RESUMEN

Cell surface molecule CD44 plays a major role in regulation of cancer stem cells CSCs on both phenotypic and functional level, however chemical inhibition approach of CD44 to targets CSCs is poorly studied. Herein, we report the discovery of certain N'-(2-oxoindolin-3-ylidene)-2-(benzyloxy)benzohydrazides as a novel inhibitor of CD44. Molecular docking study showed interference of the scaffold of these compounds with ß-catenin/TCF-4 complex, building a direct relationship between CD44 inhibition and observed well-fitted binding domain. Compound 11a, most potent member elicits inhibition effect on TCF/LEF reporter activity conformed the involvement of Wnt pathway inhibition as a mechanism of action. Furthermore, the treatment by the mentioned compound leads to inhibition of embryonic transcriptional factor Nanog but not Sox2 or Oct-4 suggested specific targeted effect. Moreover, the cytotoxicity and cell cycle effect of this series seems to be dependent on CD44 expression.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Proteínas de Homeodominio/metabolismo , Receptores de Hialuranos/metabolismo , Hidrazinas/farmacología , Transducción de Señal/efectos de los fármacos , beta Catenina/metabolismo , Antineoplásicos/química , Línea Celular Tumoral , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Humanos , Hidrazinas/química , Simulación del Acoplamiento Molecular , Proteína Homeótica Nanog , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/efectos de los fármacos

RESUMEN

The BRAF(V600E) mutation is found in approximately 40% of papillary thyroid cancers (PTC). Mice with thyroid-specific expression of Braf(V600E) (TPO-Braf(V600E)) develop PTC rapidly with high levels of serum thyroid-stimulating hormone (TSH). It is unclear to what extent the elevated TSH contributes to tumor progression. To investigate the progression of Braf(V600E)-induced PTC (BVE-PTC) under normal TSH, we transplanted BVE-PTC tumors subcutaneously into nude and TPO-Braf(WT) mice. Regression of the transplanted tumors was observed in both nude and TPO-Braf(WT) mice. They were surrounded by heavy lymphocyte infiltration and oncogene-induced senescence (OIS) was demonstrated by strong ß-gal staining and absence of Ki-67 expression. In contrast, BVE-PTC transplants continued to grow when transplanted into TPO-Braf(V600E) mice. The expression of Trp53 was increased in tumor transplants undergoing OIS. Trp53 inactivation reversed OIS and enabled tumor transplants to grow in nude mice with characteristic cell morphology of anaplastic thyroid cancer (ATC). PTC-to-ATC transformation was also observed in primary BVE-PTC tumors. ATC cells derived from Trp53 knockout tumors had increased PI3K/AKT signaling and became resistant to Braf(V600E) inhibitor PLX4720, which could be overcome by combined treatment of PI3K inhibitor LY294002 and PLX4720. In conclusion, BVE-PTC progression could be contained via p53-dependent OIS and TSH is a major disruptor of this balance. Simultaneous targeting of both MAPK and PI3K/AKT pathways offer a better therapeutic outcome against ATC. The current study reinforces the importance of rigorous control of serum TSH in PTC patients.

Asunto(s)

Carcinoma Papilar/patología , Proteínas de Neoplasias/fisiología , Neoplasias de la Tiroides/patología , Tirotropina/fisiología , Proteína p53 Supresora de Tumor/biosíntesis , Animales , Carcinoma/genética , Carcinoma/patología , Carcinoma Papilar/genética , Senescencia Celular , Cromonas/farmacología , Cromonas/uso terapéutico , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Genes p53 , Humanos , Indoles/farmacología , Indoles/uso terapéutico , Linfocitos Infiltrantes de Tumor/inmunología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Ratones Transgénicos , Morfolinas/farmacología , Morfolinas/uso terapéutico , Mutación Missense , Proteínas de Neoplasias/sangre , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/fisiología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Neoplasias de la Tiroides/genética , Tirotropina/sangre , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética

RESUMEN

BACKGROUND: A major therapeutic challenge for breast cancer is the ability of cancer cells to evade killing of conventional chemotherapeutic agents. We have recently reported the actin-bundling protein (fascin) as a major regulator of breast cancer metastasis and survival. METHODS: Survival of breast cancer patients that received chemotherapy and xenograft tumour model was used to assess the effect of chemotherapy on fascin-positive and -negative breast cancer cells. Molecular and cellular assays were used to gain in-depth understanding of the relationship between fascin and chemoresistance. RESULTS: We showed a significant correlation between fascin expression and shorter survival in breast cancer patients who received chemotherapy. In xenograft experiments, fascin-positive cancer cells displayed significantly more resistance to chemotherapy-mediated apoptotic cell death than fascin-negative counterparts. This increased chemoresistance was at least partially mediated through PI3K/Akt signalling, and was paralleled by increased FAK phosphorylation, enhanced expression of the inhibitor of apoptosis proteins (XIAP and Livin) and suppression of the proapoptotic markers (caspase 9, caspase 3 and PARP). CONCLUSIONS: This is the first report to demonstrate fascin involvement in breast cancer chemotherapeutic resistance, supporting the development of fascin-targeting drugs for better treatment of chemoresistance breast cancer.

Asunto(s)

Neoplasias de la Mama/tratamiento farmacológico , Proteínas Portadoras/fisiología , Resistencia a Antineoplásicos , Proteínas de Microfilamentos/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Desnudos , Análisis de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Vascular endothelial growth factor (VEGF) and inducible nitric-oxide synthase (iNOS) have been implicated in ischemia-induced retinal neovascularization. Retinal ischemia has been shown to induce VEGF and iNOS expression. It has been postulated that one of the crucial consequences of iNOS expression in the ischemic retina is the inhibition of angiogenesis. Furthermore, iNOS was shown to be overexpressed in Müller cells from patients with diabetic retinopathy. YC-1, a small molecule inhibitor of hypoxia-inducible factor (HIF)-1 alpha, has been shown to inhibit iNOS expression in various tissue models. Our aim was to assess the pleiotropic effects of YC-1 in an oxygen-induced retinopathy (OIR) mouse model and evaluate its therapeutic potential in HIF-1- and iNOS-mediated retinal pathologies. Dual-injections of YC-1 into the neovascular retinas decreased the total retinopathy score, inhibited vaso-obliteration and pathologic tuft formation, and concomitantly promoted physiological retinal revascularization, compared with dimethyl sulfoxide (DMSO)-treated group. Furthermore, YC-1-treated retinas exhibited a marked increase in immunoreactivities for CD31 and von Willebrand factor and displayed significant inhibition in HIF-1alpha protein expression. Furthermore, YC-1 down-regulated VEGF, erythropoietin, endothelin-1, matrix metalloproteinase-9, and iNOS message and protein levels. When hypoxic Müller and neuoroglial cells were treated with YC-1, iNOS mRNA and protein levels were reduced in a dose-dependent fashion. We demonstrate that YC-1 inhibits pathological retinal neovascularization by exhibiting antineovascular activities, which impaired ischemia-induced expression of HIF-1 and its downstream angiogenic molecules. Furthermore, YC-1 enhanced physiological revascularization of the retinal vascular plexuses via the inhibition of iNOS mRNA and protein expressions. The pleiotropic effects of YC-1 allude to its possible use as a promising therapeutic iNOS inhibitor candidate for the treatment of retinal neovascularization.

Asunto(s)

Modelos Animales de Enfermedad , Indazoles/uso terapéutico , Neovascularización Fisiológica/efectos de los fármacos , Oxígeno , Enfermedades de la Retina/prevención & control , Neovascularización Retiniana/prevención & control , Animales , Animales Recién Nacidos , Indazoles/farmacología , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/fisiopatología , Neovascularización Patológica/prevención & control , Neovascularización Fisiológica/fisiología , Oxígeno/efectos adversos , Enfermedades de la Retina/inducido químicamente , Enfermedades de la Retina/fisiopatología , Neovascularización Retiniana/fisiopatología

RESUMEN

Hypoxia-inducible factor (HIF) signaling cascade plays a critical role in angiogenesis by activating the transcription of genes encoding angiogenic growth factors. This study evaluated the effects of YC-1, a HIF-1 inhibitor, on the morphological, biochemical and molecular changes in human retinal microvascular endothelial cells. We found that YC-1 suppressed vascular endothelial cell proliferation, migration and tube formation, while it significantly increased the proteasome activity. Moreover, YC-1 induced a G(0)/G(1) cell-cycle arrest, whereas it exerted only an insignificant proapoptotic effects. Under normoxia or hypoxia, YC-1 did not alter the morphology or the cell viability. Additionally, under hypoxic conditions, YC-1 downregulated HIF-2alpha, VEGF, EPO, ET-1, and MMP-9 mRNA and protein levels, this was accompanied by a significant decrease in the MMP-9 activity. YC-1 decreased the basal expression of HIF-1alpha protein under normoxia, whereas it inhibited HIF-1alpha protein synthesis, stability, and nuclear translocation mechanisms under hypoxia. Furthermore, in a 3D collagen matrix model using mouse retinal explants cultured under normoxic and hypoxic conditions, YC-1; (1) inhibited outgrowth of new vessel sprouts; (2) reduced VEGF expression; (3) dramatically decreased the vessels immunoreactivities for CD31 and von Willebrand Factor (vWF); and (4) was highly effective in reducing the vascular density within the retina, compared to controls. These findings indicate that YC-1 possesses several antiangiogenic properties, both in vitro and ex vivo, which could be exploited as valuable therapeutic potentials to inhibit formation and the growth of new retinal vessels in the hypoxic retina.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Células Endoteliales/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Indazoles/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Vasos Retinianos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transporte Activo de Núcleo Celular , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ciclo Celular/efectos de los fármacos , Hipoxia de la Célula , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Eritropoyetina/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Microvasos/efectos de los fármacos , Microvasos/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Mensajero/metabolismo , Vasos Retinianos/citología , Vasos Retinianos/metabolismo , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor de von Willebrand/metabolismo

RESUMEN

Ribonuclease L (RNase L) is an intracellular enzyme that is vital in innate immunity, but also is a tumor suppressor candidate. Here, we show that overexpression of RNase L decreases cellular growth and downmodulates the RNA-binding protein, HuR, a regulator of cell-cycle progression and tumorigenesis. The effect is temporal, occurring in specific cell-cycle phases and correlated with the cytoplasmic localization of RNase L. Both cellular growth and HuR were increased in RNASEL-null mouse fibroblast lines when compared to wild-type cells. Moreover, the stability of HuR mRNA was enhanced in RNASEL-null cells. The HuR 3' untranslated region (UTR), which harbors U-rich and adenylate-uridylate-rich elements, was potently responsive to RNase L when compared to control 3' UTR. Our results may offer a new explanation to the tumor suppressor function of RNase L.

Asunto(s)

Endorribonucleasas/fisiología , Proteínas de Unión al ARN/antagonistas & inhibidores , Regiones no Traducidas 3'/fisiología , Animales , Antígenos de Superficie/genética , Ciclo Celular , Proliferación Celular , Regulación hacia Abajo , Proteínas ELAV , Proteína 1 Similar a ELAV , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/análisis , Proteínas de Unión al ARN/genética

RESUMEN

Anaplastic thyroid carcinoma is the most aggressive type of thyroid malignancies. Previously, we demonstrated that tumorigenicity of anaplastic thyroid carcinoma cell line ARO was significantly reduced following interleukin (IL)-12 gene transfer. We suspected that tumor target structure in ARO/IL-12 cells might be changed and such a change may make them more susceptible to be killed through mechanisms apart from natural killer-dependent pathway. To identify genes involved, we examined gene expression profile of ARO and ARO/IL-12 by microarray analysis of 3757 genes. The most highly expressed gene was cannabinoid receptor 2 (CB2), which was expressed eightfold higher in ARO/IL-12 cells than ARO cells. CB2 agonist JWH133 and mixed CB1/CB2 agonist WIN-55,212-2 could induce significantly higher rate of apoptosis in ARO/IL-12 than ARO cells. Similar results were obtained when ARO cells were transfected with CB2 transgene (ARO/CB2). A considerable regression of thyroid tumors generated by inoculation of ARO/CB2 cells was observed in nude mice following local administration of JWH133. We also demonstrated significant increase in the induction of apoptosis in ARO/IL12 and ARO/CB2 cells following incubation with 15 nM paclitaxel, indicating that tumor cells were sensitized to chemotherapy. These data suggest that CB2 overexpression may contribute to the regression of human anaplastic thyroid tumor in nude mice following IL-12 gene transfer. Given that cannabinoids have shown antitumor effects in many types of cancer models, CB2 may be a viable therapeutic target for the treatment of anaplastic thyroid carcinoma.

Asunto(s)

Carcinoma/metabolismo , Carcinoma/terapia , Terapia Genética , Interleucina-12/fisiología , Receptor Cannabinoide CB2/biosíntesis , Receptor Cannabinoide CB2/genética , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/terapia , Animales , Apoptosis/genética , Línea Celular Tumoral , Femenino , Técnicas de Transferencia de Gen , Humanos , Interleucina-12/administración & dosificación , Interleucina-12/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Receptor Cannabinoide CB2/metabolismo , Receptor Cannabinoide CB2/fisiología

RESUMEN

Human brucellosis is a zoonotic disease which is endemic in Saudi Arabia. The aim of this study was to investigate the humoral immune responses and identify the target antigens that persist at different stages in human brucellosis during antibiotic therapy. To do this, an acute case of accidental nosocomial infection was studied experimentally. Blood was collected from the patient at the time of diagnosis, and at weekly intervals during therapy until remission. IgG and IgM immunoblotting was used to characterize specific antigenic determinants, and ELISA antibody titration was performed to quantify the circulating antibodies. Results indicated that protein bands of 12-13.5 kDa bound IgG in the patient's sera but did not bind IgM on immunoblots and are probably not specific for, or important in, early stage infections. However, an 18 kDa band persisted during infection through remission. The pivotal and most important findings were that the number of protein bands seen on immunoblots, the magnitude of ELISA antibody titres and the concomitant changes in the intensity of the polypeptide bands of 42-43 kDa were positively correlated with the stage of infection. High numbers of anti-IgG and -IgM immunoblot bands coupled with high ELISA antibody titres and a concomitant increase in intensity of the 42-43 kDa bands were positively correlated with acute and severe infection. Conversely, a reduction in the number of polypeptide bands as well as a decrease in the intensity, until the complete disappearance of the 42-43 kDa bands, coupled with low (baseline) ELISA antibody titration values indicated successful treatment and remission. The routine use of the methods described here to ascertain the stage of the disease, assess the progress of antimicrobial therapy and monitor cases of relapse in human brucellosis is suggested.

Asunto(s)

Antibacterianos/uso terapéutico , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Brucella melitensis/inmunología , Brucelosis/inmunología , Enfermedad Aguda , Antibacterianos/farmacología , Antígenos Bacterianos/química , Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/aislamiento & purificación , Brucella melitensis/efectos de los fármacos , Brucella melitensis/aislamiento & purificación , Brucelosis/sangre , Brucelosis/tratamiento farmacológico , Infección Hospitalaria , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Immunoblotting , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Peso Molecular

RESUMEN

The role of innate immune cells in the recognition and activation of xenogeneic endothelium has always been considered secondary to the initial insult of xenoreactive natural antibodies (XNA) and complement. It was argued, however, that innate immune cells are capable of recognizing and activating xenogeneic endothelium in the absence XNA and complement. Here, we show that porcine aortic endothelial cells (PAECs) activate human neutrophils directly. This contact-dependent activation causes a transient calcium rise leading to increased reactive oxygen metabolite (ROM) production. Neutrophil gene-expression profiling using an adenylate uridylate-rich element-based microarray revealed a dramatic change in the neutrophil gene profiles upon exposure to PAECs. The PAEC-dependent neutrophil transcriptional activity was further confirmed by real-time polymerase chain reaction, which revealed a rapid increase in the mRNA message of a number of inflammatory cytokines. The activation of human neutrophils by PAECs was independent of galactose alpha1,3-galactose (Galalpha1,3-gal) structures, as inclusion of saturating concentrations of anti-Galalpha1,3-gal l antibodies had no significant effect. Furthermore, this activation was inhibited in the presence of the calcium chelator 1,2-bis(O-aminophenyl-ethane-ethane)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester and the ROM inhibitor diphelylene iodonium. Our data illustrate the direct activation of innate immune cells by PAECs in the absence of XNA and complement and suggest alternative recognition sites between PAECs and human innate immune cells.

Asunto(s)

Células Endoteliales/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/inmunología , Rechazo de Injerto/inmunología , Neutrófilos/inmunología , Trasplante Heterólogo/inmunología , Animales , Antígenos de Superficie/química , Antígenos de Superficie/inmunología , Aorta/citología , Aorta/inmunología , Señalización del Calcio/inmunología , Células Cultivadas , Quelantes/farmacología , Quimiotaxis de Leucocito/inmunología , Citocinas/genética , Disacáridos/inmunología , Inhibidores Enzimáticos/farmacología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Sus scrofa , Activación Transcripcional/inmunología

RESUMEN

The mortality and neurological morbidity in heatstroke have been attributed to the host's inflammatory and hemostatic responses to heat stress, suggesting that immunomodulation may improve outcome. We postulated that an experimental baboon model of heatstroke will reproduce human responses and clinical outcome to allow testing of new therapeutic strategies. Eight anesthetized juvenile baboons (Papio hamadryas) were subjected to heat stress in an incubator maintained at 44-47 degrees C until rectal temperature attained 42.5 degrees C (moderate heatstroke; n = 4) or systolic arterial pressure fell to <90 mmHg (severe heatstroke; n = 4) and were allowed to recover at room temperature. Four sham-heated animals served as a control group. Rectal temperature at the end of heat stress was 42.5 +/- 0.0 and 43.3 +/- 0.1 degrees C, respectively. All heat-stressed animals had systemic inflammation and activated coagulation, indicated by increased plasma IL-6, prothrombin time, activated partial thromboplastin time, and D-dimer levels, and decreased platelet count. Biochemical markers and/or histology evidenced cellular injury/dysfunction: plasma levels of thrombomodulin, creatinine, creatine kinase, lactic dehydrogenase, and alanine aminotransferase were increased, and varying degrees of tissue damage were present in liver, brain, and gut. No baboon with severe heatstroke survived. Neurological morbidity but no mortality was observed in baboons with moderate heatstroke. Nonsurvivors displayed significantly greater coagulopathy, inflammatory activity, and tissue injury than survivors. Sham-heated animals had an uneventful course. Heat stress elicited distinct patterns of inflammatory and hemostatic responses associated with outcome. The baboon model of heatstroke appears suitable for testing whether immunomodulation of the host's responses can improve outcome.

Asunto(s)

Golpe de Calor/fisiopatología , Respuesta al Choque Térmico/inmunología , Hemostasis/inmunología , Interleucina-6/sangre , Insuficiencia Multiorgánica/fisiopatología , Síndrome de Respuesta Inflamatoria Sistémica/fisiopatología , Animales , Modelos Animales de Enfermedad , Golpe de Calor/complicaciones , Golpe de Calor/patología , Humanos , Insuficiencia Multiorgánica/etiología , Insuficiencia Multiorgánica/patología , Papio , Índice de Severidad de la Enfermedad , Especificidad de la Especie , Síndrome de Respuesta Inflamatoria Sistémica/etiología , Síndrome de Respuesta Inflamatoria Sistémica/patología

RESUMEN

Recruitment of leukocytes from bloodstream to extrahematic sites is tightly regulated by a variety of adhesion molecules that are expressed on the leukocytes and the vessel walls. In this manuscript, we describe the interactions between natural killer (NK) cells and activated, autologous platelets under physiologic flow. We found that surface-adherent human platelets are capable of recruiting human NK cells from flow and that this recruitment is characterized by an initial tethering followed by a rolling phase. Both phases were dependent on the adhesion molecule P-selectin and its counter-ligand on the NK cells (P-selectin glycoprotein ligand 1). Activation of rolling NK cells with inflammatory mediators commonly found in atherosclerotic plaques (interleukin-12 and leukotriene B4) causes immediate cessation of the rolling process and conversion to stationary adhesion. Blocking antibodies to the adhesion molecules membrane-activated complex-1 and leukocyte function antigen-1 inhibited this conversion. Our data suggest that platelets deposited at sites of vascular injury may provide an alternative substrate to endothelial cells for initial recruitment of NK cells to the vessel wall. This may result in extravasation of the NK cells if the appropriate chemotactic signal is applied. These data implicate the P-selectin and integrin family of adhesion molecules in the recruitment of NK cells to atherosclerotic sites.

Asunto(s)

Plaquetas/inmunología , Adhesión Celular/inmunología , Quimiotaxis de Leucocito/inmunología , Mediadores de Inflamación/farmacología , Interleucina-12/farmacología , Células Asesinas Naturales/inmunología , Selectina-P/inmunología , Anticuerpos/farmacología , Plaquetas/metabolismo , Antígenos CD18/inmunología , Células Cultivadas , Humanos , Mediadores de Inflamación/inmunología , Interleucina-12/inmunología , Células Asesinas Naturales/efectos de los fármacos , Leucotrieno B4/inmunología , Leucotrieno B4/farmacología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Antígeno de Macrófago-1/inmunología

RESUMEN

BACKGROUND: Date fruit and pollen antigens share a number of cross-reactive epitopes. Date pollen has been shown to cross-react with antigens from Artemisia, cultivated rye (Secale cereale), Timothy grass (Phleum pratense), Sydney golden wattle (Acacia longifolia) and Bermuda grass (Cynodon dactylon) pollen. The present study was carried out to examine any cross-reactivities between date palm polypeptides and antigens of some common foods and vegetables that have been implicated in the oral allergy syndrome (OAS). Because most of such cross-reactivities in other allergens are attributable to the presence of carbohydrate chains and profilin, their role was also investigated. METHODS: Fresh extracts of 20 common fruits and vegetables were prepared. Putative date profilins were isolated by affinity chromatography using a poly L-proline column. Date fruit extracts were digested by various endoglycosidases and the immunoglobulin (Ig)E binding of the postdigest products was assessed in immunoblots. Rabbit antisera to whole date fruit extracts, Timothy grass profilin and putative date profilins, as well as human sera from date sensitive individuals were used in immunoblotting, ELISA and in inhibition experiments. RESULTS: IgG, ELISA and immunoblot results with the different rabbit antisera and date-sensitive atopic sera showed several antigenic cross-reactivities and similar cross-reactivities were seen with birch, date and timothy grass profilins. IgE, ELISA and immunoblot experiments with pooled date sensitive human sera showed a range of cross-reactivities with some food extracts. A number of the IgE cross-reactivities could be inhibited after preabsorption of pooled sera with date extracts. Sixty-six percent of individual date hypersensitive human sera bound IgE in putative date fruit profilin and their pooled sera bound IgE in birch pollen profilin. IgE-binding of the endoglycosidase digested date fruit extracts to atopic serum pool was restricted to only a very low molecular weight band of 6.5-8 kDa. CONCLUSION: These results indicate that date palm polypeptides share cross-reactive IgG and IgE epitopes with a number of foods implicated in the oral allergy syndrome, bind to birch and Timothy grass profilins and bind IgE through glycosyl residues. The clinical relevance of these cross-reactivities needs to be further elucidated.

Asunto(s)

Proteínas Contráctiles , Reacciones Cruzadas/inmunología , Hipersensibilidad a los Alimentos/etiología , Frutas/efectos adversos , Frutas/inmunología , Péptidos/efectos adversos , Péptidos/inmunología , Alérgenos/administración & dosificación , Alérgenos/efectos adversos , Alérgenos/inmunología , Animales , Relación Dosis-Respuesta Inmunológica , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Galectina 3/sangre , Galectina 3/efectos de los fármacos , Galectina 3/inmunología , Glicosilación/efectos de los fármacos , Humanos , Immunoblotting , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Proteínas de Microfilamentos/efectos adversos , Proteínas de Microfilamentos/inmunología , Proteínas de Microfilamentos/aislamiento & purificación , Peso Molecular , Péptidos/administración & dosificación , Polen/efectos adversos , Polen/inmunología , Profilinas , Prolina/efectos adversos , Prolina/inmunología , Prolina/aislamiento & purificación , Conejos , Síndrome

RESUMEN

Higher primates, including humans, have high levels of pre-existing naturally circulating antibodies that predominantly recognize the epitope Gal (1,3-Gal), which is highly expressed on the surface of xenogenic cells. Deposition of these antibodies on the endothelial cell surface of vascularized xenografts leads to an activation of the classical pathway of the complement system, resulting in tissue ischemia and necrosis with rapid demise of the xenograft. This hyperacute rejection (HAR) is always a major barrier in xenograft transplantation and should be minimized by accurately monitoring the naturally occurring antibodies. In the present study, we utilized a simple and rapid flow cytometric (FCM) assay to monitor the presence of these naturally occurring antibodies. We found that the FCM assay is very effective in measuring human antibodies bound to the xenogenic cells, which cause cytotoxicity. This assay could be useful in the pre- and post-xenotransplantation monitoring of xenoantibodies, thus, helping in the development of strategies to block the binding of preformed human antibodies to the xenograft in order to overcome the problem of HAR.

Asunto(s)

Anticuerpos Heterófilos/sangre , Supervivencia Celular , Endotelio Vascular/citología , Adulto , Animales , Aorta , Células Cultivadas , Disacáridos/inmunología , Epítopos/inmunología , Citometría de Flujo/métodos , Humanos , Persona de Mediana Edad , Porcinos

RESUMEN

BACKGROUND: Vaccinia virus complement control protein (VCP) was the first secretory microbial protein shown to have structural similarity to the family of complement control proteins. VCP can block both the classical and alternate complement pathways. Recently, VCP has been shown to bind to heparin, and this property contributes to separate functions, making the molecule a multifunctional protein. METHODS: VCP prepared from a natural infection of RK-13 cells with vaccinia virus was purified to homogeneity. Cultured pig aortic endothelial cells (PAECs) were mixed with human serum, anti-Gal alpha1,3 Gal antibody, neutrophils, or natural killer (NK) cells in the presence or absence of VCP and either direct binding of FITC-labeled antibody or killing by cytotoxic cells was estimated. RESULTS: Our cell culture studies demonstrate that VCP blocks complement-mediated killing of PAECs by human serum in a dose-dependent manner. We also demonstrate that VCP is capable of blocking Gal alpha1,3 Gal binding sites on PAECS. Surprisingly, VCP effectively blocked interactions between PAECs and cytotoxic cells such as human naive neutrophils and NK cells. CONCLUSION: VCP is a novel protein amongst the complement control protein family and can, not only block xenorejection by inhibiting complement but also by blocking killing by cytotoxic cells.

Asunto(s)

Endotelio Vascular/citología , Linfocitos T Citotóxicos/inmunología , Proteínas Virales/farmacología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Aorta/citología , Citotoxicidad Inmunológica , Rechazo de Injerto/prevención & control , Humanos , Porcinos , Trasplante Heterólogo/inmunología

RESUMEN

Fluorinated analogues of propranolol, namely trifluoroethyl propranolol (F3), pentafluoropropyl propranolol (F5), and heptafluorobutyl propranolol (F7), were found to induce reactive oxygen metabolite (ROM) production in human neutrophils in a dose-dependent manner. Preincubation of neutrophils with the calcium chelator BAPTA-AM or the tyrosine kinase inhibitor genistein inhibited this ROM production. Direct measurements of intracellular calcium revealed that these analogues caused a transient increase in intracellular calcium. In addition, these fluorinated analogues of propranolol caused a transient increase in actin polymerization. The effects of these compounds were found to be dependent upon the degree of fluorination of the parent compound. Propranolol, on the other hand, had no direct effect on ROM, calcium, or actin polymerization when added alone to neutrophils, although it did modify responses of cells to various stimuli. Whereas ROM production induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine was enhanced in a dose-dependent manner, the response to the particulate stimulus, latex beads, was abolished.

Asunto(s)

Calcio/metabolismo , Neutrófilos/efectos de los fármacos , Propranolol/farmacología , Especies Reactivas de Oxígeno/metabolismo , Actinas/metabolismo , Flúor/química , Homeostasis/efectos de los fármacos , Humanos , Técnicas In Vitro , Neutrófilos/metabolismo , Oxidantes/química , Oxidantes/farmacología , Propranolol/análogos & derivados , Propranolol/química

RESUMEN

BACKGROUND: Date fruits are allergenic and standardized extracts are required for diagnosis and therapy of this allergy. Since there are several cultivars of dates, this study was carried out to assess the allergenicity of different cultivars in order to select suitable source material for standardization. METHODS: The protein profiles of 18 of the most commonly sold varieties were compared by SDS-PAGE and their relative allergenicity assessed by SPT and IgE-based ELISA and immunoblotting. Thirty-two date fruit-sensitive patients were skin tested with a pooled extract from all the cultivars. Six of the patients with high SPT results (> or =3+) who volunteered were further tested with the 18 cultivars and their sera used in ELISA and immunoblotting. RESULTS: Six of the cultivars gave high SPT-positive reactions in > or =4 of patients. Five of these high SPT-reactive cultivars gave high IgE ELISA scores (> or =0.58) but individual cultivars varied in their number of IgE immunoblot bands. Cultivar-specific IgE-binding patterns indicated that only certain cultivars bound IgE at molecular weights of < or =14.3 and 27-33 kDa whilst all cultivars bound to a 54-58 kDa doublet. Cultivars that bind to the < or =14.3 and 27-33 kDa bands appeared to form the majority of the high SPT-reactive cultivars. When individual sera of 24 of the 32 SPT-positive patients were used in IgE immunoblots with the pooled cultivar extract, all sera bound IgE at < or =14.3 and 27-33 kDa and about 60% of sera bound to a 54-58 kDa doublet bands. CONCLUSIONS: These results indicate that allergenicity of date fruits is a cultivar-specific phenomenon. Sixty to 100% of sera from date fruit-allergic patients bind IgE to three major allergens of < or =14.3, 27-33 and 54-58 kDa. Five of the cultivars that evoke high SPT reactions, high IgE ELISA scores and bind IgE to the major allergens, can be selected for the preparation of 'in-house' allergen extracts and for allergen standardization.

Asunto(s)

Alérgenos/análisis , Epítopos/inmunología , Hipersensibilidad a los Alimentos/etiología , Frutas/efectos adversos , Inmunoglobulina E/inmunología , Adulto , Alérgenos/química , Alérgenos/inmunología , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Hipersensibilidad a los Alimentos/sangre , Hipersensibilidad a los Alimentos/epidemiología , Frutas/inmunología , Humanos , Immunoblotting , Inmunoglobulina E/sangre , Masculino , Peso Molecular , Extractos Vegetales/análisis , Extractos Vegetales/inmunología , Arabia Saudita/epidemiología , Pruebas Cutáneas

RESUMEN

BACKGROUND: Interaction between vascularized xenograft and host immune system is thought to occur via Galactose alpha (1,3) Galactose (Gala 1,3 gal) structures decorating the xenograft. METHODS: We raised anti-Gala 1,3 gal-BSA polyclonal antibodies in baboons and investigated effect(s) of these antibodies as well as soluble Gala 1,3 gal-BSA on human naive natural killer (NK) cell interactions with porcine aortic endothelial cells. RESULTS: We demonstrate that human naive (unstimulated) NK cells recognize xenogeneic endothelial cells under conditions where binding to the Gala 1,3 gal structures is minimized by the presence of blocking anti-Gala 1,3 gal IgG or soluble Gala 1-3 gal and in the absence of xenoreactive natural antibodies and complement. After xenogeneic encounter both endothelial cells and human NK cells are activated. Endothelial cell activation is rapid and is manifested initially by an intraendothelial calcium transient and subsequently by expression of P-selectin and vascular endothelial cell adhesion molecule-1 on the xenoendothelium surface. NK cell activation is manifested by increased expression of perforin and increased cytotoxicity towards the xenoendothelium. Neither recognition nor activation of the xenoendothelium was affected by the introduction of either anti-Gala 1,3 gal IgG or soluble Gala 1-3 gal. CONCLUSION: Our data provide evidence that innate immune cells, such as NK cells, recognize and activate xenoendothelial cells independently of Gala 1-3 gal structures and raise the possibility of novel interactive sites on both human naive NK cells and discordant xenogeneic endothelium.

Asunto(s)

Endotelio Vascular/citología , Galactosa/farmacología , Células Asesinas Naturales/citología , Células Asesinas Naturales/trasplante , Trasplante Heterólogo/patología , Animales , Anticuerpos/sangre , Calcio/metabolismo , Trasplante de Células , Galactosa/inmunología , Humanos , Activación de Linfocitos/efectos de los fármacos , Papio , Ovinos , Porcinos

RESUMEN

As barriers to xenotransplantation are surmounted, such as suppression of hyperacute rejection allowing improved graft survival, it becomes important to define longer-term host-xenograft interactions. To this end we have prepared in baboons high titer anti-alpha-Galactosyl (alphaGal) and anti-porcine aortic endothelial cell antibodies, similar to human natural xenoantibodies and reactive with epitopes of thyroglobulin, laminin, and heparan sulfate proteoglycans. When injected into pigs with a protocol similar to that used in the rat to show the nephritogenic potential of heterologous anti-laminin and anti-heparan sulfate proteoglycan antibodies, baboon immunoglobulins bound first to renal vascular endothelium, and later to interstitial cells, especially fibroblasts and macrophages, and to antigens in basement membranes and extracellular matrix, where they colocalized with laminin- and heparan sulfate proteoglycan-antibodies, and with bound Griffonia simplicifolia B4. A similar binding was observed in other organs. The pigs did not develop an acute complement-dependent inflammation, but rather chronic lesions of the basement membranes and the extracellular matrix. Incubation of renal fibroblasts with baboon anti-alpha-Galactosyl antibodies resulted in increased synthesis of transforming growth factor-beta and collagen, suggesting a possible basis for the fibrotic response. The results demonstrate that in this experimental model a consequence of alphaGal antibody interaction with porcine tissues, is immunoreactivity with alphaGal on matrix molecules and interstitial cells, priming mechanisms leading to fibrosis resembling that in chronic allograft rejection. The possibility that similar lesions may develop in long-surviving pig xenografts is discussed.

Asunto(s)

Anticuerpos Heterófilos/inmunología , Especificidad de Anticuerpos , Trisacáridos/inmunología , Animales , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Especificidad de Órganos , Papio , Ratas , Especificidad de la Especie , Porcinos

RESUMEN

Endoplasmic reticulum-associated amyloid beta-peptide (Abeta)-binding protein (ERAB)/L-3-hydroxyacyl-CoA dehydrogenase type II (HADH II) is expressed at high levels in Alzheimer's disease (AD)-affected brain, binds Abeta, and contributes to Abeta-induced cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the NADH-dependent reduction of S-acetoacetyl-CoA with a Km of approximately 68 microM and a Vmax of approximately 430 micromol/min/mg. The contribution of ERAB/HADH II enzymatic activity to Abeta-mediated cellular dysfunction was studied by site-directed mutagenesis in the catalytic domain (Y168G/K172G). Although COS cells cotransfected to overexpress wild-type ERAB/HADH II and variant beta-amyloid precursor protein (betaAPP(V717G)) showed DNA fragmentation, cotransfection with Y168G/K172G-altered ERAB and betaAPP(V717G) was without effect. We thus asked whether the enzyme might recognize alcohol substrates of which the aldehyde products could be cytotoxic; ERAB/HADH II catalyzed oxidation of a variety of simple alcohols (C2-C10) to their respective aldehydes in the presence of NAD+ and NAD-dependent oxidation of 17beta-estradiol. Addition of micromolar levels of synthetic Abeta(1-40) to purified ERAB/HADH II inhibited, in parallel, reduction of S-acetoacetyl-CoA (Ki approximately 1.6 microM), as well as oxidation of 17beta-estradiol (Ki approximately 3.2 microM) and (-)-2-octanol (Ki approximately 2.6 microM). Because micromolar levels of Abeta were required to inhibit ERAB/HADH II activity, whereas Abeta binding to ERAB/HADH II occurred at much lower concentrations (Km approximately 40-70 nM), the latter more closely simulating Abeta levels within cells, Abeta perturbation of ERAB/HADH II was likely to result from mechanisms other than the direct modulation of enzymatic activity. Cells cotransfected to overexpress ERAB/HADH II and betaAPP(V717G) generated malondialdehyde-protein and 4-hydroxynonenal-protein epitopes, which were detectable only at the lowest levels in cells overexpressing either ERAB/HADH II or betaAPP(V717G) alone. Generation of such toxic aldehydes was not observed in cells contransfected to overexpress Y168G/K172G-altered ERAB and betaAPP(V717G). We conclude that the generalized alcohol dehydrogenase activity of ERAB/HADH II is central to the cytotoxicity observed in an Abeta-rich environment.

Asunto(s)

3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Péptidos beta-Amiloides/metabolismo , Proteínas Portadoras/metabolismo , Alcoholes/metabolismo , Animales , Células COS , Supervivencia Celular , Oxidación-Reducción , Proteínas Recombinantes/metabolismo , Fracciones Subcelulares/enzimología , Especificidad por Sustrato , Células Tumorales Cultivadas