such as cystic fibrosis and influenza infection. However, there is a lack of knowledge about the porcine respiratory immune system. Here we segregated and studied six populations of pig lung dendritic cells (DCs)/macrophages (Mθs) as follows: conventional DCs (cDC) 1 and cDC2, inflammatory monocyte-derived DCs (moDCs), monocyte-derived Mθs, and interstitial and alveolar Mθs. The three DC subsets present migratory and naive T-cell stimulation capacities. As observed in human and mice, porcine cDC1 and cDC2 were able to induce T-helper (Th)1 and Th2 responses, respectively. Interestingly, porcine moDCs increased in the lung upon influenza infection, as observed in the mouse model. Pig cDC2 shared some characteristics observed in human but not in mice, such as the expression of FCÉRIα and Langerin, and an intra-epithelial localization. This work, by unraveling the extended similarities of the porcine and human lung DC/Mθ networks, highlights the relevance of pig, both as an exploratory model of DC/Mθ functions and as a model for human inflammatory lung pathologies.
Asunto(s)
Células Dendríticas/inmunología , Gripe Humana/inmunología , Macrófagos Alveolares/inmunología , Macrófagos/inmunología , Infecciones por Orthomyxoviridae/inmunología , Orthomyxoviridae/inmunología , Sistema Respiratorio/inmunología , Animales , Antígenos CD/metabolismo , Células Cultivadas , Células Dendríticas/virología , Modelos Animales de Enfermedad , Humanos , Lectinas Tipo C/metabolismo , Activación de Linfocitos , Macrófagos/virología , Macrófagos Alveolares/virología , Lectinas de Unión a Manosa/metabolismo , Ratones , Receptores de IgE/metabolismo , Porcinos , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
OBJECTIVES: Atherosclerosis is an inflammatory disease of the arterial wall that leads to myocardial infarction and stroke. Regulatory T cells (Tregs) and IL-10 exert significant anti-atherogenic effects in experimental models of atherosclerosis by modulating vascular inflammation. We have previously shown that Mycobacterium bovis BCG killed by extended freeze-drying (EFD BCG) decreases lung and colon inflammation by recruiting IL-10-producing Tregs. Therefore, the aim of this study was to investigate the effect of EFD BCG on the development of atherosclerosis. DESIGN: We used two strains of atherosclerosis-prone mice: Ldlr(-/-) (four or six EFD BCG injections) and Apoe(-/-) (six injections). RESULTS: In both models, EFD BCG significantly reduced the size of atherosclerotic lesions, increased IL-10 production and reduced the serum levels of pro-inflammatory cytokines (IL-6, IL-13, KC and tumour necrosis factor-α). Shortly after treatment with EFD BCG, the number of plasmacytoid dendritic cells (pDCs) and Foxp3(+) Tregs in the draining lymph nodes increased. EFD BCG also led to accumulation of Tregs, but not of pDCs in the spleen, and reduced activity of NF-κB and increased activity of PPAR-γ in both the spleen and vascular tissue of treated mice. CONCLUSION: EFD BCG has atheroprotective effects through IL-10 production and Treg expansion. These findings support a novel approach to the prevention and treatment of atherosclerosis.
Asunto(s)
Aterosclerosis , Vacuna BCG/farmacología , Interleucina-10/metabolismo , Mycobacterium bovis/inmunología , Linfocitos T Reguladores , Animales , Aterosclerosis/inmunología , Aterosclerosis/prevención & control , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/metabolismo , Liofilización/métodos , Fenómenos del Sistema Inmunológico/efectos de los fármacos , Ratones , PPAR gamma/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
As an alternative strategy to classical inactivated viral vaccine against FMDV, naked DNA vaccine is attractive because of safety, flexibility and low cost. However DNA vaccination is usually poorly efficient in target species. Indeed we found that naked DNA plasmids encoding for P1-2A3C3D and GM-CSF proteins did not induce any detectable immunity against FMDV in sheep. Interestingly, we demonstrate herein that formulations of DNA on poly(D,L-lactide-co-glycolide) (PLG) or in lipofectin triggered divergent types of immune responses: PLG stimulated a T cell response and could elicit significant neutralising antibody titers, whereas lipofectin generated even higher antibody titers but no significant T cell response. The DNA/PLG regimen used in five sheep protected against clinical symptoms and viraemia and prevented the carrier state in four of them. Thus formulated DNA can be remarkably efficient against FMDV in a ruminant species that is usually refractory to DNA vaccination.
Asunto(s)
Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Enfermedades de las Ovejas/prevención & control , Vacunas Virales/inmunología , Animales , Formación de Anticuerpos/inmunología , Portador Sano , Ensayo de Inmunoadsorción Enzimática , Excipientes , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Hipersensibilidad Tardía/inmunología , Inmunidad Celular/inmunología , Interferón gamma/biosíntesis , Ácido Láctico , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Fosfatidiletanolaminas , Plásmidos/genética , Plásmidos/inmunología , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros , Ovinos , Linfocitos T/inmunología , Transfección , Vacunas de ADN/inmunología , Viremia/sangre , Viremia/inmunología , Viremia/virologíaRESUMEN
Two populations of B lymphocytes, B-1 (CD5+ and/or CD11b+) and B-2 (CD5- and CD11b-) cells have been described. In mice, which is the species of reference for B-1 and B-2 cell studies, these two subsets present different developmental schemes, phenotypes, antibody repertoires, localization and behaviours. Interestingly, in sheep, B cells rearrange their immunoglobulin (Ig) loci around the neonatal period, similarly to murine B-1 cells. However, the phenotype of the sheep B cells has not been characterized with regards to their developmental pathway. In this report, we show that two sheep B-cell subsets can be distinguished on the basis of CD11b expression. Relative to CD11b- B cells, the CD11b+ B cells frequently co-express CD5, CD11c, higher levels of surface IgM (sIgM), show larger cell size and higher cell-cycling activity, and thus present a B-1-like phenotype. However, unlike murine B-1 cells, sheep B-1 like cells mainly localize in blood, display a higher propensity to spontaneous apoptosis relative to B-2-like cells, and proliferate after sIgM stimulation. Our data show that despite neonatal immunoglobulin loci rearrangements, sheep B cells do not all express a B-1-like phenotype. However, B-1-and B-2-like cells co-exist and present phenotypic and behavioural specificities. Nevertheless, sheep B-1-and B-2-like cells differ from the murine B-1 and B-2 cells in their cell behaviour. These subsets can thus not be considered as true homologues among species.
Asunto(s)
Linfocitos B/inmunología , Antígenos CD5 , Activación de Linfocitos/fisiología , Antígeno de Macrófago-1 , Ovinos/inmunología , Animales , Apoptosis , Linfocitos B/citología , Ciclo Celular , Femenino , Reordenamiento Génico de Linfocito B , Inmunoglobulina M/inmunología , Inmunofenotipificación , Integrina alfaXbeta2 , Receptores de Complemento 3d , Receptores Inmunológicos/metabolismo , Especificidad de la EspecieRESUMEN
In this study, we show that bovine leukemia virus (BLV)-induced persistent lymphocytosis (PL) results from the in vivo expansion of the CD11b+ B-lymphocyte population. This subset shares phenotypic characteristics with murine and human B-1 cells. BLV interactions with the sheep B-1-like subset were explored. We found that B-1- and B-2-like cells are initially infected to similar extents. However, in long-term-infected sheep, the viral load is higher in B-1-like cells and only B-1- and not B-2-like cells show increased ex vivo survival compared to that in uninfected sheep. Ex vivo viral expression was found in both B-1- and B-2-like cells, indicating that both cell types support viral replication. Finally, cycloheximide and a protein kinase C inhibitor (H7) that blocks the ex vivo activation of viral expression did not affect the increased survival in B-1-like cells, suggesting that resistance to apoptosis is acquired in vivo. Collectively, these results indicate a peculiar susceptibility of sheep B-1-like cells to BLV transforming effects and further support the involvement of increased survival in BLV pathogenesis.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Leucosis Bovina Enzoótica/inmunología , Virus de la Leucemia Bovina/inmunología , Linfocitosis/inmunología , Antígeno de Macrófago-1/inmunología , Animales , Apoptosis , Bovinos , Supervivencia Celular , Leucosis Bovina Enzoótica/fisiopatología , Leucosis Bovina Enzoótica/virología , Linfocitosis/fisiopatología , Linfocitosis/virología , Ovinos , Factores de Tiempo , Carga ViralRESUMEN
Human bladder carcinomas often express high levels of the epidermal growth factor (EGF) receptor. In three human bladder carcinoma cell lines (OBR, T24, and 647V), we show that two EGF receptor ligands, namely EGF and transforming growth factor alpha, enhanced the apoptosis due to serum starvation on cells cultured as monolayers. Conversely, EGF and transforming growth factor alpha prevented apoptosis when the same serum-starved cells were cultured as three-dimensional spheroids. Both stimulation and inhibition of apoptosis by EGF were associated with p21 WAF1/CIP1 overexpression. In 647V spheroids, EGF protection against radiation-induced apoptosis was negated by genistein and tyrphostin AG1478, suggesting that blockade of the EGF signal transduction in patients with bladder cancer may improve the radiotherapy efficacy.
Asunto(s)
Apoptosis/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Proteínas de Neoplasias/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Factor de Crecimiento Transformador alfa/farmacología , Tirfostinos , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Apoptosis/efectos de la radiación , Medio de Cultivo Libre de Suero , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Genisteína , Humanos , Isoflavonas/farmacología , Nitrilos/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinazolinas/farmacología , Factores de Tiempo , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Experimental inoculation of sheep with bovine leukaemia virus (BLV), a retrovirus homologous to the human T-lymphotropic virus type 1 (HTLV-1), induces a chronic expansion of the B lymphocyte population (persistent lymphocytosis) and the development of a B cell leukaemia/lymphosarcoma syndrome. To gain insight into the mechanisms of BLV-induced lymphocytosis, we tested B cell survival capacity and cycling activity in peripheral blood mononuclear cells (PBMCs) from lymphocytotic, asymptomatic and control sheep. Interestingly, B cells from lymphocytotic sheep presented a lower level of spontaneous apoptosis (29%) in ex vivo cultures compared to that obtained with infected asymptomatic (42%) and control (57%/o) sheep PBMCs. Virus capsid (CA) synthesis was mainly found among surviving B cells and the percentage of CA-producing B cells correlated with the extent of B cell survival, indicating that BLV replication in B lymphocytes may promote protection from cell death. B cell survival was not linked with increases in expression of Bcl-2 mRNA or membrane leukosialin (CD43), although both are documented to be involved in some aspects of the B cell life-span. Finally, cell cycle analyses in freshly isolated PBMCs from lymphocytotic sheep revealed a slightly increased proportion of B cells in S phase compared to controls. Altogether, these data suggest that both BLV-induced B cell proliferation and extended survival are involved in the lymphocytotic stage encountered in BLV infection in sheep.
Asunto(s)
Apoptosis , Linfocitos B/inmunología , Leucosis Bovina Enzoótica/inmunología , Virus de la Leucemia Bovina , Linfocitosis/inmunología , Animales , Linfocitos B/citología , Linfocitos B/patología , Bovinos , Ciclo Celular , Supervivencia Celular , Células Cultivadas , Leucosis Bovina Enzoótica/fisiopatología , Citometría de Flujo , Virus Linfotrópico T Tipo 1 Humano , Humanos , Linfocitosis/virología , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , ARN Mensajero , Valores de Referencia , Ovinos , Transcripción GenéticaRESUMEN
Bovine immunodeficiency virus (BIV) is a lentivirus whose serologic prevalence is worldwide. Little is known about its impact on animal health status, pathogenesis and mode of transmission. Understanding BIV biology implies isolation of new viral strains and long-term studies on experimentally-infected cows and surrogate hosts such as rabbits.
Asunto(s)
Enfermedades de los Bovinos , Síndromes de Inmunodeficiencia/veterinaria , Infecciones por Lentivirus/veterinaria , Lentivirus/clasificación , Animales , Bovinos , Femenino , Variación Genética , Genoma Viral , Síndromes de Inmunodeficiencia/epidemiología , Lentivirus/genética , Lentivirus/aislamiento & purificación , Infecciones por Lentivirus/epidemiología , Infecciones por Lentivirus/transmisión , Prevalencia , Conejos , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Direct inoculation of a cloned bovine leukemia virus (BLV) provirus into sheep has allowed study of the viral infectivity of genetic mutants in vivo. Three BLV variants cloned from BLV-induced tumors and 12 in vitro-modified proviruses were isolated and analyzed for viral expression in cell culture. The proviruses were then inoculated into sheep in order to assess viral infectivity in vivo. Of three variants cloned from BLV-induced tumors (344, 395, and 1345), one (344) was found infectious in vivo. This particular provirus was used to engineer 12 BLV mutants. A hybrid between the 5' region of the complete but noninfectious provirus 395 and the 3' end of mutant 344 was infectious in vivo, suggesting that the tax/rex sequences were altered in virus 395. As expected, several regions of the BLV genome appeared to be essential for viral infection: the protease, pol, and env genes. Even discrete modifications in the fusion peptide located at the NH2 end of the transmembrane gp30 glycoprotein destroyed the infectious potential. In contrast, mutations and deletions in the X3 region present between the env gene and the 3' tax/rex region did not interfere with viral infection in vivo. This region of unknown function could thus be used to introduce foreign sequences. A BLV recombinant carrying a ribozyme directed against the tax/rex sequences was still infectious in vivo. Cotransfection of two noninfectious mutants carrying deletions led to infection in two of four independent injections, the infectious virus being then a recombinant between the two deletants. The experimental approach described here should help to gain insight into essential mechanisms such as in vivo viral replication, cooperation between deletants for viral infectivity, and viral superinfections. The gene products in the X3 and X4 region which are dispensable for in vivo infection could be involved in leukemogenesis, and thus proviruses deleted in these sequences could constitute the basis for a live attenuated vaccine.
Asunto(s)
Virus de la Leucemia Bovina/crecimiento & desarrollo , Ovinos/microbiología , Replicación Viral , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , Análisis Mutacional de ADN , Virus de la Leucemia Bovina/genética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Proteínas de los Retroviridae/genética , Proteínas de los Retroviridae/metabolismo , TransfecciónRESUMEN
Assessment of the function of putative dominantly-acting oncogenes or recessive tumor-suppressor genes in human tumor development and progression must ultimately involve xenografting experiments using immune deficient animals such as nude mice. Most human tumor xenograft experiments have employed conventional subcutaneous injection procedures. However, despite the simplicity of this procedure, it poses some serious potential drawbacks as most types of human tumor will not readily grow or metastasize from a subcutaneous ('ectopic') site of injection. In contrast, 'orthotopic' injection procedures will often enhance the tumorigenic and/or metastatic ability of tumor cell populations. An example of this is summarized in the context of human malignant melanoma where the effects of subcutaneous versus subdermal injection are compared. Despite the seeming subtle and minor change in injection site, superior growth of human melanomas can be obtained by the latter, orthotopic-like, route of injection. It therefore follows that induction of tumorigenic or metastatic properties in a given human cell population by gene transfection may not be detected if the transfected cells are assayed in vivo only by subcutaneous injection procedures. An example of this is provided by experiments involving transfection of normal or mutated ras genes into a low-grade, well-differentiated human bladder carcinoma cell line, called RT-4. Thus overexpression of normal or mutated (valine 12) c-H-ras resulted in acquisition of a clinical-like invasive phenotype. However, this was clearly seen only if the cells were injected into the bladders (i.e. 'intravesically') of nude mice. In contrast, conventional subcutaneous injection of the high ras expressing transfected RT-4 cell lines did not reveal acquisition of invasive properties: all cell lines grew locally as well-encapsulated tumor masses. It is argued that similar orthotopic injection procedures should be employed when assessing the suppressive effects of various wild-type tumor-suppressor genes on human tumor growth in vivo. Utilization of subcutaneous injection procedures may grossly exaggerate the growth suppressive effects of such genes. This could explain the paradox of why, on the one hand, alterations involving many different genes (including different suppressor genes) appear to be involved in human carcinoma tumorigenesis while on the other hand, complete suppression of tumorigenicity can be caused by transfer of a single wild-type suppressor gene. Such complete suppressions might be observed only after ectopic (usually subcutaneous) injection procedures.
Asunto(s)
Genes Supresores de Tumor , Neoplasias/patología , Oncogenes , Transfección , Animales , Genes Recesivos , Ratones , Ratones Desnudos , Invasividad Neoplásica , Trasplante de Neoplasias/métodos , Neoplasias/genética , Trasplante Heterólogo , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Recent studies have shown that orthotopic (transurethral) transplantation of human bladder cancer cell lines into nude mice permits tumor growth that more accurately reflects their clinical malignant status in the original host. We have previously demonstrated that transfection and overexpression of normal or mutated c-Ha-ras genes into a noninvasive human papillary transitional cell carcinoma cell line confer upon these cells an invasive phenotype in vivo with behavior remarkably similar to the clinical behavior of high grade bladder carcinomas. Since elevated expression levels of the epidermal growth factor receptor (EGF-R), in addition to that of c-Ha-ras, have been correlated with transitional cell carcinoma progression, we sought to determine whether up-regulation of the EGF-R had occurred in the invasive high ras expressors and if so, what functional significance this might have. Our results show that invasive cell lines which overexpress the c-Ha-ras gene also have increased epidermal EGF-R expression. This was found to occur at both the protein and mRNA levels, and analysis of the EGF-R promoter/enhancer sequences has revealed a putative AP-1 site which may possibly enhancer sequences has revealed a putative AP-1 site which may possibly serve as a ras response element. In addition, we found that the cells overexpressing the EGF-R had acquired a positive sensitivity to the stimulatory mitogenic effects of EGF. Hence, the results obtained suggest a role for either a normal or a mutated overexpressing Ha-ras in up-regulating the surface EGF-R, possibly through an AP-1 site during human bladder carcinoma progression; they also highlight the potential that EGF may have in cooperating with this EGF-R up-regulation to help mediate enhanced tumor growth.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Genes ras , Northern Blotting , Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/patología , División Celular/efectos de los fármacos , Línea Celular , Receptores ErbB/genética , Humanos , Invasividad Neoplásica , Fenotipo , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Regulación hacia Arriba , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
It is known from a variety of experimental systems that the ability of tumor cells to grow locally and metastasize can be affected by the presence of adjacent normal tissues and cells, particularly mesenchymally derived stromal cells such as fibroblasts. However, the comparative influence of such normal cell-tumor cell interactions on tumor behavior has not been thoroughly investigated from the perspective of different stages of tumor progression. To address this question we assessed the influence of normal dermal fibroblasts on the growth of human melanoma cells obtained from different stages of tumor progression. We found that the in vitro growth of most (4 out of 5) melanoma cell lines derived from early-stage radial growth phase or vertical growth phase metastatically incompetent primary lesions is repressed by coculture with normal dermal fibroblasts, suggesting that negative homeostatic growth controls are still operative on melanoma cells from early stages of disease. On the other hand, 9 out of 11 melanoma cell lines derived from advanced metastatically competent vertical growth phase primary lesions, or from distant metastases, were found to be consistently stimulated to grow in the presence of dermal fibroblasts. Evidence was obtained to show that this discriminatory fibroblastic influence is mediated by soluble inhibitory and stimulatory growth factor(s). Taken together, these results indicate that fibroblast-derived signals can have antithetical growth effects on metastatic versus metastatically incompetent tumor subpopulations. This resultant conversion in responsiveness to host tissue environmental factors may confer upon small numbers of metastatically competent cells a growth advantage, allowing them to escape local growth constraints both in the primary tumor site and at distant ectopic tissue sites.
Asunto(s)
Melanoma/patología , Tejido Adiposo/citología , Tejido Adiposo/fisiología , Tejido Adiposo/efectos de la radiación , Animales , Bovinos , Comunicación Celular , División Celular , Células Cultivadas , Replicación del ADN , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Recién Nacido , Masculino , Piel/citología , Fenómenos Fisiológicos de la PielRESUMEN
During the natural history of a tumor, cancer cells become more and more aggressive and their increasing malignancy leads usually to the patient's death. The expression of malignant properties by tumor cells is manifested by the occurrence of metastases and is the result of an overexpression of molecules that are normally or barely non expressed by the normal cell progenitors. These molecules can be involved in cell attachment (receptor to the extracellular matrix), in proteolysis (collagenases), in angiogenesis (b FGF), in adhesion to endothelial cells, in resistance to the immune system. The genetic instability of tumor cells favors the amplification, mutation and gene translocation events, resulting in the activation of some genes or/and oncogenes which might direct the expression of the malignant properties. Finally, metastatic cells have been shown to have a growth advantage over non metastatic cells, so that metastatic cell population becomes ultimately numerously dominant in the primary tumor. The current knowledge about the malignant cell properties allow us to begin to understand how a cancer cell becomes metastatic and how the metastatic dissemination is usually an ineluctable process.
Asunto(s)
Metástasis de la Neoplasia/patología , Animales , Ratones , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/inmunología , Metástasis de la Neoplasia/fisiopatología , Neovascularización Patológica , FenotipoRESUMEN
We have previously shown, using tumor cell populations genetically tagged by random integrations of plasmid DNA, that metastatically-competent clonal cell variants have a strong growth advantage within primary tumors over their non-metastatic counterparts. As a result, primary tumors can become overgrown by the progeny of such cells, a process referred to as "clonal dominance" of primary tumors by metastatically-competent cells. Because of the well-known "metastatic inefficiency" of the multi-step cascade process of spread and growth, clonal dominance within primary tumors may be necessary for distant metastatic spread or increase the probability of its occurrence. To examine this hypothesis mice were inoculated s.c. with mixture of non-metastatic and genetically tagged, metastatically-competent mouse mammary carcinoma cells in defined ratios, but always containing an excess of the unmarked non-metastatic population. Progressive overgrowth of the metastatic subpopulation was monitored as a function of time by Southern analysis of DNA obtained from mixed primary tumors. This allowed us to evaluate the effects that surgical removal of the primary tumor had before, during and after effective clonal dominance, and what influence this had on the subsequent formation of distant metastases. Surgical removal of primary tumors before metastatic clonal dominance resulted in a low (0.25%) frequency of lung metastases, whereas removal just 1 or 2 weeks later during or after clonal dominance was achieved resulted in a high (75-100%) frequency of such metastases. Our results support the hypothesis that dominance of primary tumors by metastatically competent cells may be necessary for distant metastatic spread, and also suggest that clonal interactions play a significant role in modulating the metastatic ability of tumor cells in vivo.
Asunto(s)
Adenocarcinoma/patología , Neoplasias Mamarias Experimentales/patología , Metástasis de la Neoplasia , Animales , Southern Blotting , ADN/análisis , Femenino , Marcadores Genéticos , Técnicas In Vitro , Ratones , Ratones Endogámicos CBARESUMEN
Recent studies have shown that orthotopic (transurethral) transplantation of human bladder cancer cell lines into nude mice permits tumor growth that accurately reflects their clinical malignant status in the original host. Thus, such a system allows a unique opportunity to analyze the genetic events involved in the conversion of low-grade bladder cancer, the vast majority of which are curable, to the high-grade life-threatening form of the disease. Since 5-10% of transitional cell carcinomas (TCCs) have been shown to contain a mutated HRAS gene, and protein expression levels of all forms of HRAS have been correlated with TCC progression, we chose to study the contribution of the HRAS oncogene in bladder tumor progression. We evaluated the effects of transfection of normal or mutated HRAS genes into a human TCC, called RT-4, that behaves as a superficial noninvasive papillary tumor after transurethral orthotopic inoculation into athymic nude mice. We found that overexpression of either transfected normal or mutated HRAS genes converted RT-4 cells to express an invasive phenotype remarkably similar in nature to the clinical behavior of high-grade bladder carcinomas. These results suggest a role for overexpressed normal or mutated RAS genes in human bladder carcinoma progression, and highlight the importance of using orthotopic inoculation systems for evaluation of the contribution of oncogenes to malignant tumor progression.
Asunto(s)
Carcinoma de Células Transicionales/genética , Transformación Celular Neoplásica/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias de la Vejiga Urinaria/genética , Animales , Northern Blotting , Carcinoma de Células Transicionales/patología , Expresión Génica , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , ARN Mensajero/genética , Transfección , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Cell surface carbohydrate structures acting as ligands for tissue specific mammalian lectins have been implicated in cell-cell interactions during embryogenesis, lymphocyte homing, and tumor cell metastasis. In this report, we provide evidence that beta 1-4 linked galactose (Gal) residues in N-linked oligosaccharides on the surface of blood born tumor cells serve as a ligand for binding to microvascular endothelial cells. D36W25, a class 1 glycosylation mutant of the MDAY-D2 lymphoreticular tumor cell line, lacks sialic acid and Gal in cellular glycans due to a defect in the Golgi UDP-Gal transporter. Using UDP-Gal and bovine galactosyltransferase in vitro, beta 1-4 Gal was restored to the surface of the cells and 70% of the galactosylated glycans persisted for 8 h in vitro at 37 degrees C. Compared to mock-treated D36W25 cells, galactosylated D36W25 cells showed an 80% increase in binding to microvascular endothelial cell monolayers in vitro. The enhanced binding of galactosylated D36W25 cells to endothelial cell was inhibited by the addition of lactosamine-conjugated albumin to the assay. Consistent with these observations, swainsonine and castinospermine, two inhibitors of N-linked processing that result in loss of lactosamine antennae inhibited the binding of wild-type MDAY-D2 cells to endothelial cells in vitro. Injection of radiolabeled tumor cells into the circulation of syngeneic mice, showed that galactosylation of D36W25 cells resulted in 2-3 more tumor cells retained in the lungs and livers. In addition, galactosylation of D36W25 cells increased by 30-fold the number of visible liver metastases on inspection 4 wk after tumor cell injection. These results suggest that beta 1-4Gal-binding lectins on microvascular endothelial cells can contribute to retention and secondary tumor formation of blood born tumor cells. With the increasing availability of purified glycosyltransferases, reconstruction of a variety of carbohydrate sequences on the surface of class 1 mutants provides a controlled means of studying carbohydrate-lectin interactions on viable cells.
Asunto(s)
Endotelio Vascular/fisiología , Galactosa , Indolizinas , Lectinas , Oligosacáridos , Células Tumorales Cultivadas/fisiología , Alcaloides/farmacología , Animales , Bovinos , Adhesión Celular , Línea Celular , Membrana Celular/fisiología , Endotelio Vascular/citología , Citometría de Flujo , Glicósido Hidrolasas/antagonistas & inhibidores , Cinética , Linfoma no Hodgkin , Ratones , Ratones Endogámicos DBA , Mutación , Swainsonina , Células Tumorales Cultivadas/citologíaAsunto(s)
Neoplasias/genética , Animales , Evolución Biológica , Southern Blotting , Células Clonales/patología , Elementos Transponibles de ADN , Marcadores Genéticos , Vectores Genéticos , Humanos , Metástasis de la Neoplasia , Neoplasias/etiología , Neoplasias/patología , Plásmidos , Provirus/genética , Retroviridae/genéticaRESUMEN
Transplantation of human tumors into the organ or tissue of their origin (orthotopic transplantation) in nude mice can result in significant enhancement of tumor growth and metastases, compared with sc (ectopic) transplantation. Because melanocytes are normally found in the epidermal-dermal junction, intradermal inoculation of melanoma cells might be expected to improve their potential for malignant growth as xenografts. The purpose of our study was to examine this possibility. We found that because mouse epidermis and dermis are so thin, it was not possible to inject a bolus of tumor cells intradermally; instead the cells were actually deposited in the most superficial layer of the subcutis (i.e., subdermally). We evaluated the behavior of cells from a human melanoma cell line after sc or subdermal inoculation into National Institutes of Health Swiss athymic nude mice. The cells used were from (1) the predominantly amelanotic human malignant melanoma cell line MeWo, originally established from a melanotic lymph node metastasis, and (2) two MeWo variants resistant to wheat germ agglutinin (WGAr), which were selected for altered malignant capacities. Whereas 5 X 10(5) MeWo cells were required to achieve 100% tumor take with sc injection, only 2 x 10(4) cells were required with subdermal inoculation. Subdermal injection of the MeWo cells resulted in the development of highly melanotic and nonencapsulated primary tumors, which grew quickly into the dermis and epidermis and metastasized at high frequency to draining lymph nodes. In contrast, the tumors that developed after sc injection were found in the deepest layer of the subcutis and were predominantly amelanotic and encapsulated; they rarely metastasized to lymph nodes.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Melanoma/patología , Neoplasias Cutáneas/patología , Animales , Femenino , Humanos , Inyecciones Intradérmicas , Inyecciones Subcutáneas , Melaninas/biosíntesis , Melanoma/metabolismo , Melanoma/secundario , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Tumorales Cultivadas , Aglutininas del Germen de TrigoRESUMEN
Bovine leukemia virus-infected sheep were demonstrated to possess a plasma factor that specifically suppresses in vitro virus expression in lymphocyte cultures. This blocking activity is observed even after phytohemagglutinin (PHA) stimulation and is independent of cellular proliferation. Such a factor may play a critical role in the regulation of BLV expression.
Asunto(s)
Virus de la Leucemia Bovina/crecimiento & desarrollo , Leucemia Experimental/microbiología , Retroviridae/crecimiento & desarrollo , Animales , Antígenos Virales/análisis , Linfocitos B/microbiología , Regulación de la Expresión Génica , Virus de la Leucemia Bovina/genética , Leucemia Experimental/sangre , Activación de Linfocitos , Ovinos , Factores de Tiempo , Interferencia Viral , Replicación ViralRESUMEN
Bovine leukemia virus (BLV) replication in B lymphocytes from experimentally infected sheep depends on three conditions. First, viral production is detected only when the infected animals exhibit blood lymphocytosis. Second, it requires in vitro cultivation but is never observed in vivo. Third, enhancement of virus expression after phytohemaglutinin (PHA) or concavalin A stimulation is observed in lymphocyte cultures for all infected animals, whereas specifically B cell mitogens are inefficient. The PHA effect is linked to the presence of T lymphocytes. In addition, the conditioned medium prepared from PHA-stimulated normal lymphocytes greatly enhances BLV production. Altogether, these results establish that T lymphocytes, through a lymphokine production, are important and may be essential for BLV growth in B lymphocytes.