Effect of the earthquake-tsunami (Chile, 2010) on toxic metal content in the Chilean abalone mollusc Concholepas concholepas.

Toxic metal content were measured in samples of mollusc Concholepas concholepas obtained from the Chilean coast. Samples were collected during two periods, one before and one after the earthquake-tsunami which occurred in the Maule Region, Chile, February 27th, 2010 as a result of an earthquake with a magnitude of 8.8. Quantification of toxic metals in samples of hepatopancreas and muscle tissue was performed using flame atomic absorption spectroscopy. The analytical methodology was validated with certified reference material. The content means measured in muscle tissue for January 2010 were Cu: 10.3; Cr: 0.7; Cd: < 0.1; Mn: 3.6 and Zn: 38.6 µg g-1 (dry weight). For October 2014, the means were Cu: 8; Cr: 2.4; Cd: < 0.1; Mn: 5.6 and Zn: 45.4 µg g-1 (dry weight). In hepatopancreas tissue, the content means were Cu: 14.8; Cr: 2.4; Cd: 246.2; Mn: 4.4 and Zn: 1552.9 µg g-1 (dry weight). For October 2014, the means were Cu: 53.7; Cr: 3.5; Cd: 118; Mn: 13.4 and Zn: 1352.3 µg g-1 (dry weight). Cd, Cr, Cu, Mn and Zn content in the samples of hepatopancreas were generally higher than those recorded in muscle tissue however they were not always statistically significant. Statistical analysis showed that Cu and Mn content in the post-tsunami period increased in the hepatopancreas tissue. The concentrations of Cd, Cr, Cu, Mn, and Zn measured in the muscular tissue (edible part) of the species Conchalepas concholepas, were lower than the maximum contents allowed by the current legislation (FAO/WHO, 2004; EU, 2001) and its consumption is not a risk to human health.

Human erythrocytes and neuroblastoma cells are in vitro affected by sodium orthovanadate.

Research on biological influence of vanadium has gained major importance because it exerts potent toxic, mutagenic, and genotoxic effects on a wide variety of biological systems. However, hematological toxicity is one of the less studied effects. The lack of information on this issue prompted us to study the structural effects induced on the human erythrocyte membrane by vanadium (V). Sodium orthovanadate was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence in order that orthovanadate interacted with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies it was observed that morphological changes on human erythrocytes were induced; b) fluorescence spectroscopy experiments in isolated unsealed human erythrocyte membranes (IUM) showed that an increase in the molecular dynamics and/or water content at the shallow depth of the lipids glycerol backbone at concentrations as low as 50µM was produced; c) X-ray diffraction studies showed that orthovanadate 0.25-1mM range induced increasing structural perturbation to DMPE; d) somewhat similar effects were observed by differential scanning calorimetry (DSC) with the exception of the fact that DMPC pretransition was shown to be affected; and e) fluorescence spectroscopy experiments performed in DMPC large unilamellar vesicles (LUV) showed that at very low concentrations induced changes in DPH fluorescence anisotropy at 18°C. Additional experiments were performed in mice cholinergic neuroblastoma SN56 cells; a statistically significant decrease of cell viability was observed on orthovanadate in low or moderate concentrations.

Fish cell cultures as in vitro models of inflammatory responses elicited by immunostimulants. Expression of regulatory genes of the innate immune response.

We report the differential expression of various genes related to the regulation of the innate immune responses, including pro-inflammatory (IL-1ß1, IL-8, TNF-α1, TNF-α2) and immune-suppressing (IL-10) cytokines, interferon-induced Mx-1 protein, enzymes regulating nitric oxide (inducible nitric oxide synthase, arginase-2) and eicosanoid (COX-2) production, and Toll-like pathogen pattern-recognition receptors TLR-3, TLR-5 and TLR-9, in two lympho-haematopoietic stromal cell lines derived from the spleen (trout splenic stroma, TSS) and the pronephros (trout pronephric stroma-2, TPS-2) of rainbow trout (Oncorhynchus mykiss), as well as in primary cultures of rainbow trout head kidney macrophages, after their exposure to the well-known immunostimulants LPS, levamisole and poly I:C. Although there were differences in the responses between the two stromal cell lines, using reverse transcription followed by real time polymerase chain reaction (RT-qPCR) we demonstrated that exposure to the immunostimulants, particularly poly I:C and LPS, resulted in significant changes in the expression of the immunoregulatory genes in the two stromal cell lines in many cases their responses resembling in fold change magnitudes and in response profiles to those observed in the primary macrophage cultures. Exposure to poly I:C and, with lower fold change values, to LPS produced upregulation of the pro- (IL-1ß, IL-8, TNF-α) and anti-inflammatory (IL-10) cytokine genes, as well as of the Mx-1 gene. Furthermore, the immunostimulation elicited the upregulation of COX-2, iNOS and arginase-2 genes in the cell lines. Likewise, the TSS and TPS-2 cell lines significantly upregulated the expression of TLR-3, TLR-5 and TLR-9 genes after exposure to the immunostimulants, thus explaining the ability of the stromal cells to recognise and respond to the immunostimulants. Such results give support to an important role of lympho-haematopoietic stromal cells in the development and control of pro-inflammatory responses in fish. The upregulation of genes of pro-inflammatory cytokines and of mediators of the innate immune responses correlates well with the previously demonstrated functional capacities, including phagocytosis, microbicidal activity and NO production, exhibited by the TSS and TPS-2 stromal cell lines when exposed to the same immunostimulants. On the other hand, the expression of immunosuppressing genes (IL-10, COX-2 and arginase-2) demonstrate that the lympho-haematopoietic stromal cells are also able to contribute to the control of inflammatory responses. This study reinforce the possibility of using histotypic cell cultures, as those formed by the TSS and TPS-2 cell lines, formed by heterogeneous cell populations that partially replicates the cell-cell and cell-extracellular matrix interactions, to develop cost-effective and repetitive in vitro systems for the screening of immunostimulant candidates for aquaculture, as they are able to replicate in vitro immune regulatory networks occurring in vivo.

Fish cell cultures as in vitro models of pro-inflammatory responses elicited by immunostimulants.

We have tested the elicitation of innate defence-related responses in two stromal cell lines derived from the spleen (trout splenic stroma, TSS) and the pronephros (trout pronephric stroma-2, TPS-2) of rainbow trout (Oncorhynchus mykiss) after they were exposed to different concentrations of lipopolysaccharide (LPS), levamisole, or polyinosinic polycytidylic acid (poly-I:C). For comparison, cultures of rainbow trout head kidney macrophages were also included in the study, and the effect of the immunostimulants on the phagocytic activity, the intracellular and extracellular reactive oxygen species and nitric oxide production were assayed. Although the responses varied depending upon the concentration of the immunostimulants and the particular cell line, our results demonstrate that those activities were enhanced in the TSS and TPS-2 cell lines after exposure to any of the immunostimulants. These results indicate that the stromal cells of the main lympho-haemopoietic organs of O. mykiss develop innate defence responses, which are enhanced by well-known immunostimulants. In addition, such enhancement of the defence responses in the TSS and TPS-2 cell lines could be also elicited when they were exposed to conditioned supernatants from levamisole- or poly I:C-stimulated HK macrophage cultures, thus demonstrating that the haemopoietic stromal cells respond to macrophage-derived factors. Moreover, we demonstrate that the stromal cell lines constitutively expressed the Toll-like receptors TLR3, TLR5 and TLR9 genes. The results are discussed considering the role of the lympho-haemopoietic stromal cells in the innate immune responses, and the possibility of using histiotypic cell cultures of non-leucocyte cells of the haemopoietic organs to develop in vitro methods to select new immunostimulant candidates for aquaculture.

Is eating wild rainbow trout safe? The effects of different land-uses on heavy metals content in Chile.

Cu, Mn, Fe, Zn, Cd and Pb levels were measured in liver and muscle samples of Rainbow Trout Oncorhynchus mykiss collected from three watersheds with different land-uses: native forest, exotic plantation, and agriculture in Chile, during January, April, July, and October 2012. Cd and Pb levels were not detected in the liver and muscle, probably since they are under the detection limits. Higher metal concentrations (liver-muscle tissues) were detected in samples from agriculture and exotic plantation streams, whereas trout from native forest streams had lower metal concentrations. Higher metal concentrations were detected in liver tissue compared to muscle tissue, and both negatively correlated to the length and weight of the fish. This suggest the liver had higher ability to accumulate Cu, Mn, Fe and Zn compared to muscle tissue. The concentration range of Fe and Zn recorded in the muscle are within the range reported by other authors, whereas Mn and Cu concentrations are higher than reported in the literature. However, at all sites the concentration of selected metals were below the limits permitted by current legislation (FAO), and therefore did not put the human population at risk, suggesting that is eating wild rainbow trout safe in Chile.

Simulated FDG-PET studies for the assessment of SUV quantification methods.

AIM: To study in detail the accuracy and repeatability of three commonly used methods for SUV estimation in solitary pulmonary nodules. MATERIAL AND METHODS: We have designed a realistic framework based on simulated FDG-PET acquisitions from an anthropomorphic activity model that included solitary pulmonary nodules (different sizes) of well-known SUV. This framework enables us to compare the SUV values obtained from the reconstructed PET images with the real SUV values. Three commonly used methods (SUVmax, SUVmean and SUV50) were used to estimate the tumor activity. RESULTS: Our results showed the tumor activity was overestimated using SUVmax and clearly subestimated using SUVmean. Instead, the quantification of SUV50 showed great agreement with the simulated tumor activity and only slight subestimation was found for very small lesions. On the other hand, SUVmean showed better performance than SUV50 in terms of repeatability, providing variabilities below 5% for all tumor sizes and for injected doses as low as 111 MBq. CONCLUSIONS: Our findings showed that SUV50 provided better performance for estimating accurately tumor SUV values in pulmonary nodules, but SUVmean showed better results in terms of repeatability.

Dolor postoperatorio en cirugía de reparación del manguito rotador mini abierto y artroscópico bajo protocolo de analgesia multimodal / Postoperative pain after mini open and arthroscopic rotator cuff repair surgery under multimodal analgesia protocol

Introducción La cirugía de manguito rotador ha mejorado los síntomas y la función asociada a las lesiones que no han respondido al manejo conservador, sin embargo, puede presentarse dolor severo e incomodidad en el postoperatorio requiriendo una adecuada analgesia. Se busca evaluar el dolor durante el postoperatorio de acuerdo a la técnica utilizada: artroscópica o mini abierta bajo el protocolo de analgesia multimodal. Materiales y métodos Fueron incluidos 60 pacientes en el estudio, de los cuales se llevaron 24 pacientes a cirugía mini abierta y 36 pacientes a cirugía artroscópica entre mayo 2017 y junio 2018. Se realizo seguimiento hasta el primer mes postoperatorio. El desenlace primario es el dolor de acuerdo a la escala visual análoga (EVA) en los diferentes grupos de acuerdo a los diferentes momentos del postoperatorio. Resultados La mediana de dolor postoperatorio fue menor en los pacientes llevados a cirugía mini-abierta con una diferencia estadísticamente significativa en la primera (0 vs 4 (p = 0.001)), segunda hora de postoperatorio (0 vs 1 (p=0.016)) y en el control al mes postoperatorio (3 vs 5 (p=0.043)). Conclusión El dolor en el postoperatorio es menor para el grupo de cirugía mini abierta, con diferencia estadísticamente significativa en las dos primeras horas del postoperatorio y al mes de la cirugía. Estos resultados podrían atribuirse a la inflamación del hombro dada por la hidratación de los tejidos con la artroscopia y también por que las lesiones eran de mayor tamaño en el grupo de cirugía artroscópica. Nivel de Evidencia: II

Background Rotator cuff surgery improve symptoms and function in patients who have not responded to a conservative treatment. However, pain can be severe and uncomfortable after surgery requiring adequate analgesia. We aimed to evaluate pain during the postoperative period after rotator cuff surgery in two different groups: arthroscopic and mini open surgical repair both under a multimodal analgesia protocol. Methods Sixty patients were included in the study, 24 patients underwent mini-open surgery and 36 patients underwent arthroscopic surgery between May 2017 and June 2018. Final follow-up was done on postoperative day 30. The primary outcome is pain according to the visual analog scale (VAS) in the two different groups during different postoperative moments. Results Median postoperative pain was lower in patients undergoing mini-open surgery than in patients undergoing arthroscopic surgery on the first postoperative hour (0 vs 4 (p=0.001)), during the second postoperative hour (0 vs 1 (p=0.016)) and at final follow up (3 vs 5 (p=0.043)). Conclusion Pain during the postoperative period was lower for patients undergoing mini-open surgery, with a statistically significant difference during the first two postoperative hours and at final follow up. These results can be attributed to the local inflammation of the shoulder caused by the hydration of the tissues with arthroscopy surgery and bigger size tears in the group of arthroscopy surgery. Evidence Level: II.

The in vitro infection of the hematopoietic stroma of trout kidney by hemorrhagic septicemia rhabdovirus.

Viral hemorrhagic septicaemia virus (VHSV) infected the hematopoietic stromal cells (7,8) derived from pronephritic tissue of the rainbow trout, Oncorhynchuss mykiss, W., at their ninth passage in vitro. Viral infection resulted in the development of lytic cytopathic effects on confluent in vitro tridimensional network stromal cell cultures. Replication of VHSV in the stromal cell cultures was demonstrated by the increase in infectivity by epithelioma papulosum cyprini (EPC) cell culture assays and by the increase of the nucleoprotein antigen of VHSV by ELISA. By using anti-VHSV monoclonal antibodies (MAbs), flow cytometry studies demonstrated that only the infected stromal cells contained cytoplasmic viral antigens. The lytic infection of trout hematopoietic stromal cells in vitro could be relevant to the hemorrhagic pathology seen in the kidney of fish infected with VHSV.

Correlation between production of acyl homoserine lactones and proteases in an Aeromonas hydrophila aroA live vaccine.

Aeromonas hydrophila is a pathogen that causes disease in a wide range of homeothermic and poikilothermic hosts due to its multifactorial virulence. We have previously described the characterisation and use of an auxotrophic aroA mutant of the A. hydrophila AG2 strain as a live attenuated vaccine against A. hydrophila infections in rainbow trout (Oncorhynchus mykiss). In this study we report the expression of extracellular proteolytic activities and of quorum-sensing molecules by this mutant grown under different culture conditions, and in vaccine inocula. The aroA strain expresses extracellular proteases efficiently during in vitro growth and this ability is retained in vaccine inocula that were prepared by washing the bacterial cultures and resuspending the cells in phosphate-buffered saline. Since proteases are considered to be major bacterial antigens, the expression of these enzymes in the live attenuated vaccine may contribute to the superior protection afforded by these kind of vaccines. On the other hand, the production of serine- and metalloprotease activities in A. hydrophila has been described as controlled in a cell density-dependent fashion, through a mechanism known as quorum sensing. A microtiter method was developed that allowed correlation of the production of quorum-sensing molecules and of proteases produced by the aroA strain during in vitro growth and in the vaccine inocula. The production of both products was related to the type of culture medium and conditions used to grow the aroA mutant, whereas there was no correlation between the concentration of acyl homoserine lactones and protease production.

Alterations in the peripheral lymphoid organs and differential leukocyte counts in Saprolegnia-infected brown trout, Salmo trutta fario.

The number of circulating leukocytes and the structure of splenic and renal lymphoid tissue were comparatively analysed in healthy and Saprolegnia-infected wild brown trout, Salmo trutta fario. Sick trout showed lymphopenia, mainly due to decreased numbers of circulating small lymphocytes, and heterophilia. The splenic and renal lymphoid tissue of infected trout exhibited similar changes, consisting of cellular depletion, lymphoid cell degeneration, and vascular alterations with blood vessel enlargement and hypertrophy of sinusoidal endothelial cells. Furthermore, the endothelial cells in the spleen and kidney of the infected trout contained cytoplasmic vesicles filled with material of possible fungal origin. The absence of a reticular sheath was also evident in the splenic ellipsoids. These results suggest some immunodepression in Saprolegnia-infected trout which might favour the course of the disease.

Reporte de caso de enfermedad mano-pie-boca con lesiones mucocutáneas que evolucionan a necrosis / A case report of hand, foot, and mouth disease with necrotizing mucocutaneous lesions

La enfermedad de mano-pie-boca es una patología originada en la mayoría de los casos por el virus coxsackie A tipo 16, aunque también puede ser ocasionada por otras cepas de la familia de los coxsackievirus. Dicho virus se propaga principalmente por vía fecal oral y, en menor proporción, por secreciones. Se presenta principalmente en verano, siendo frecuente en niños menores de 10 años. Dentro de dicha enfermedad las lesiones mucocutáneas que evolucionen en necrosis son poco frecuentes, constituyéndose en una complicación severa que requiere hospitalización. En el presente artículo se reporta un caso con diagnóstico de enfermedad mano-pie-boca, que evolucionó hacia lesiones mucocutáneas necróticas, mostrando una respuesta favorable a una terapia de soporte de aciclovir, líquidos y electrolitos.

In most cases, the cause of hand, foot, and mouth disease (HFMD) is coxsackievirus A type 16. The infection can also be caused by other strains of coxsackievirus, spreading mainly by the oral-fecal route, while it is less likely to be transmitted through secretions. HFMD occurs mainly in summer and is more common in children under ten. Skin lesions develop during the disease but rarely become necrotic. When present, they are a severe complication requiring hospitalization. This paper reports the case of a patient with HFMD who developed necrotic mucocutaneous lesions that responded favorably to intravenous acyclovir, fluids, and electrolyte support therapy.

Detection of novel Human papilloma virus type 82 in laryngeal cancer: case report.

Human papilloma virus infection is thought to play a role in laryngeal carcinogenesis; the variable association reported in literature may be due to wide range of HPV genotypes. We report the case of a 51-year-old man affected by laryngeal squamous cell carcinoma; analysis of DNA extracted by cancer cells by an innovative molecular virology assay (INNO-LiPA HPV Genotyping Extra) showed the presence of two high-risk HPV genotypes, HPV-73 and -82. Immunohistochemical examination confirmed positivity for both capsid protein and viral oncogenic protein E7. Such association has never been reported in literature so far, and a brief discussion on the importance of assessing HPV status in laryngeal cancer is provided.

Estudios simulados de PET FDG para la evaluación de diferentes métodos de cuantificación de SUV / Simulated FDG-PET studies for the assessment of SUV quantification methods

Objetivo. estudiar en detalle la precisión y la repetitividad de tres métodos de uso común en la estimación del SUV de nódulos pulmonares solitarios. Material y métodos. hemos diseñado una metodología de trabajo basada en la simulación de adquisiciones de estudios PET-FDG a partir de modelos antropomórficos de actividad, que incluyen nódulos pulmonares de diferente tamaño y valor de SUV conocido. Esta metodología nos permite comparar el SUV estimado a partir de la imagen PET con el SUV teórico. La actividad del tumor fue estimada mediante tres métodos conocidos: SUVmax, SUVmean y SUV50. Resultados. nuestros resultados muestran que, por un lado SUVmax sobreestima la actividad en el tumor, mientras SUV50 subestima el valor de actividad de manera muy significa. En cambio, la cuantificación de SUV50 mostró un buen acuerdo con los valores de SUV teóricos o simulados, y únicamente mostró una ligera subestimación para lesiones muy pequeñas. Por otro lado, SUVmean mostró un mejor comportamiento que SUV50 en términos de repetitividad, proporcionando variabilidades por debajo del 5% para todos los tamaños de lesión y para dosis inyectadas tan bajas como 111 MBq. Conclusiones. nuestros hallazgos mostraron que SUV50 proporciona el mejor comportamiento para estimar la actividad en nódulos pulmonares, pero SUVmean mostró mejores resultados en términos de repetitividad (AU)

Aim. To study in detail the accuracy and repeatability of three commonly used methods for SUV estimation in solitary pulmonary nodules. Material and methods. We have designed a realistic framework based on simulated FDG-PET acquisitions from an anthropomorphic activity model that included solitary pulmonary nodules (different sizes) of well-known SUV. This framework enables us to compare the SUV values obtained from the reconstructed PET images with the real SUV values. Three commonly used methods (SUVmax, SUVmean and SUV50) were used to estimate the tumor activity. Results. Our results showed the tumor activity was overestimated using SUVmax and clearly subestimated using SUVmean. Instead, the quantification of SUV50 showed great agreement with the simulated tumor activity and only slight subestimation was found for very small lesions. On the other hand, SUVmean showed better performance than SUV50 in terms of repeatability, providing variabilities below 5% for all tumor sizes and for injected doses as low as 111 MBq. Conclusions. Our findings showed that SUV50 provided better performance for estimating accurately tumor SUV values in pulmonary nodules, but SUVmean showed better results in terms of repeatability (AU)

Modulation of the immune response to an Aeromonas hydrophila aroA live vaccine in rainbow trout: effect of culture media on the humoral immune response and complement consumption.

The Aeromonas hydrophila aroA is an attenuated strain that has been assessed as a live vaccine in rainbow trout, Oncorhynchus mykiss. In this study the effects of different culture media used to grow the strain on its survival after in vitro exposure to rainbow trout serum, and on its immunogenicity in rainbow trout were compared. Four culture media were tested: Luria broth (LB), Luria broth with 0.25% glucose, trypticase soy broth (TSB), and brain-heart infusion broth (BHIB). Bacteria grown in culture media with glucose (TSB, BHIB and LB with 0.25% glucose) showed reduced complement consumption and a lower serum susceptibility. O. mykiss vaccinated with inocula prepared with BHIB- and LB-grown aroA cells resuspended in phosphate-buffered saline (PBS) showed higher and longer-lasting serum agglutinating antibody titres than those vaccinated with TSB-grown bacteria. Thus, a direct relationship between serum resistance and immunogenicity could not be established, but BHIB and LB culture media were the most effective in increasing the immunogenicity of the A. hydrophila aroA vaccine.

Spontaneous in vitro angiogenesis in a trout pronephric stromal cell line (TPS), and in TPS-haemopoietic co-cultures.

This study describes angiogenic processes taking place in the in vitro micro-environment of a trout pronephric stroma cell line (TPS) under specific culture conditions, in which fetal calf serum, horse serum and hydrocortisone-sodium-21-hemisuccinate were used as supplements to the culture medium. When TPS cultures were kept in the same flask, i.e. without passages, for longer than 7 months, epithelioid cells differentiated into endothelial cells. Early stages of such differentiation were characterised by the presence of intracellular tubular vacuoles in clusters of neighbouring epithelioid cells. Subsequently, the endothelial cells reorganised and gave rise to microvascular structures, which branched over and into the TPS multilayers. The lining cells of the microvasculature showed typical characteristics of endothelial cells, such as ovoid or cubical shape, bundles of microfilaments and microtubules, and particularly numerous small vesicles at the apical pole, some of them fused to the plasma membrane. Similar angiogenic processes were also observed in long-term haemopoietic co-cultures formed by the TPS cell line and trout pronephric cell suspensions. Developing haemopoietic cells were observed at the basal pole of the vessels, and in the vascular lumen, where some immature cells appeared in close contact with the endothelium. These results indicate that the TPS cell line contains endothelial cell precursors, which are able to differentiate under certain culture conditions.

Post-hatching development of the thymic epithelial cells in the rainbow trout Salmo gairdneri: an ultrastructural study.

This study reports the ultrastructure of subpopulations of epithelial cells of the thymic parenchyma during the post-hatching development of the rainbow trout, Salmo gairdner, kept at 14 degrees C. At hatching, the thymus contained a small number of medium and large thymocytes interspersed among three different types of epithelial cells: (1) epithelial cells adjacent to the connective tissue capsule; (2) ramified dark epithelial cells with electron-dense cytoplasm; and (3) pale electron-lucent epithelial cells displaying secretory-like features. All these cells types were anchored to one another by desmosomes and had apparently differentiated from the pharyngeal epithelium. At 4 days after hatching, the thymus enlarged, and numerous gaps occurred between the cell processes of contiguous epithelial cells adjacent to the capsular connective tissue. In 21-day-old trout, thymic trabeculae developed carrying blood vessels, and a subcapsular zone became evident containing lymphoblasts and large subcapsular epithelial cells. In 30-day-old trout, an outer thymic zone developed consisting of spindle-shaped epithelial cells which formed a dense network. At this stage, scattered cystic cells, which apparently differentiated from the pale epithelial cells, were present.

Tissue distribution and structure of barrier cells in the hematopoietic and lymphoid organs of salmonids.

BACKGROUND: Barrier cells have been recognized as a discrete group of fibroblastic- or myofibroblastic-like cells located in the lymphoid and hematopoietic organs of mammals. This paper reports the results of a morphological study of the main lymphoid organs of three salmonid species, in which cells structurally similar to the mammalian barrier cells were observed in healthy animals. METHODS: The spleen, kidney, and thymus of fingerlings of rainbow trout, Oncorhynchus mykiss, and Coho salmon, Oncorhynchus kisutch, and of adult brown trout, Salmo trutta fario, were processed for electron microscopy study using various fixation methods. Semithin sections were used for the Periodic Acid-Schiff (PAS) staining technique, and for the demonstration of the endogenous peroxidase activity. RESULTS: The kidney and spleen of all the species contained a variable, but usually low, number of electron-dense, elongated, and branched cells, ultrastructurally similar to the mammalian barrier cells. They also occurred in the thymus of some brown trout and Coho salmon, but not of rainbow trout. The electron density of this cell type was present after the various types of fixation procedures. They show numerous ribosomes, well-developed secretory organelles, electron-clear vesicles, large granules, and microfilaments. In all the salmonid species, barriers cells were positive for PAS staining and for endogenous peroxidase activity. A small number of barrier cells were in mitosis. In the different organs barrier cells appeared as isolated cells, or forming syncytial networks. They were found lining the blood sinusoids of the splenic red pulp and of the renal hematopoietic tissue, in the periellipsoidal sheaths, and closely associated with erythropoietic and plasmacytopoietic foci. CONCLUSIONS: Our results demonstrate the presence of cells closely resembling mammalian barrier cells in the hematopoietic and lymphoid organs of salmonids. The structure and tissue distribution of the salmonid barrier cells are discussed in relation to the functional roles described for this cell type in mammals.

Molecular characterization of the Aeromonas hydrophila aroA gene and potential use of an auxotrophic aroA mutant as a live attenuated vaccine.

The aroA gene of Aeromonas hydrophila SO2/2, encoding 5-enolpyruvylshikimate 3-phosphate synthase, was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829, and the nucleotide sequence was determined. The nucleotide sequence of the A. hydrophila aroA gene encoded a protein of 440 amino acids which showed a high degree of homology to other bacterial AroA proteins. To obtain an effective attenuated live vaccine against A. hydrophila infections in fish, the aroA gene was inactivated by the insertion of a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of A. hydrophila AG2 by means of the suicide vector pSUP202. The A. hydrophila mutant AG2 aroA::Ka(r) was highly attenuated when inoculated intraperitoneally into a rainbow trout, with a 50% lethal dose of >2 x 10(8) CFU. The mutants were not recoverable from the internal organs after 48 h postinoculation. Immunohistochemical studies demonstrated that immunopositive materials, but not whole cells, reacting with a polyclonal antiserum against A. hydrophila were present in the kidney and spleen 9 days postinjection. Vaccination of rainbow trout with the AroA mutant as a live vaccine conferred significant protection against the wild-type strain of A. hydrophila.

In vitro and in situ characterization of fish thymic nurse cells.

We present an enzyme- and immuno-cytochemical, and ultrastructural characterization of trout thymic nurse cells (TNCs). Our data suggest that isolated trout thymic multicellular complexes are epithelial cells with acidic compartments that may be involved in the processing of antigens and in the generation of the MHC-II proteins that these cell express, and also that isolated TNCs are the in vitro equivalent of the pale and intermediate electronlucent epithelial cells located in the inner zone of the trout thymus, constituting indirect evidence of the phylogenetical relationships of the inner zone of the teleost thymus with the thymic cortex of higher vertebrates.

Enzyme- and immuno-histochemical study of the thymic stroma in the rainbow trout, Salmo gairdneri, Richardson.

Owing to the lack of data about thymic non-lymphoid cells in fish we decided to perform a histochemical characterization of these cells in order to ascertain their relationships to other thymic components. In the present study we analyze the enzyme-histochemical patterns for acid phosphatase, alkaline phosphatase, non-specific sigma-naphthyl acetate esterase and 5' nucleotidase activities, as well as the presence of keratin demonstrated by immunoperoxidase staining, in the non-lymphoid cell populations of the thymus of the rainbow trout, Salmo gairdneri. According to their location in the organ, morphology and histochemical reactivities, we were able to define seven different subpopulations of keratin-positive epithelial cells: 1) Epithelial cells limiting with the capsular and septal connective tissues; 2) Subcapsular epithelial cells; 3) Stellate epithelial cells of the inner thymic zone; 4) Large, ovoid epithelial cells of the inner thymic zone; 5) Acidophilic epithelial cells of the outer thymic zone; 6) Cystic cells; and 7) Goblet cells. The significance of the heterogeneity of the epithelial cell (EC) population, its specific distribution in the organ, which apparently conforms distinct cell microenvironments, as well as the possible phylogenetical relationships between these microenvironments and the classical cortex and medulla of the mammalian thymus, are discussed.