RESUMEN
Vulvovaginal candidiasis is a mucosal infection affecting many women, but the immune mechanisms operating against Candida albicans at the mucosal level remain unknown. A rat model was employed to further characterize the contribution of B and T cells to anti-Candida vaginal protection. Particularly, the protective role of vaginal B cells was studied by means of adoptive transfer of vaginal CD3(-) CD5(+) IgM(+) cells from Candida-immunized rats to naïve animals. This passive transfer of B cells resulted into a number of vaginal C. albicans CFU approximately 50% lower than their controls. Sorted CD3(-) CD5(+) IgM(+) vaginal B lymphocytes from Candida-infected rats proliferated in response to stimulation with an immunodominant mannoprotein (MP) antigen of the fungus. Importantly, anti-MP antibodies and antibody-secreting B cells were detected in the supernatant and cell cultures, respectively, of vaginal B lymphocytes from infected rats incubated in vitro with vaginal T cells and stimulated with MP. No such specific antibodies were found when using vaginal B cells from uninfected rats. Furthermore, inflammatory and anti-inflammatory cytokines, such as interleukin-2 (IL-2), IL-6 and IL-10, were found in the supernatant of vaginal B cells from infected rats. These data are evidence of a partial anti-Candida protective role of CD3(-) CD5(+) IgM(+) vaginal B lymphocytes in our experimental model.
Asunto(s)
Traslado Adoptivo , Linfocitos B/inmunología , Candida albicans/inmunología , Candidiasis Vulvovaginal/prevención & control , Animales , Anticuerpos Antifúngicos/biosíntesis , Antígenos Fúngicos/inmunología , Linfocitos B/química , Complejo CD3/análisis , Antígenos CD5/análisis , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Femenino , Proteínas Fúngicas/inmunología , Humanos , Inmunoglobulina M/biosíntesis , Glicoproteínas de Membrana/inmunología , Ratas , Ratas Wistar , Vagina/microbiologíaRESUMEN
Herein, we provide evidence on the expression of transient receptor potential vanilloid type 1 (TRPV1) on human urothelial cancer (UC) cells and its involvement in the apoptosis induced by the selective agonist capsaicin (CPS). We analyzed TRPV1 messenger RNA and protein expression on human UC cell lines demonstrating its progressive decrease in high-grade UC cells. Treatment of RT4 cells with CPS induced cell cycle arrest in G(0)/G(1) phase and apoptosis. These events were associated with rapid co-ordinated transcription of pro-apoptotic genes including Fas/CD95, Bcl-2 and caspase families and ataxia telangiectasia mutated (ATM)/CHK2/p53 DNA damage response pathway. CPS induced Fas/CD95 upregulation, but more importantly Fas/CD95 ligand independent, TRPV1-dependent death receptor clustering and triggering of both extrinsic and intrinsic mitochondrial-dependent pathways. Moreover, we observed that CPS activates ATM kinase that is involved in Ser15, Ser20 and Ser392 p53 phosphorylation as shown by the use of the specific inhibitor KU55933. Notably, ATM activation was also found to control upregulation of Fas/CD95 expression and its co-clustering with TRPV1 as well as RT4 cell growth and apoptosis. Altogether, we describe a novel connection between ATM DNA damage response pathway and Fas/CD95-mediated intrinsic and extrinsic apoptotic pathways triggered by TRPV1 stimulation on UC cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Capsaicina/farmacología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Canales Catiónicos TRPV/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Receptor fas/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Invasividad Neoplásica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fármacos del Sistema Sensorial/farmacología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/metabolismo , Urotelio/efectos de los fármacos , Urotelio/metabolismoRESUMEN
Novel 1,4-dioxane compounds structurally related to WB 4101 (1) were prepared in order to investigate the possibility that the quite planar 1,4-benzodioxane template of 1 might be replaced by the less conformationally constrained 1,4-dioxane ring. The biological profiles of the new compounds were assessed using binding assays at human cloned alpha 1-adrenoreceptor (alpha 1-AR) subtypes and 5-HT 1A receptors, expressed in Chinese hamster ovary and HeLa cell membranes, respectively, and by functional experiments in isolated rat vas deferens (alpha 1A), spleen (alpha 1B), and aorta (alpha 1D). Moreover, the cytotoxic effects of the novel compounds were determined in PC-3 prostate cancer cells. The results showed that the properly substituted 1,4-dioxane nucleus proved to be a suitable scaffold for selective alpha 1D-AR antagonists (compound 14), potential anticancer agents (compound 13), and full 5-HT 1A receptor agonists (compound 15). In particular, compound 15 may represent a novel lead in the development of highly potent 5-HT 1A receptor full agonists useful as antidepressant and neuroprotective agents.
Asunto(s)
Antagonistas de Receptores Adrenérgicos alfa 1 , Benceno/química , Dioxanos/química , Dioxanos/farmacología , Etilaminas/química , Etilaminas/farmacología , Receptor de Serotonina 5-HT1A/metabolismo , Agonistas de Receptores de Serotonina/química , Agonistas de Receptores de Serotonina/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Modelos Moleculares , Estructura Molecular , Receptores Adrenérgicos alfa 1/metabolismo , Relación Estructura-ActividadRESUMEN
Climacostol (5-(Z)-non-2-enyl-benzene-1,3-diol) is a natural toxin isolated from the freshwater ciliated protozoan Climacostomum virens and belongs to the group of resorcinolic lipids, compounds that show antimicrobial, antiparasitic and antitumor activities. We investigated the cytotoxic activity of the chemically synthesized toxin on: (1) human tumor squamous carcinoma A431 cells, (2) human promyelocytic leukaemia HL60 cells, and (3) human non-tumor endothelial EA.hy926 cells. The results showed that climacostol effectively inhibited the growth of tumor cell lines in a dose-dependent manner by inducing programmed cell death, with non-tumor cells proving significantly more resistant to the toxin.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias/patología , Resorcinoles/farmacología , Acetilcisteína/farmacología , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 8/efectos de los fármacos , Caspasa 8/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Fosfatidilserinas/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The activity of substance P (SP) in the rat thymus seems to be tightly controlled by its bioavailability. In this study, we provide evidence for the expression of the SP-degrading enzyme, neutral endopeptidase (NEP)/CD10, by rat thymocyte subsets, and we illustrate its involvement in the in vivo SP/neurokinin-1 receptor (NK(1)R)-mediated regulation of thymocyte survival and proliferation. NEP/CD10 was expressed at both mRNA and protein levels on a substantial portion (45.5%) of CD5(+) thymocytes, namely on the CD4(+)CD8(+) (double positive; DP) and CD4(+) subsets. Continuous administration of thiorphan, a specific NEP/CD10 inhibitor, by means of miniosmotic pumps, enhanced rat thymocyte preprotachykinin-A (PPT-A) and NK(1)R mRNA expression as well as SP and NK(1)R protein levels in an NK(1)R-dependent manner. Thiorphan increased CD10(+)CD4(+) and CD10(+)DP thymocyte numbers, and an NK(1)R antagonist, (S)1-{2-[3(3-4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)-piperidine-3-yl]ethyl}-4-pheny-1-azoniabicyclo[2.2.2]octane, chloride (SR140333), abrogated these stimulatory effects. In addition, the NEP/CD10 inhibitor stimulated interleukin (IL)-2 production, IL-2 receptor alpha chain expression, and concanavalin A-induced proliferation of CD5(+) thymocytes, and it inhibited spontaneous and NK(1)R-dependent thymocyte apoptosis. The thiorphan-protective antiapoptotic and proliferative effects involved the activation of Akt serine-threonine kinase, subsequent up-regulation of survivin mRNA, down-regulation of procaspase-3 mRNA levels, and suppression of caspase-3 activity, which were inhibited by SR140333 and mimicked by exogenous SP administration. Overall, our findings suggest that by controlling SP availability, NEP/CD10 negatively regulates thymocyte homeostasis and development.
Asunto(s)
Inhibidores de Caspasas , Activación de Linfocitos/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Linfocitos T/efectos de los fármacos , Tiorfan/farmacología , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Interleucina-2/biosíntesis , Subunidad alfa del Receptor de Interleucina-2/análisis , Masculino , Neprilisina/genética , Neprilisina/fisiología , Fosforilación , ARN Mensajero/análisis , Ratas , Ratas Wistar , Receptores de Neuroquinina-1/análisis , Receptores de Neuroquinina-1/genética , Sustancia P/genética , Survivin , Linfocitos T/metabolismoRESUMEN
The good results obtained as potential antitumor drugs with aza-anthracenediones and aza-anthrapyrazoles, e.g. pixantrone, 1a, and 1b (Chart 1), prompted us to design and synthesize a series of symmetrical bis derivatives, compounds 7-10 (Chart 1). These compounds are dimers of different aza-anthracenedione and aza-anthrapyrazolone monomers connected by the linker found to be the most appropriate among potential bis intercalators synthesized by us. The DNA-binding properties of bis derivatives 7 and 8 have been examined using fluorometric techniques: these target compounds are excellent DNA ligands, with a clear binding site preference for AT-rich duplexes. In vitro cytotoxic activity of all target compounds 7-10 and of reference compound pixantrone toward human cancer adenocarcinoma cell line HT29 is also described. Two selected compounds have been investigated for their capacity of inducing early apoptosis.
Asunto(s)
Antracenos/síntesis química , Antineoplásicos/síntesis química , Compuestos Aza/síntesis química , Pirazoles/síntesis química , Antracenos/química , Antracenos/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis , Compuestos Aza/química , Compuestos Aza/farmacología , ADN/química , Ensayos de Selección de Medicamentos Antitumorales , Perfilación de la Expresión Génica , Células HT29 , Humanos , Pirazoles/química , Pirazoles/farmacología , ARN Mensajero/biosíntesisRESUMEN
OBJECTIVE: To evaluate the expression of transient receptor potential vanilloid type 2 (TRPV2) in normal human bladder and urothelial carcinoma (UC) tissues. METHODS: Bladder specimens were obtained by transurethral resection or radical cystectomy. TRPV2 mRNA expression in normal human urothelial cells (NHUCs), UC cell lines, and formalin-fixed paraffin-embedded normal (n=6) and cancer bladder tissues (n=58) was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (RT-PCR). TRPV2 protein expression was assessed by cytofluorimetric and confocal microscopy analyses in NHUCs and UC cells and by Western blotting and immunohistochemistry in normal and UC tissues. RESULTS: Enhanced TRPV2 mRNA and protein expression was found in high-grade and -stage UC specimens and UC cell lines. Both the full-length TRPV2 (hTRPV2) and a short splice-variant (s-TRPV2) were detected in NHUC and normal bladder specimens, whereas a progressive decline of s-TRPV2 in pTa, pT1, and pT2 stages was observed, up to a complete loss in pT3 and pT4 UC specimens. CONCLUSIONS: Normal human urothelial cells and bladder tissue specimens express TRPV2 at both the mRNA and protein levels. A progressive loss of s-TRPV2 accompanied by a marked increase of hTRPV2 expression was found in high-grade and -stage UC tissues.
Asunto(s)
Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/patología , Canales Catiónicos TRPV/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Urotelio/metabolismo , Urotelio/patología , Análisis de Varianza , Biomarcadores de Tumor/análisis , Western Blotting , Carcinoma de Células Transicionales/metabolismo , Distribución de Chi-Cuadrado , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Microscopía Confocal , Estadificación de Neoplasias , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
The good results obtained in the past decade with various types of potential bisintercalating agents, e.g., LU 79553, DMP 840, BisBFI, MCI3335, WMC-26, BisAC, BisPA, and the asymmetrical derivative WMC-79 (Chart 1), prompted us to investigate a new series of asymmetrical bisintercalators, compounds 1a-t (Chart 2), which can combine the potentiality of bisintercalation with a possible different mechanism of action due to two diverse chromophores. The DNA-binding properties of these compounds have been examined using fluorometric techniques: target compounds are excellent DNA ligands, with a clear preference for binding to AT-rich duplexes. In vitro cytotoxicity of these derivatives toward human hormone-refractory prostate adenocarcinoma cell line (PC-3) is described. Apoptosis assays of four selected compounds are also reported. Very potent cytotoxic compounds, some of them capable of inducing early apoptosis, have been identified.
Asunto(s)
Acridinas/síntesis química , Antineoplásicos/síntesis química , Sustancias Intercalantes/síntesis química , Acridinas/química , Acridinas/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Unión Competitiva , Línea Celular Tumoral , ADN/química , Ensayos de Selección de Medicamentos Antitumorales , Compuestos Heterocíclicos de 4 o más Anillos/química , Humanos , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , Masculino , Neoplasias de la Próstata , Solubilidad , Relación Estructura-ActividadRESUMEN
This study analyzes the phenotype of vaginal dendritic cells (VDCs), their antigenic presentation and activation of T-cell cytokine secretion, and their protective role in a rat model of Candida vaginitis. Histological observation demonstrated a significant accumulation of OX62(+) VDCs in the mucosal epithelium of Candida albicans-infected rats at the third round of infection. We identified two subsets of OX62(+) VDCs differing in the expression of CD4 molecule in both noninfected and Candida-infected rats. The OX62(+) CD4(+) subset of VDCs displayed a lymphoid cell-like morphology and expressed the T-cell antigen CD5, whereas the OX62(+) CD4(-) VDC subset exhibited a myeloid morphology and was CD5 negative. Candida infection resulted in VDC maturation with enhanced expression of CD80 and CD134L on both CD4(+) and CD4(-) VDC subsets at 2 and 6 weeks after Candida infection. CD5(-) CD4(-) CD86(-) CD80(-) CD134L(+) VDCs from infected, but not noninfected, rats spontaneously released large amounts of interleukin-12 (IL-12) and tumor necrosis factor alpha, whereas all VDC subsets released comparable levels of IL-10 and IL-2 cytokines. Furthermore, OX62(+) VDCs from infected rats primed naïve CD4(+) T-cell proliferation and release of cytokines, including gamma interferon, IL-2, IL-6, and IL-10, in response to staphylococcal enterotoxin B stimulation in vitro. Adoptive transfer of highly purified OX62(+) VDCs from infected rats induced a significant acceleration of fungal clearance compared with that in rats receiving naive VDCs, suggesting a protective role of VDCs in the anti-Candida mucosal immunity. Finally, VDC-mediated protection was associated with their ability to rapidly migrate to the vaginal mucosa and lymph nodes, as assessed by adoptive transfer of OX62(+) VDCs labeled with 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester.
Asunto(s)
Candida albicans/inmunología , Candidiasis/inmunología , Candidiasis/patología , Células Dendríticas/inmunología , Vaginitis/inmunología , Vaginitis/patología , Traslado Adoptivo , Animales , Candidiasis/prevención & control , Proliferación Celular , Células Cultivadas , Células Dendríticas/patología , Células Dendríticas/trasplante , Femenino , Inmunidad Mucosa , Inmunofenotipificación , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Ratas , Ratas Wistar , Vagina/inmunología , Vagina/patología , Vaginitis/prevención & controlRESUMEN
A series of new alpha1-adrenoreceptor antagonists (5-18) was prepared by introducing various substituents (Topliss approach) into the ortho, meta, and para positions of the benzyloxy function of the phendioxan open analogue 4 ("openphendioxan"). All the compounds synthesized were potent antagonists and generally displayed, similarly to 4, the highest affinity values at alpha1D- with respect to alpha1A- and alpha1B-AR subtypes and 5-HT1A subtype. By sulforhodamine B (SRB) assay on human PC-3 prostate cancer cells, the new compounds showed antitumor activity (estimated on the basis of three parameters GI50, TGI, LC50), at low micromolar concentration, with 7 ("clopenphendioxan") exhibiting the highest efficacy. Moreover, this study highlighted for the first time alpha1D- and alpha1B-AR expression in PC3 cells and also demonstrated the involvement of these subtypes in the modulation of apoptosis and cell proliferation. A significant reduction of alpha1D- and alpha1B-AR expression in PC3 cells was associated with the apoptosis induced by 7. This depletion was completely reversed by norepinephrine.
Asunto(s)
Antagonistas Adrenérgicos alfa/síntesis química , Antineoplásicos/síntesis química , Apoptosis , Proliferación Celular , Éteres Fenílicos/síntesis química , Neoplasias de la Próstata/patología , Receptores Adrenérgicos alfa 1/fisiología , Antagonistas Adrenérgicos alfa/química , Antagonistas Adrenérgicos alfa/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Masculino , Éteres Fenílicos/química , Éteres Fenílicos/farmacología , Ensayo de Unión Radioligante , Relación Estructura-ActividadRESUMEN
A series of potential DNA-binding antitumor agents, 2-[omega-(alkylamino)alkyl]-9-methoxy-5-nitro-2,6-dihydroindazolo[4,3-bc][1,5]naphthyridines (2a-f), 10-aza derivatives of PZA, has been prepared by condensation of 9-chloro-2-methoxy-6-nitro-5,10-dihydrobenzo[b][1,5]naphthyridin-10-one (6) with the appropriate (omega-aminoalkyl)hydrazine in tetrahydrofuran/methanol. Compound 6 was obtained by heating at 100 degrees C in H(2)SO(4)5, yielded by the condensation of 2,6-dichloro-3-nitrobenzoic acid (4) and 6-methoxy-3-pyridinamine (3). The non-covalent DNA-binding properties of 2 have been examined using a fluorometric technique. In vitro cytotoxic potencies of these derivatives against human hormone-refractory prostate adenocarcinoma cell line (PC-3) are described and compared to that of parent drug PZA. We selected the most cytotoxic target derivatives 2c,d, the in vitro inactive 2f, and reference compound PZA to investigate whether in vitro treatment with these drugs was able to induce necrotic and/or apoptotic cell death. To this purpose, we evaluated the percentage of apoptotic cells in PC-3 treated with the target compounds 2c,d,f and reference compound PZA, by Annexin V staining and Propidium iodide (PI)/Annexin V, biparametric flow cytometric analysis and agarose gel electrophoresis.
Asunto(s)
Antineoplásicos/clasificación , Antineoplásicos/síntesis química , Naftiridinas/clasificación , Naftiridinas/síntesis química , Animales , Antineoplásicos/farmacología , Bovinos , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos/métodos , Humanos , Naftiridinas/farmacologíaRESUMEN
Herein we provide evidence that substance P (SP) and its neurokinin-1 receptor (NK-1R) expressed on thymocytes counteract thymus depletion induced by neonatal capsaicin (CPS) treatment by affecting thymocyte proliferation and apoptotic death. SP administration reversed the CPS-mediated inhibitory effects on the total thymocyte number and subset distribution, namely CD4+ and CD4- CD8- cells, through its interaction with NK-1R as shown by concomitant NK-1R (SR140333) antagonist administration. SP-induced enhancement of thymus cellularity parallels its ability of inhibiting the thymocyte apoptotic program. Indeed, exogenously administered SP completely nullified CPS-induced apoptosis, and SR140333 abrogated the SP-mediated protective effect. SP administration also stimulated concanavalin A (Con A)-induced thymocyte proliferation of CPS-treated rats, completely reversing the CPS-induced inhibition. The SP-mediated stimulation of Con A-induced thymocyte proliferation was NK-1R dependent as shown by concomitant administration of SP and SR140333 to CPS-treated rats. Our results also demonstrate that CPS treatment induces a marked decrease of thymocyte PPT-A mRNA level and endogenous SP content as evaluated by quantitative RT-PCR, in situ hybridization and cytofluorimetric analysis. By contrast, NK-1R mRNA levels were increased in thymocytes from CPS-treated rats. Exogenous SP administration augmented PPT-A, SP and NK-1R thymocyte expression in CPS-treated rats, and this enhancement was antagonized by SR140333 administration. Overall, our results strongly suggest that the immunomodulatory effects of neonatal CPS treatment on rat thymocyte functions are dependent on vanilloid-mediated regulation of SP and NK-1R functional expression by neuronal and immune cells.
Asunto(s)
Capsaicina/farmacología , Receptores de Neuroquinina-1/metabolismo , Sustancia P/metabolismo , Linfocitos T/efectos de los fármacos , Timo/efectos de los fármacos , Adyuvantes Inmunológicos/farmacología , Animales , Animales Recién Nacidos , Atrofia/inducido químicamente , Atrofia/inmunología , Atrofia/fisiopatología , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/inmunología , Sitios de Unión/efectos de los fármacos , Sitios de Unión/inmunología , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , División Celular/efectos de los fármacos , División Celular/inmunología , Concanavalina A/farmacología , Sustancias de Crecimiento/inmunología , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/farmacología , Masculino , Neuroinmunomodulación/efectos de los fármacos , Neuroinmunomodulación/inmunología , Antagonistas del Receptor de Neuroquinina-1 , Piperidinas/farmacología , Quinuclidinas/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Neuroquinina-1/agonistas , Sustancia P/genética , Sustancia P/farmacología , Linfocitos T/inmunología , Linfocitos T/patología , Timo/inmunología , Timo/fisiopatología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
The protective roles of different lymphocyte subsets were investigated in a rat vaginal candidiasis model by adoptive transfer of vaginal lymphocytes (VL) or sorted, purified CD3(+) T cells, CD4(+) or CD8(+) T cells, or CD3(-) CD5(+) B cells from the vaginas of naïve or immune rats following three rounds of Candida albicans infection. The adoptive transfer of total VL from nonimmune animals did not alter the course of vaginal candidiasis of the recipient rats. In contrast, the animals receiving total VL or CD3(+) T cells from immune rats showed a highly significant acceleration of fungus clearance compared with animals which received nonimmune VL. The animals with vaginal CD3(-) CD5(+) B cells transferred from immune rats also had fewer Candida CFU than the controls, but fungal clearance was significantly retarded with respect to the animals administered immune T cells. Sorted, purified CD4(+) and CD8(+) vaginal T cells from immune rats were also adoptively transferred to naïve animals. Although both populations were seen to accelerate the clearance of the fungus from the vagina, CD4(+) T cells were much more effective than CD8(+) T cells. Overall, there was no difference between the antifungal effects of immune vaginal CD4(+) T cells and those achievable with the transfer of whole, immune VL. Histological observations of the vaginal tissues of rats with adoptively transferred immune T cells demonstrated a remarkable accumulation of lymphocytes in the subepithelial lamina propria and also infiltrating the mucosal epithelium. These results strongly suggest that distinct vaginal lymphocyte subsets participate in the adaptive anti-Candida immunity at the vaginal level, with the vaginal CD4(+) T cells probably playing a major role.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Candidiasis Vulvovaginal/inmunología , Candidiasis Vulvovaginal/prevención & control , Subgrupos Linfocitarios/inmunología , Traslado Adoptivo , Animales , Subgrupos de Linfocitos B/inmunología , Complejo CD3/metabolismo , Antígenos CD5/metabolismo , Linfocitos T CD8-positivos/inmunología , Candidiasis Vulvovaginal/microbiología , Candidiasis Vulvovaginal/patología , Femenino , Ratas , Ratas Wistar , Subgrupos de Linfocitos T/inmunología , Vagina/inmunología , Vagina/microbiología , Vagina/patologíaRESUMEN
The signaling pathways triggered by adherence of Candida albicans to the host cells or extracellular matrix are poorly understood. We provide here evidence in C. albicans yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain C. albicans yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited C. albicans yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of alpha v beta 3 and alpha v beta 5 integrin-like on C. albicans either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in C. albicans yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a C. albicans mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that C. albicans Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells.
Asunto(s)
Candida albicans/enzimología , Cadenas beta de Integrinas , Proteínas Tirosina Quinasas/metabolismo , Tirosina/metabolismo , Vitronectina/metabolismo , Antígenos Bacterianos/biosíntesis , Antígenos CD/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Adhesión Celular , Línea Celular , Endotelio Vascular/citología , Activación Enzimática , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Integrina alfaV , Integrina beta3 , Integrinas/metabolismo , Mutagénesis , Fosforilación , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores de Vitronectina/metabolismoRESUMEN
Herein, we provide evidence of the expression and function of substance P (SP) and neurokinin-1 receptor (NK-1R) in the rat thymus. In situ hybridization evidenced NK-1R mRNA mainly in the thymic medulla, and Northern blot analysis of mRNA from FACS-sorted thymocytes identified NK-1R on CD4+, CD8+ and double-positive subpopulations. With flow cytometry, it could be seen that NK-1R was expressed on the majority of CD5+ thymocytes, and it was identified by Western blot analysis as two bands migrating at 44 and 54 kD. SP administration rescues thymocytes from spontaneous and NK-1R antagonist (SR140333)-induced apoptosis and stimulates concanavalin A (ConA)-induced thymocyte proliferation, CD25 expression and IL-2 production, whereas SR140333 exerts inhibitory effects on these functions. We also demonstrated the expression of mRNA for the SP precursor preprotachykinin-A in the thymic medulla and purified CD5+ thymocytes. SP protein was detected on 40% of CD5+ thymocytes and identified as a band of 1.3 kD by Western blot analysis. Finally, thymocytes spontaneously released SP, which was increased upon ConA or CD3 stimulation.