RESUMEN
Vaccination against human cytomegalovirus (CMV) infection remains high priority. A recombinant form of a protein essential for CMV entry, glycoprotein B (gB), demonstrated partial protection in a clinical trial (NCT00299260) when delivered with the MF59 adjuvant. Although the antibody titre against gB correlated with protection poor neutralising responses against the 5 known antigenic domains (AD) of gB were evident. Here, we show that vaccination of CMV seronegative patients induces an antibody response against a region of gB we term AD-6. Responses to the polypeptide AD-6 are detected in >70% of vaccine recipients yet in <5% of naturally infected people. An AD-6 antibody binds to gB and to infected cells but not the virion directly. Consistent with this, the AD-6 antibody is non-neutralising but, instead, prevents cell-cell spread of CMV in vitro. The discovery of AD-6 responses has the potential to explain part of the protection mediated by gB vaccines against CMV following transplantation.
Asunto(s)
Infecciones por Citomegalovirus , Vacunas contra Citomegalovirus , Humanos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Citomegalovirus , Infecciones por Citomegalovirus/prevención & control , Vacunas contra Citomegalovirus/inmunología , Proteínas del Envoltorio ViralRESUMEN
Tunable electromagnets and corresponding devices, such as magnetic lenses or stigmators, are the backbone of high-energy charged particle optical instruments, such as electron microscopes, because they provide higher optical power, stability, and lower aberrations compared to their electric counterparts. However, electromagnets are typically macroscopic (super-)conducting coils, which cannot generate swiftly changing magnetic fields, require active cooling, and are structurally bulky, making them unsuitable for fast beam manipulation, multibeam instruments, and miniaturized applications. Here, we present an on-chip microsized magnetic charged particle optics realized via a self-assembling micro-origami process. These micro-electromagnets can generate alternating magnetic fields of about ±100 mT up to a hundred MHz, supplying sufficiently large optical power for a large number of charged particle optics applications. That particular includes fast spatiotemporal electron beam modulation such as electron beam deflection, focusing, and wave front shaping as required for stroboscopic imaging.
RESUMEN
Barrett's esophagus (BE) is a premalignant condition associated with the development of esophageal adenocarcinoma (EAC). Despite the low risk of progression to EAC, evidence highlights the notably poor survival rates of this malignancy. The mainstay form of diagnosis of BE is endoscopy and biopsy sampling. However, research emphasizes limitations with regards to the histological detection of BE and associated dysplasia. The aim of this study is to evaluate the clinical significance of CEACAM6 as a potential biomarker for the diagnosis of BE and beyond. Retrospective tissue samples were obtained from columnar lined esophagus without goblet cells (n = 27), BE (n = 18), BE associated dysplasia (n = 16), and EAC (n = 24). Standardized immunohistochemistry for CEACAM6 was performed followed by quantitative staining analysis. Statistical analysis across the BE spectrum for CEACAM6 was undertaken and a P value <0.05 was considered significant. CEACAM6 expression increased from columnar lined epithelium (CLE) to BE with a subsequent decrease to dysplasia and adenocarcinoma. The expression of CEACAM6 was significant from CLE to BE at p 0.001, CLE to dysplasia at p 0.001, BE to dysplasia at p 0.006, CLE to adenocarcinoma at p 0.001 and BE to adenocarcinoma at p 0.001. There was no significant difference in expression between dysplasia and adenocarcinoma (P = 0.15). Our findings highlight the increasing expression of CEACAM6 from CLE to BE with a subsequent decrease to dysplasia and adenocarcinoma. In view of this, we advocate the utilization of this marker for the enhanced diagnosis of BE and for the distinction of BE and dysplasia.
Asunto(s)
Adenocarcinoma/metabolismo , Antígenos CD/metabolismo , Esófago de Barrett/diagnóstico , Esófago de Barrett/metabolismo , Moléculas de Adhesión Celular/metabolismo , Neoplasias Esofágicas/metabolismo , Anciano , Esófago de Barrett/patología , Biomarcadores/metabolismo , Biopsia , Esófago/metabolismo , Esófago/patología , Femenino , Proteínas Ligadas a GPI/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Curva ROC , Estudios RetrospectivosRESUMEN
Off-axis electron holography is a well-established transmission electron microscopy technique, typically employed to investigate electric and magnetic fields in and around nanoscale materials, which modify the phase of the reconstructed electron wave function. Here, we elaborate on a detailed analysis of the two characteristic intensity terms that are completing the electron hologram, the conventional image intensity and the interference fringe intensity. We show how both are related to elastic and inelastic scattering absorption at the sample and how they may be separated to analyze the chemical composition of the sample. Since scattering absorption is aperture dependent, a quantitative determination of the corresponding attenuation coefficients (reciprocal mean free path lengths) requires the use of holographic image modi with well-defined objective aperture stops in the back-focal plane of the objective lens. The proposed method extends quantitative electron holography to a correlated three-in-one characterization of electric and magnetic fields, Z-contrast and dielectric losses in materials.
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Ras-driven tumorigenesis is assumed to depend on Raf for ERK activation and proliferation; yet, an in vivo requirement for Raf as MEK/ERK activator in this setting has not been demonstrated to date. Here, we show that epidermis-restricted B-Raf ablation restrains the onset and stops the progression of established Ras-driven tumors by limiting MEK/ERK activation and proliferation. Concomitant elimination of B-Raf and Raf-1 enforces the abrupt regression of established tumors owing to the decrease in ERK activation and proliferation caused by B-Raf ablation combined with the ERK-independent increase in Rho-dependent kinase (Rok) signaling and differentiation triggered by Raf-1 inactivation. Thus, B-Raf and Raf-1 have non-redundant functions in Ras-driven tumorigenesis. Of note, Raf kinase inhibitors achieve impressive results in melanomas harboring oncogenic BRAF, but are ineffective against Ras-driven tumors; moreover, therapy-related skin tumors driven by a paradox ERK activation as well as primary and acquired resistance have been reported. Our results suggest that therapies targeting both Raf kinase-dependent and -independent pathways may be effective against a broader range of malignancies and reduce the risks of adverse effects and/or resistance.
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Transformación Celular Neoplásica/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Neoplasias Cutáneas/enzimología , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-raf/genética , Transducción de Señal , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patologíaRESUMEN
The epidermis is the outermost layer of the body and protects it from environmental insults. This crucial function is sustained by a continuous process of self-renewal involving the carefully balanced proliferation and differentiation of progenitor cells constantly replacing the mature cells at the surface of the epidermis. Genetic changes in the signalling pathways controlling keratinocyte proliferation and differentiation disrupt this balance and lead to pathological changes including carcinogenesis. This review discusses the role of Ras, an oncogene critically involved in the development of skin neoplasia, and its downstream effector Raf in epidermal homeostasis and tumourigenesis. In particular, we will focus on the recently established role of Raf-1 as the decisive element that, by restraining keratinocyte differentiation, allows the development and maintenance of Ras-driven tumours.
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Transformación Celular Neoplásica , Epidermis/crecimiento & desarrollo , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Quinasas raf/fisiología , Animales , HumanosRESUMEN
Multi-colour flow cytometry is the only technological platform that can analyse the highly complex cellular composition of the immune system in parallel and at a single cell resolution. Analysis of the T cell compartment, in particular, requires the simultaneous measurement of multiple markers in order to account for lineage, phenotype and function. Flow cytometry also enables the analysis of intracellular signalling events. By combining the expression of surface markers, intracellular cytokines, phosphorylated versus unphosphorylated kinases, cell proliferation and DNA profile, mechanistic and kinetic information of subset-specific signalling may be obtained: this has not previously been achieved. Here we present a protocol which permits all of these aspects to be explored simultaneously. By comparing basic procedures previously described we were able to optimise different variables, including the choice of antibody/fluorochrome pairs, permeabilisation, fixation and labelling time, to obtain the best DNA staining of different cell types. We applied this method to study subset-specific signalling related to cytokine production and DNA synthesis in T cells responding to specific antigens.
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Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Citocinas/metabolismo , Citometría de Flujo , Inmunofenotipificación/métodos , Activación de Linfocitos , Fosfoproteínas/metabolismo , Subgrupos de Linfocitos T/metabolismo , Biomarcadores/metabolismo , Brefeldino A/farmacología , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Replicación del ADN , Enterotoxinas/farmacología , Femenino , Humanos , Cinética , Activación de Linfocitos/efectos de los fármacos , Masculino , Transducción de Señal , Coloración y Etiquetado , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunologíaRESUMEN
The myelin and lymphocyte protein (MAL) is a raft-associated membrane protein predominantly expressed by oligodendrocytes and Schwann cells. Here we show that MAL regulates myelination in the peripheral nervous system. In mice overexpressing MAL, myelination was retarded and fibers were hypomyelinated, whereas myelination in MAL knockout mice was accelerated. This was not due to impaired Schwann cell proliferation, differentiation or axonal sorting. We found that the expression level of p75 neurotrophin receptor mRNA and protein was strongly reduced in developing sciatic nerves in MAL-overexpressing mice. This reduction is well correlated with the observed alterations in myelination initiation, speed of myelination and alterations in Remak bundle development. Our results suggest a functional role for MAL in peripheral myelination by influencing the expression of membrane components that mediate axon-glia interaction during ensheathment and myelin wrapping.
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Proteínas de Transporte de Membrana/metabolismo , Proteínas de la Mielina/metabolismo , Vaina de Mielina/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Nervios Periféricos/metabolismo , Proteolípidos/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Animales , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Microdominios de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Transgénicos , Proteínas de la Mielina/genética , Vaina de Mielina/ultraestructura , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito , Fibras Nerviosas Mielínicas/ultraestructura , Nervios Periféricos/crecimiento & desarrollo , Nervios Periféricos/ultraestructura , Proteolípidos/genética , ARN Mensajero/metabolismo , Receptor de Factor de Crecimiento Nervioso/genética , Células de Schwann/metabolismoAsunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Endorribonucleasas/farmacología , Activación de Linfocitos/efectos de los fármacos , Antineoplásicos/inmunología , Antineoplásicos/farmacología , Biomarcadores/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Células Cultivadas , Citocinas/inmunología , Evaluación Preclínica de Medicamentos , Endorribonucleasas/inmunología , Humanos , Activación de Linfocitos/inmunología , Ribonucleasas/inmunología , Ribonucleasas/farmacologíaRESUMEN
Fibroblast growth factor (FGF) signaling can bypass the requirement for estrogen receptor (ER) activation in the growth of ER-positive (ER+) breast cancer cells. Fibroblast growth factor-1 stimulation leads to phosphorylation of the adaptor protein Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT-1) on C-terminal tyrosine residues, whereas it is constitutively bound through its N-terminal phosphotyrosine-binding domain (PTB) to FGF receptors (FGFRs). By expressing the PTB domain of SNT-1 (SNT-1 PTB) in an inducible manner in an ER+ breast carcinoma line, ML20, we asked whether we could uncouple FGFR activation from its downstream signaling components and abrogate FGF-1-induced antiestrogen-resistant growth. Induction of SNT-1 PTB resulted in a significant decrease of FGF-1-dependent tyrosine phosphorylation of endogenous SNT-1, strong inhibition of complex formation between SNT-1, Gab-1 and Sos-1, and reduced activation of Ras, mitogen-activated protein kinase (MAP kinase), and Akt. SNT-1 PTB also inhibited the phosphorylation of p70S6K on Thr421/Ser424 and Ser411, which may result from the abrogation of MAP kinase activity. Moreover, we also observed a decreased phosphorylation of the MAP kinase-independent site Thr389. This may reflect both inhibition of PI-3 kinase pathways and mammalian target of rapamycin (mTOR)-dependent signaling, as the phosphorylation of Thr389 site was sensitive to treatment with the PI3-K and mTOR inhibitors, LY294002 and rapamycin, respectively. Collectively these results suggest that SNT-1 plays a pivotal role in FGF-dependent activation of the Ras-MAP kinase, PI-3 kinase, and mTOR pathways in these cells. Fibroblast growth factor-1 dependent colony formation of ML20 cells in media containing the pure antiestrogen ICI 182,780 was also markedly inhibited upon induction of SNT-1 PTB, suggesting that blockade of FGFR-SNT-1 interactions might abrogate FGF-mediated antiestrogen resistance in breast cancers.
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Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Neoplasias de la Mama/enzimología , Carcinoma/enzimología , Moduladores de los Receptores de Estrógeno/farmacología , Factor 1 de Crecimiento de Fibroblastos/fisiología , Proteínas de la Membrana/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfotirosina/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Carcinoma/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Doxiciclina/farmacología , Resistencia a Antineoplásicos/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de la Membrana/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Transducción de Señal/genéticaRESUMEN
"Individualized therapy strategies" involve strategies that allow treatment to be guided by patient-specific conditions. For this, robust biomarkers are needed. Examples of biomarker-guided therapies already in use are the treatment of insulin-dependent diabetes (biomarker: blood glucose level) or the treatment of hypertension (biomarker: blood pressure). By contrast, most immunomodulatory therapies are given according to the patient's body weight or the patient's drug blood level rather than according to biomarkers indicating the patient's state of the immune system. Herein we report on new biomarkerguided studies in the immunosuppressive treatment of transplant patients and patients with autoimmune disease and we discuss its benefits and pitfalls.
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Enfermedades Autoinmunes/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Trasplante de Órganos , Animales , Infecciones por Citomegalovirus/tratamiento farmacológico , HumanosRESUMEN
BACKGROUND: Microbiological infections are considered to be of pathophysiological importance in atopic dermatitis (AD). As yet, no information is available regarding cytomegalovirus (CMV) infection in this disease. This, however, is of interest because of the high prevalence of latent infections in the general population, the frequent reactivation in inflammatory diseases, and the immunomodulating capacity of CMV. OBJECTIVES: To investigate the prevalence of latent CMV infection, the frequency of active CMV infection, and the immune response to CMV in patients with moderate to severe AD. Methods To detect active infection we analysed CMV antigen expression by peripheral blood mononuclear cells (PBMC) from 27 patients with moderate to severe AD in comparison with 53 healthy volunteers. We used three monoclonal antibodies recognizing different CMV-encoded antigens and immunocytological staining (alkaline phosphatase-antialkaline phosphatase technique). RESULTS: Patients with AD had a higher mean frequency of CMV-positive PBMC: 2.25 per 10 000 vs. 0.74 per 10 000 in controls (P = 0.001) as well as a higher incidence of CMV antigenaemia: 29.6% vs. 7.5% (P < 0.01). Seropositivity for anti-CMV IgG antibodies indicated subclinical activation of latent infection. Remarkably, a clearance of CMV antigenaemia was observed during anti-eczematous treatment. Significantly higher plasma levels of tumour necrosis factor-alpha, which is involved in CMV reactivation, and interleukin-12, which is crucial for an antiviral cellular immune response, were observed in AD patients in comparison with healthy volunteers. Furthermore, a significantly enhanced frequency of circulating activated HLA-DR+ T cells especially in CMV-seropositive AD patients (19.3% vs. 13.5% in seronegative AD patients vs. 10.2% in controls) suggested that the active CMV infection triggers a cellular immune response. This was also supported by a high frequency of CMV-specific interferon-gamma-producing T cells in CMV-seropositive patients with AD. CONCLUSIONS: Our data suggest that active, subclinical CMV infection is more frequent in patients with moderate to severe AD and may have immunopathophysiological relevance.
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Antígenos Virales/análisis , Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , Dermatitis Atópica/virología , Latencia del Virus , Adulto , Estudios de Casos y Controles , Infecciones por Citomegalovirus/inmunología , Dermatitis Atópica/inmunología , Femenino , Antígenos HLA-DR/análisis , Humanos , Interferón gamma/biosíntesis , Interleucina-12/sangre , Activación de Linfocitos , Recuento de Linfocitos , Masculino , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Flow-cytometry can be used in different ways in order to analyze or enumerate antigen specific T-cells. The three basic principles are direct staining of the T-cell receptor using so called tetramer reagents, staining intracellular cytokines following antigen-specific ex vivo T-cell activation or staining with dyes that are incorporated (increase in staining) or distributed between daughter cells (decrease in staining) upon proliferation in response to a specific antigen challenge. Each system has its advantages and disadvantages. Here we demonstrate that tetramer staining, cytokine flow cytometry and staining with CFDA-SE can be combined permitting the analysis of proliferation and cytokine production with a subset of T-cells specific for a single peptide antigen.
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Citocinas/biosíntesis , Citometría de Flujo/métodos , Linfocitos T/citología , Antígenos/biosíntesis , Antígenos/química , División Celular , Citocinas/metabolismo , Humanos , Interferón gamma/biosíntesis , Activación de Linfocitos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The effect of xymedone, a non-glucoside analog of pyridine nucleosides, on the apoptosis of human CD4+ T cells of the Jurkat line was studied by laser flow cytometry method. Xymedone decreased the membrane expression of phosphatidylserine and suppressed the increase in permeability of the cytoplasmic membrane, thus inhibiting the onset of a degradation stage of the apoptotic cascade. Possible mechanisms of the antiapoptogenic effect of xymedone within the framework of a (cytochrome C/caspase 3)-dependent signal pathway of the apoptosis are discussed.
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Apoptosis/efectos de los fármacos , Sustancias de Crecimiento/deficiencia , Fosfatidilserinas/metabolismo , Pirimidinas/farmacología , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Regulación hacia Abajo , Citometría de Flujo , Humanos , Células Jurkat , Fosfatidilserinas/genéticaRESUMEN
Recently we reported about a possible involvement of extrarenal systemic cytomegalovirus (CMV) infection in graft deteriorating immune processes. We now examined whether Epstein-Barr virus (EBV) may also be associated with late renal graft injury. We analyzed the expression of early antigen-, viral capsid antigen-, and a latency-associated EBV-RNA-transcript, which is not translated into protein in peripheral blood mononuclear cells of kidney transplant patients with histologically proven late acute rejection and no signs of CMV or any other infection (A), patients with stable graft function (B), and healthy probands (C). A total of 40% in group A vs. 5 and 0% in groups B and C, respectively, expressed early antigen-mRNA (P<0.05) suggesting an activation of lytic EBV infection. Response to steroid bolus therapy in group A was comparably poor with that observed in CMV-related graft injury. Our data suggest that extrarenal lytic EBV infection may also be involved in the pathogenesis of late graft injury. A controlled ganciclovir trial may prove the significance of our observation.
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Infecciones por Virus de Epstein-Barr/complicaciones , Rechazo de Injerto/virología , Trasplante de Riñón , Enfermedad Aguda , Adulto , Antígenos Virales/análisis , Cápside/inmunología , Femenino , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/inmunología , Humanos , Masculino , Persona de Mediana Edad , ARN Viral/análisis , Factores de TiempoRESUMEN
OBJECTIVE: To determine the value of procalcitonin (PCT) monitoring in transplant patients receiving pan-T-cell antibody therapy. DESIGN: Retrospective clinical study. SETTING: A collaborative study between the Institute of Medical Immunology, the Department of Nephrology and Internal Intensive Care, both Charite, Humboldt University Berlin, and the Department of Laboratory Medicine, Friedrichshain Hospital, Berlin, Germany. PATIENTS AND INTERVENTIONS: Thirty-one patients were included in the study: 8 kidney transplant patients with acute rejection episodes, 5 receiving OKT3 monoclonal antibody therapy, 3 receiving steroid bolus therapy; 21 patients undergoing renal transplantation, 11 receiving ATG perioperatively, 10 without ATG administration; 2 patients undergoing renal transplantation and receiving anti-IL-2R mAb. MEASUREMENTS AND RESULTS: Procalcitonin (PCT) and tumor necrosis factor (TNF) alpha plasma levels were measured in infection-free transplant patients treated with the pan-T-cell antibodies ATG or OKT3. We found PCT plasma concentrations up to 600 ng/ml (reference < 0.5 ng/ ml), which are comparable to those seen in severe sepsis. Increases in TNF-alpha plasma levels preceded the rises in PCT. After peaking on day 1 of therapy the PCT plasma concentrations returned to normal values independently of further antibody administration. In contrast, steroid bolus therapy or anti-interleukin 2 receptor mAb administration did not increase plasma PCT or TNF-alpha levels. CONCLUSIONS: PCT monitoring for evaluating infectious complications in kidney transplant patients must be very careful during pan-T-cell antibody therapy.
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Suero Antilinfocítico/uso terapéutico , Calcitonina/sangre , Inmunosupresores/uso terapéutico , Trasplante de Riñón , Muromonab-CD3/uso terapéutico , Precursores de Proteínas/sangre , Linfocitos T/inmunología , Péptido Relacionado con Gen de Calcitonina , Humanos , Estudios Retrospectivos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
High steady-state frequencies of CMV-specific CD4(+) memory T cells are maintained in CMV-exposed subjects, and these cells are thought to play a key role in the immunologic control of this permanent infection. However, the essential components of this response are poorly defined. Here, we report the use of a step-wise application of flow cytometric and molecular techniques to determine the number and size of the TCR Vbeta-defined clonotypes within freshly obtained CMV-specific CD4(+) memory T cell populations of four healthy, CMV-exposed human subjects. This analysis revealed a stable clonotypic hierarchy in which 1-3 dominant clonotypes are maintained in concert with more numerous subdominant and minor clonotypes. These dominant clonotypes accounted for 10-50% of the overall CMV response, and comprised from 0.3 to 4.0% of peripheral blood CD4(+) T cells. Two subjects displayed immunodominant responses to single epitopes within the CMV matrix phosphoprotein pp65; these single epitope responses were mediated by a single dominant clonotype in one subject, and by multiple subdominant and minor clonotypes in the other. Thus, the CMV-specific CD4(+) T cell memory repertoire in normal subjects is characterized by striking clonotypic dominance and the potential for epitope focusing, suggesting that primary responsibility for immunosurveillance against CMV reactivation rests with a handful of clones recognizing a limited array of CMV determinants. These data have important implications for the understanding of mechanisms by which a genetically stable chronic viral pathogen such as CMV is controlled, and offer possible insight into the failure of such control for a genetically flexible pathogen like HIV-1.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Citomegalovirus/inmunología , Memoria Inmunológica , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/virología , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Linfocitos T CD4-Positivos/metabolismo , Ligando de CD40/biosíntesis , Células Clonales , Citocinas/biosíntesis , Epítopos de Linfocito T/biosíntesis , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Citometría de Flujo/métodos , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Humanos , Epítopos Inmunodominantes/biosíntesis , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Memoria Inmunológica/genética , Lectinas Tipo C , Masculino , Familia de Multigenes/inmunología , Fosfoproteínas/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/metabolismo , Proteínas de la Matriz Viral/inmunologíaRESUMEN
ERalpha-negative breast tumors tend to overexpress growth factor receptors such as epidermal growth factor receptor or c-erbB-2. Raf-1 is a key intermediate in the signal transduction pathways of these receptors. High levels of constitutive Raf kinase (Deltaraf) activity imparts ERalpha- positive MCF-7 breast cancer cells with the ability to grow in the absence of estrogen. Deltaraf transfectants maintained in estrogen-depleted media showed greatly diminished responses to 17beta-estradiol or the pure antiestrogen ICI 182,780. Western blotting, ligand binding, and immunohistochemistry assays revealed a loss of ERalpha protein expression, and ribonuclease protection assays indicated that this correlated with loss of ERalpha message. In examining the basal expression of estrogen-induced genes in the stable transfectants or in transient cotransfection assays with an estrogen-response element- reporter construct and Deltaraf or constitutively active MAPK kinase (DeltaMEK), no ligand- independent activation of ERalpha was observed. Transient expression of Deltaraf and double-label immunostaining showed ERalpha was lost in those cells that transiently expressed Deltaraf. Abrogation of Raf signaling via treatment with the MEK inhibitors PD 098059 or U0126 resulted in reexpression of ERalpha. Similar studies performed with MCF-7 cells overexpressing epidermal growth factor receptor or c-erbB-2 confirmed that hyperactivation of MAPK resulted in down-regulation of ERalpha that was reversible by MEK inhibition or transfection with dominant negative ERK1 and ERK2 constructs. These data suggest that the hyperactivation of MAPK in epidermal growth factor receptor- or c-erbB-2-overexpressing breast cancer cells is directly responsible for generation of an ERalpha-negative phenotype and, more importantly, that this process may be abrogated by inhibiting these pathways, thus restoring ERalpha expression.