Mulero Méndez, Victoriano Francisco

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Mulero Méndez, Victoriano Francisco

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Mulero Méndez, Victoriano Francisco

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Universidad de Murcia. Departamento de Biología Celular e Histología

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Open Access
Diseño de un protocolo para el aislamiento de células cebadas del exudado peritoneal de Sparus aurata para la identificación de receptores de estrógenos
(2020-04-13) Gómez-González, Nuria Esther; García-García, Érick; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Facultades, Departamentos, Servicios y Escuelas::Departamentos de la UMU::Biología Celular e Histología
RESUMEN. La contaminación de las aguas por disruptores endocrinos, y en concreto de estrógenos sintéticos, crece diariamente pudiendo cambios en el sistema inmunitario en los animales que en ellas habitan al presentar en las células que lo componen receptores de estrógenos. La inflamación es la primera respuesta de los animales frente a una infección o daño en el tejido. La histamina tiene un papel muy importante en la regulación de la inflamación en vertebrados capaces de almacenarla en células especializadas inmunitarias llamadas células cebadas. Sparus aurata presenta histamina en las células cebadas de tejidos mucosos. El exudado peritoneal de S. aurata está enriquecido en células cebadas. Hemos identificado que el exudado peritoneal de dorada está compuesto por linfocitos, granulocitos acidófilos, macrófagos y células cebadas. Hemos diseñado un protocolo de separación de las células cebadas de exudado peritoneal de S. aurata consiguiendo purezas superiores al 95% cuando se mide por citometría. Estos datos fueron confirmados mediante microscopía electrónica. Este protocolo de separación servirá como un paso hacia adelante para realizar estudios in vivo e in vitro orientándonos en la evolución de los mecanismos inflamatorio de vertebrados. También hemos demostrado por citometría de flujo y western-blot utilizando los anticuerpos adecuados que las células cebadas de esta especie presentan el receptor de estrógenos acoplado a la proteína G (GPER). Además, con la idea de poder estudiar en investigaciones futuras el efecto que altas concentraciones de estrógenos causan en el sistema inmune de S. aurata intentamos estudiar la capacidad de liberación de histamina en células cebadas tras su estimulación con diferentes PAMPs (patrones moleculares asociados a patógenos) lipopolisacáridos (LPS), Poly I:C (análogo sintético de la doble cadena de ARN (dsRNA)) y AND genómico de Vibrio angillarum (VaDNA), aunque ninguno era capaz de inducir la liberación de histamina en estas células.
Open Access
Efecto de la testosterona en la espermatogénesis de machos de dorada (Sparus aurata L.)
(2020-04-13) Sánchez-Hernández, Miriam; Chaves-Pozo, Elena; Cabas, Isabel; García-Alcázar, Alicia; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Facultades, Departamentos, Servicios y Escuelas::Departamentos de la UMU::Biología Celular e Histología
RESUMEN Los andrógenos pueden inducir la espermatogénesis en peces teleósteos inmaduros. Sin embargo, su función no se conoce con exactitud en los ejemplares adultos. Para conocer el efecto in vivo de la testosterona (T) sobre la espermatogénesis de dorada (Sparus aurata L. 1758), especie hermafrodita protándrica de interés económico alto en la región de Murcia, inyectamos un implante (in situ forming microparticle system) a ejemplares que se encontraban en la etapa de involución testicular del segundo ciclo reproductor. Se trabajó con tres grupos de ejemplares: i) inyectados con implantes con 1 mg T/Kg de peso corporal, ii) inyectados con implantes sin T y iii) no inyectados. Se tomaron muestras de sangre, gónada e hígado a los 7, 14, 21 y 28 días después de haberlos inyectado para realizar un análisis hormonal, morfológico, inmunocitoquímico y de expresión génica. El objetivo de nuestro trabajo era determinar el efecto de la T, y de sus derivados, sobre la fisiología de la gónada analizando la progresión de la espermatogénesis, la tasa de proliferación celular y de apoptosis, la presencia de leucocitos y el perfil de expresión de genes relevantes en la respuesta inmunitaria. Los implantes con T provocaron un aumento en los niveles de T, alcanzando niveles supra-fisiológicos. El perfil de expresión génica, en gónada e hígado de algunas enzimas implicadas en la síntesis y el metabolismo de esteroides, descartó la transformación de T en compuestos estrogénicos. Por otra parte la T parece inhibir la respuesta inmunitaria en la gónada ya que observamos una disminución de los niveles de expresión de los genes que codifican para los TLR, lo que podría provocar una disminución de su capacidad para reconocer y responder a los patógenos. Sin embargo se observó un aumento de granulocitos acidófilos y linfocitos lo que nos sugiere que los andrógenos participan en el flujo migratorio de leucocitos al interior de la gónada.
Open Access
Comunicaciones Póster.-Effects of 17α-ethinylestradiol in the immune system of the gilthead seabream (Sparus aurata L.) through mast cells
(2020-05-29) Gómez González, Nuria E.; García-Alcázar, A.; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar; Facultades, Departamentos, Servicios y Escuelas::Departamentos de la UMU::Biología Celular e Histología
Open Access
17α-Ethynylestradiol alters the peritoneal immune response of gilthead seabream
(Elsevier, 2017) Gómez González, Nuria Esther; Cabas, Isabel; Rodenas, María del Carmen; Arizcun, Marta; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Biología Celular e Histología
17α-Ethynylestradiol (EE2), a synthetic estrogen used in most oral contraceptives pills and hormone replacement therapies, is found in many water bodies, where it can modulate the fish immune response. EE2 acts as an endocrine disruptor in gilthead seabream, Sparus aurata L., a marine teleost fish of great economic value in Mediterranean aquaculture, as it induces hepatic vitellogenin gene (vtg) expression. Moreover, EE2 also alters the capacity of gilthead seabream to appropriately respond to infection although it does not behave as an immunosuppressor. Nevertheless, these previous studies have mainly focused on the head kidney leukocytes and no information exists on peritoneal leukocytes, including mast cells. In the present work, juvenile gilthead seabream fish were fed a pellet diet supplemented with EE2 for 76 days and intraperitoneally injected with hemocyanin plus imject alum adjuvant at the end of EE2 treatment and 92 days later, and the peritoneal immune response was analyzed. EE2 supplementation induced vtg expression but returned to basal levels by 3 months post-treatment. Interestingly, gilthead seabream peritoneal leukocytes express the genes encoding for the nuclear estrogen receptor α and the G protein-coupled estrogen receptor 1 and the dietary intake of EE2 induced these expression. Moreover, EE2 induced an inflammatory response in the peritoneal cavity in unvaccinated fish, which was largely maintained for several months after the cessation of the treatment. However, the impact of EE2 in vaccinated fish was rather minor and transient. Taken together, the study provides fresh information about endocrine immune disruption, focusing on peritoneal leukocytes.
Open Access
Histamine and mast cell activator compound 48/80 are safe but inefficient systemic adjuvants for gilthead seabream vaccination
(Elsevier, 2017-02-10) Gómez González N.E., Cabas I.; Montero J., García Alcázar A; Mulero Méndez, Victoriano Francisco; García Ayala, Alfonsa; Biología Celular e Histología
Histamine has a key role in the regulation of inflammatory and innate immune responses in vertebrates. Gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost of great commercial value, was the first fish species shown to possess histamine-containing mast cells (MCs) at mucosal tissues. MCs are highly abundant in the peritoneal exudate of gilthead seabream and compound 48/80 (Co 48/80), often used to promote MC activation and histamine release, is able to promote histamine release from gilthead seabream MCs in vitro and in vivo. The aim of the present study was to analyze the effect of histamine and Co 48/80 on the immune responses of gilthead seabream. For this purpose, histamine and Co 48/80 were intraperitoneally injected alone or combined with 109 heat-killed Vibrio anguillarum cells and their effects on head kidney and peritoneal exudate were analyzed. The results indicated that although histamine and Co 48/80 were both able to alter the percentage of peritoneal exudate and head kidney immune cell types, only Co 48/80 increased reactive oxygen species production by peritoneal leukocytes. In addition, histamine, but not Co 48/80, was able to slightly impair the humoral adaptive immune response, i.e. production of specific IgM to V. anguillarum. Notably, both histamine and Co 48/80 reduced the expression of the gene encoding histamine receptor H2 in peritoneal exudate leukocytes. These results show for the first time in fish that although systemic administration of histamine and Co 48/80 is safe, neither compound can be regarded as an efficient adjuvant for gilthead seabream vaccination.
Restricted
Characterization of macrophages from the bony fish gilthead seabream using an antibody against the macrophage colony-stimulating factor receptor
(Elsevier, 2008-04-07) Mulero Méndez, Iván; Meseguer Peñalver, J.; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
Two major professional phagocyte populations have been described in fish, namely granulocytes and monocytes/macrophages. Although the distribution and localization of macrophages have been documented in several teleost species using mainly light and/or electron microscopy, the lack of appropriate markers for these cells has hampered our in-depth knowledge of their biology. We report here the generation of a monospecific rabbit polyclonal antibody against the gilthead seabream macrophage colony-stimulating factor receptor (Mcsfr), which is an excellent marker of macrophages in mammals and the zebrafish. The anti-Mcsfr has been found to be very useful in immunohistochemistry (IHC) to specifically immunostain the purified macrophages (adherent cells) obtained from the head-kidney as well as different cell populations in paraffin-embedded organs, including the head-kidney, spleen, thymus, gills and liver. Unexpectedly, however, no Mcsfr immunoreactive (Mcsfr+) cells were observed in the brain and intestine of the gilthead seabream. We also show that the distribution of Mcsfr+ cells in the head-kidney and the spleen is unaltered following infection with the fish pathogenic bacterium Vibrio anguillarum and that the Il1b-producing cells in these two organs after infection are exclusively acidophilic granulocytes. Finally, as the epitope recognized by the anti-Mcsfr is well conserved, we illustrate the potential usefulness of this antibody in other teleost species, such as the European seabass.
Open Access
Comunicaciones Orales.-Effects of Endocrine Disruptors Chemicals on the immune response of gilthead seabream (Sparus aurata L.)
(2020-05-06) Rodenas Bleda, María del Carmen; García-Alcázar, A.; Meseguer, J.; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Facultades, Departamentos, Servicios y Escuelas::Departamentos de la UMU::Biología Celular e Histología
ABSTRACT Endocrine disruptors chemicals (EDCs) exert their effects via agonistic/antagonistic interactions with hormone receptors or by interfering with the normal synthesis, transport, metabolism, and secretion of endogenous hormones [1]. EDCs, from the pharmaceutical industry, such as 17α-ethinylestradiol (EE2) and tamoxifen (Tmx,), have been founded in the aquatic environment. EE2 is used in most oral contraceptive pills and in hormone replacement therapy, while Tmx is an estrogen receptor ligand used in cancer therapy. This study aims to investigate the effects of the dietary intake of EE2 (5 μg/g food) or Tmx (100 μg/g food) on the immune response of gilthead seabream species, a marine protandrous teleost, with a great commercial value in the Mediterranean area.
Open Access
Acidophilic granulocytes of the marine fish gilthead seabream (Sparus aurata L.) produce interleukin-1b following infection with Vibrio anguillarum
(Springer, 2004) Chaves-Pozo, Elena; García-Castillo, Jesús; Meseguer, José; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Pelegrín Vivancos, Pablo; Bioquímica y Biología Molecular B e Inmunología
The fish immune response to Gram-negative bacteria is poorly understood. In this study, we use a monoclonal antibody (mAb) specific to acidophilic granulocytes from the marine fish gilthead seabream (Sparus aurata L.), together with an antiserum specific to interleukin-1b (IL-1b) from this species, in order to investigate whether these cells are involved in the immune response against the pathogenic bacterium Vibrio anguillarum and, in particular, in the production of the pro-inflammatory cytokine IL-1b. We found that gilthead seabream head- kidney, peritoneal exudate and peripheral blood leukocytes accumulated proIL-1b intracellularly when challenged in vitro with V. anguillarum, whereas only peritoneal exudate and blood leukocytes were able to accumulate proIL-1b following infection. Importantly, the blood leukocytes from infected animals that accumulated proIL-1b were shown to be the acidophilic granulocytes. A rapid mobilization of such cells from the head-kidney to the site of inflammation following infection with V. anguillarum was also observed.
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Specific and non-overlapping functions of testosterone and 11-ketotestosterone in the regulation of professional phagocyte responses in the teleost fish gilthead seabream
(Elsevier, 2012-09-06) Águila Martínez, Sonia; Castillo Briceño, P.; Sánchez, M.; Cabas, Isabel; García Alcázar, Alicia; Meseguer, José; Mulero Méndez, Victoriano Francisco; García Ayala, Alfonsa; Biología Celular e Histología; ; Medicina
Sex hormones, both estrogens and androgens, have a strong impact on immunity in mammals. In fish, the role of androgens in immunity has received little attention and contradictory conclusions have been obtained. However, it is well known that sex steroids are involved in fish growth, osmoregulation and gonad remodelation. In this study, we examine the in vitro effects of testosterone and 11-ketotestosterone, the two main fish androgens, on the professional phagocytes of the teleost fish gilthead seabream (Sparus aurata L.). Although both testosterone and 11-ketotestosterone failed to modulate the respiratory burst of seabream phagocytes, testosterone but not 11-ketotestosterone was able to increase the phagocytic ability of non-activated phagocytes. Curiously, 11-ketotestosterone was more powerful than testosterone at inducing the expression of its own receptor, namely androgen receptor b (ARb), in acidophilic granulocytes (AGs), but none of them affected the basal ARb expression levels in macrophages (MØ). Furthermore, although physiological concentrations of testosterone exerted a pro-inflammatory effect on both AGs and MØs, 11-ketotestosterone showed an anti-inflammatory effect in AGs and a strong pro-inflammatory effect in MØs. Interestingly, both androgens modulated the expression of toll-like receptors in these two immune cell types, suggesting that androgens might regulate the sensitivity of phagocytes to pathogens and damage signals. Testosterone and 11-ketotestosterone have a competitive effect, at least, on the modulation of the expression of some genes. Therefore, our results show for the first time a non-overlapping role for testosterone and 11-ketotestosterone in the regulation of professional phagocyte functions in fish.