In vivo toxicity of the ribosome-inactivating lectin ebulin f in elderly mice.

Ebulin f is a ribosome-inactivating protein (RIP) present in green fruits of the dwarf elder (Sambucus ebulus L). Since dwarf elder fruits are used for food and as a medicine, we assessed the study of toxicological effects and safety of ebulin f in elderly mice, comparing these results with those reported in young animals and with other RIPs. Female Swiss mice aged 6 and 12 months of age were intraperitoneally injected with a single dose from 1.4 to 4.5 mg/kg ebulin f. Heart, stomach, intestines, lung, kidney, liver, spleen, pancreas, adrenal gland, uterus, ovary and brain were studied. Histology analysis was carried out by staining with hematoxylin and eosin and Masson's trichrome observed with a light microscope, or apoptosis detection by TUNEL method observed with a confocal laser microscope. Treated animals injected with the lower dose could recover their weights, but after 14 days half of them died. The higher dose caused a progressive loss of body weight leading to death. In the animals of the experimental groups it was found atrophy of Lieberkühn's crypts, pneumonia, nephronal degeneration, myocardial atrophy, centrolobular hepatic necrosis, splenic white pulp necrosis foci and increased rate of apoptosis in the intestines and liver, in which apoptoses were mainly located in the vicinity of the lobular central vein. We conclude that ebulin f affects vital organs in elderly mice.

ACUTE RETINAL DAMAGE AFTER USING A TOXIC PERFLUORO-OCTANE FOR VITREO-RETINAL SURGERY.

PURPOSE: To describe a series of retinal acute toxicity cases with severe visual loss after intraocular use of a toxic perfluoro-octane (PFO). The clinical presentation is described, and the likely causes are analyzed. New biological methods for testing safety of intraocular medical devices are proposed. METHODS: Information regarding a series of eyes suffering acute severe events after intraocular use of a toxic PFO was analyzed. Four types of spectroscopy, nuclear magnetic resonance, and chromatography were used to identify the potential PFO contaminants. Cultures of human retinal pigment epithelial cells (ARPE-19) and porcine neuroretina were used to quantify the toxicity of the suspect PFO lots. RESULTS: Of 117 cases of intraocular toxicity, 96 were considered clearly related to the use of PFO. Fifty-three cases had no light perception, and 97 had no measurable visual acuity. Retinal necrosis (n = 38) and vascular occlusion (n = 33) were the most characteristic findings. Two hydroxyl compounds, perfluorooctanoic acid and dodecafluoro-1-heptanol, and benzene derivatives were identified as the suspected toxic agents. While existing toxicity testing failed, we proposed new tests that demonstrated clear toxicity. CONCLUSION: Protocols to determine cytotoxicity of intraocular medical devices should be revised to assure safety. Acute toxic events should be reported to health authorities and scientific media.

A novel coculture model of porcine central neuroretina explants and retinal pigment epithelium cells.

PURPOSE: To develop and standardize a novel organ culture model using porcine central neuroretina explants and RPE cells separated by a cell culture membrane. METHODS: RPE cells were isolated from porcine eyes, expanded, and seeded on the bottom of cell culture inserts. Neuroretina explants were obtained from the area centralis and cultured alone (controls) on cell culture membranes or supplemented with RPE cells in the same wells but physically separated by the culture membrane. Finally, cellular and tissue specimens were processed for phase contrast, cyto-/histological, and immunochemical evaluation. Neuroretina thickness was also determined. RESULTS: Compared to the neuroretinas cultured alone, the neuroretinas cocultured with RPE cells maintained better tissue structure and cellular organization, displayed better preservation of photoreceptors containing rhodopsin, lower levels of glial fibrillary acidic protein immunoexpression, and preservation of cellular retinaldehyde binding protein both markers of reactive gliosis. Neuroretina thickness was significantly greater in the cocultures. CONCLUSIONS: A coculture model of central porcine neuroretina and RPE cells was successfully developed and standardized. This model mimics a subretinal space and will be useful in studying interactions between the RPE and the neuroretina and to preclinically test potential therapies.

Toxicity of the anti-ribosomal Lectin Ebulin f in lungs and intestines in elderly mice.

All parts of dwarf elder (Sambucus ebulus L.) studied so far contain a ribosome-inactivating protein with lectin activity (ribosome-inactivating lectin; RIL), known as ebulin. Green fruits contain ebulin f, the toxicity of which has been studied in six-week-old mice, where it was found that the intestines were primary targets for it when administered intraperitoneally (i.p.). We performed experiments to assess whether ebulin f administration to six- and 12-month-old mice would trigger higher toxicity than that displayed in six-week-old mice. In the present report, we present evidence indicating that the toxicological effects of ebulin f after its i.p. administration to elderly mice are exerted on the lungs and intestines by an increased rate of apoptosis. We hypothesize that the ebulin f apoptosis-promoting action together with the age-dependent high rate of apoptosis result in an increase in the lectin's toxicity, leading to a higher lethality level.

Elderberries: a source of ribosome-inactivating proteins with lectin activity.

Sambucus (Adoxaceae) species have been used for both food and medicine purposes. Among these, Sambucus nigra L. (black elder), Sambucus ebulus L. (dwarf elder), and Sambucus sieboldiana L. are the most relevant species studied. Their use has been somewhat restricted due to the presence of bioactive proteins or/and low molecular weight compounds whose ingestion could trigger deleterious effects. Over the last few years, the chemical and pharmacological characteristics of Sambucus species have been investigated. Among the proteins present in Sambucus species both type 1, and type 2 ribosome-inactivating proteins (RIPs), and hololectins have been reported. The biological role played by these proteins remains unknown, although they are conjectured to be involved in defending plants against insect predators and viruses. These proteins might have an important impact on the nutritional characteristics and food safety of elderberries. Type 2 RIPs are able to interact with gut cells of insects and mammals triggering a number of specific and mostly unknown cell signals in the gut mucosa that could significantly affect animal physiology. In this paper, we describe all known RIPs that have been isolated to date from Sambucus species, and comment on their antiviral and entomotoxic effects, as well as their potential uses.

Ebulin from dwarf elder (Sambucus ebulus L.): a mini-review.

Sambucus ebulus L. (dwarf elder) is a medicinal plant, the usefulness of which also as food is restricted due to its toxicity. In the last few years, both the chemistry and pharmacology of Sambucus ebulus L. have been investigated. Among the structural and functional proteins present in the plant, sugar-binding proteins (lectins) with or without anti-ribosomal activity and single chain ribosome-inactivating proteins (RIPs) have been isolated. RIPs are enzymes (E.C. 3.2.2.22) that display N-glycosidase activity on the 28S rRNA subunit, leading to the inhibition of protein synthesis by arresting the step of polypeptide chain elongation. The biological role of all these proteins is as yet unknown. The evidence suggests that they could be involved in the defense of the plant against predators and viruses or/and a nitrogen store, with an impact on the nutritional characteristics and food safety. In this mini-review we describe all the isoforms of ebulin that have to date been isolated from dwarf elder, as well as their functional characteristics and potential uses, whilst highlighting concern regarding ebulin toxicity.

Time course modifications in organotypic culture of human neuroretina.

The purpose of this study was to characterize organ culture of human neuroretina and to establish survival and early degeneration patterns of neural and glial cells. Sixteen neuroretina explants were prepared from 2 postmortem eyes of 2 individuals. Four explants were used as fresh retina controls, and 12 were evaluated at 3, 6, and 9 days of culture. Neuroretina explants (5 × 5 mm) were cultured in Transwell(®) dishes with the photoreceptor layer facing the supporting membrane. Culture medium (Neurobasal A-based) was maintained in contact with the membrane beneath the explant. Cryostat and ultrathin sections were prepared for immunohistochemistry and electron microscopy. Neuroretinal modifications were evaluated after toluidine blue staining and after immunostaining for neuronal and glial cell markers. Ultrastructural changes were analyzed by electron microscopy. From 0 to 9 days in culture, there was progressive retinal degeneration, including early pyknosis of photoreceptor nuclei, cellular vacuolization in the ganglion cell layer, decrease of both plexiform layer thicknesses, disruption and truncation of photoreceptor outer segments (OS), and marked reduction in the number of nuclei at both nuclear layers where the cells were less densely packed. At 3 days there was swelling of cone OS with impairment of pedicles, loss of axons and dendrites of horizontal and rod bipolar cells that stained for calbindin (CB) and protein kinase C (PKC-α), respectively. After 9 days, horizontal cells were pyknotic and without terminal tips. There were similar degenerative processes in the outer plexiform layer for rod bipolar cells and loss of axon terminal lateral varicosities in the inner plexiform layer. Glial fibrillary acidic protein (GFAP) staining did not reveal a dramatic increase of gliosis in Müller cells. However, some Müller cells were CB immunoreactive at 6 days of culture. Over 9 days of culture, human neuroretina explants underwent morphological changes in photoreceptors, particularly the OS and axon terminals, and in postsynaptic horizontal and bipolar cells. These early changes, not previously described in cultured human samples, reproduce some celullar modifications after retinal damage. Thus, this model may be suitable to evaluate therapeutic agents during retinal degeneration processes.

MRT letter: a fast and easy method for general fluorescent staining of cultured cells on transparent or opaque supports.

Development of cell-based therapy entails the use of different types of materials as support for cultured cells. Some of these materials are opaque. For a general microscope study of cell cultures prepared on transparent supports, Giemsa stain with bright field microscopy is useful. With opaque supports or scaffolds, epifluorescence microscopy is necessary. The method the authors describe uses eosin Y to stain cytoplasm and DAPI to stain nuclei under fluorescence microscopy. This method provides easy and fast fluorescent staining for a general morphological study of cultured cells on transparent or opaque supports.

Histopathological changes in gerbil liver and kidney after aluminum subchronic intoxication.

Young gerbil livers and kidneys were analyzed by means of light and electron microscope to assess the histopathological changes caused by prolonged systemic aluminum (Al) administration. The experimental group was injected with AlCl3 i.p. for 5 weeks, while litter mates received PBS as sham-injected controls or served as untouched controls. Mortality occurred in 33% of experimental and 12.5% of sham-injected groups. The animals were perfused intracardially with 1% glutaraldehyde plus 1% paraformaldehyde and samples of liver and kidneys were processed for aluminum and iron histochemistry and conventional light- and transmission electron microscopy. White deposits composed of cellular debris appeared on the surface of liver and kidneys and in the mesentery as a consequence of Al treatment. Adherences of Glisson capsule to the diaphragm, as well as scattered small foci of hepatocyte necrosis with non-caseificant microgranulomas and mild portal inflammation, developed in the experimental group. Sham-injected animals also exhibited these granulomas but to a lesser degree. Al deposits were found in experimental animal granulomas and inside macrophages cytoplasm scattered throughout the liver. Iron deposition appeared in pericentral hepatocytes of experimental animals, in granulomas and in portal spaces of the three groups of animals. Ultrastructurally, hepatocytes of experimental animals showed mitochondria hyalinization, disintegration of endoplasmic reticulum and clustering of ribosomes. Phagolysosomes appeared larger and occurred more frequently in both hepatocytes and Kupffer cells of experimental animals. In 2 out of the 6 experimental animals studied, tubular atrophy was present in the renal cortical region, the kidneys of the remaining animals appearing normal. Al and iron were found very occasionally in the kidney parenchyma of experimental animals, while isolated mesangial cells showed iron deposits in a few glomeruli of both experimental and the two groups of control animals.

Elastin-like recombinamers as substrates for retinal pigment epithelial cell growth.

The aim of this study is to investigate the use of elastin-like recombinamers (ELRs) as a substrate that can maintain the growth, phenotype, and functional characteristics of retinal pigment epithelial (RPE) cells efficiently and as a suitable carrier for the transplantation of autologous RPE cells for treatment of age-related macular degeneration (AMD). ELR films containing a bioactive sequence, RGD (ELR-RGD), and one with no specific sequence (ELR-IK) as control, were obtained by solvent-casting onto glass and subsequent cross-linking. ARPE19 cells were seeded on sterilized ELR films as well as on the control surfaces. Cells were analysed after 4, 24, 72, and 120 h to study cell adhesion, proliferation, cell viability, morphology, and specificity by staining with Trypan blue, DAPI, Rhodamin-Phalloidin and RPE65, ZO-1 antibodies and observing under fluorescence as well as electron microscope. ARPE19 cells seeded on both ELR films and controls were 100% viable and maintained their morphology and set of characteristics at the different time points studied. Cell proliferation on ELR-RGD was significantly higher than that found on ELR-IK at all time points, although it was less than the growth rate on polystyrene. ARPE19 cells grow well on ELR-RGD maintaining their phenotype. These results should be extended to further studies with fresh human RPE cells and in vivo studies to determine whether this ELR-RGD matrix could be used as a Bruch's membrane prosthesis and carrier for transplantation of RPE cells in patients suffering with AMD.

Sialic acid-binding dwarf elder four-chain lectin displays nucleic acid N-glycosidase activity.

Sialic acid-binding dwarf elder agglutinin (SEA) present only in rhizomes of the medicinal plant Sambucus ebulus L., was found to be a tetrameric glycoprotein consisting of two covalently-associated dimers of an enzymic A chain with rRNA N-glycosidase activity (EC 3.2.2.22) linked to a B chain with agglutinin properties. The lectin inhibited protein synthesis by a cell-free system and depurinated ribosomes. Cloning of the corresponding gene and molecular modeling of the deduced amino acid sequence demonstrated that SEA has a three-dimensional structure which resembles that reported for other two tetrameric type 2 RIPs from Sambucus (SNAI and SSA). The lectin agglutinated red blood cells and displayed sugar affinity for sialic acid residues apart from d-galactose, binding to the mucin-producing gut goblet cells. Since sialic acid is present in animal cells, especially in epithelial lining gut cells, but not in plants, SEA could play a role in the defense against insect attack. The nucleotide sequence reported in this paper has been submitted to the GenBank nucleotide database under accession number AM981401.

[Interleukin-8 expression in lung tissue during ischemia-reperfusion]. / Estudio de la expresión de interleucina-8 en el tejido pulmonar durante la isquemia-reperfusión.

OBJECTIVE: Local cytokine production is a pathogenic factor in ischemia-reperfusion injury in early graft dysfunction. This study analyzed interleukin 8 (IL-8) messenger RNA (mRNA) expression in lung tissue and the association between IL-8 mRNA levels and interstitial lung changes in an experimental model of warm lung ischemia-reperfusion. MATERIAL AND METHODS: We studied 16 New Zealand rabbits divided into 3 groups: control, ischemia (tissue taken from right lower lobe after 1, 2, or 3 hours of ischemia), and reperfusion (tissue taken from right upper and middle lobes after 1 hour of ischemia and 1, 2, or 3 hours of reperfusion). Expression of IL-8 mRNA was determined by reverse transcription and polymerase chain reaction. Interstitial infiltration by polymorphonuclear neutrophils was determined. The Mann-Whitney U-test was used for statistical comparisons, with P< .05 considered to indicate a significant result. RESULTS: During ischemia, IL-8 mRNA levels were elevated at the end of hour 1 (P=.009) with respect to the control group, but not thereafter. Interstitial changes were minimal. IL-8 mRNA levels during reperfusion were similar to those observed during ischemia, with a slight increase at the end of hour 2. There were no significant differences between hours 1, 2, and 3. Polymorphonuclear neutrophil recruitment occurred at the beginning of reperfusion (P=.014), but no significant differences were observed at hours 2 or 3. Progressive thickening of alveolar septa and edema was documented. CONCLUSIONS: Changes in IL-8 mRNA expression during ischemia precede interstitial infiltration by polymorphonuclear neutrophils during reperfusion, suggesting that the 2 processes are related. Quantification of IL-8 mRNA expression could facilitate early diagnosis of graft dysfunction.

Estudio de la expresión de interleucina-8 en el tejido pulmonar durante la isquemia-reperfusión / Interleukin-8 expression in lung tissue during ischemia­ reperfusion

Objetivo: La generación local de citocinas es un factor patogénico en el daño por isquemia-reperfusión en la disfunción precoz del injerto. Este estudio analiza la expresión en tejido pulmonar de ARN mensajero de interleucina-8 (ARNm de IL-8) y su relación con los cambios intersticiales pulmonares en un modelo experimental de isquemia-reperfusión pulmonar normotérmica. Material y métodos: Se estudiaron 16 conejos de la raza Nueva Zelanda en 3 grupos de estudio: a) basal; b) isquemia (lóbulo inferior derecho tras isquemia de 1, 2 o 3 h), y c) reperfusión (lóbulos superior y medio derechos tras 1 h de isquemia y 1, 2 o 3 h de reperfusión). Se determinó la expresión del ARNm de IL-8 mediante transcripción inversa y reacción en cadena de la polimerasa y estudió la infiltración intersticial por polimorfonucleares (PMN). Para el análisis estadístico se empleó el test de la U de Mann-Whitney aceptando como significativo un valor de p < 0,05. Resultados: Durante el período de isquemia se observó respecto al basal elevación del ARNm de IL-8 al final de la primera hora (p = 0,009), pero no durante el resto del período isquémico. Los cambios intersticiales fueron mínimos. Durante la reperfusión los valores de ARNm de IL-8 fueron semejantes a los observados durante la isquemia, con una ligera elevación al final de segunda hora; no hubo diferencias significativas entre la primera, segunda y tercera horas. Hubo reclutamiento de PMN al inicio de la reperfusión (p = 0,014), sin observarse diferencias significativas en la segunda y tercera horas. Se objetivó un engrosamiento progresivo de los tabiques interalveolares y edema. Conclusiones: Los cambios en la expresión del ARNm de IL-8 durante la isquemia preceden a la infiltración intersticial de PMN durante la reperfusión, lo que señala una relación entre ambos procesos. La cuantificación del ARNm de IL-8 podría ser un procedimiento para el seguimiento diagnóstico de la disfunción precoz del injerto

Objective: Local cytokine production is a pathogenic factor in ischemia­reperfusion injury in early graft dysfunction. This study analyzed interleukin 8 (IL-8) messenger RNA (mRNA) expression in lung tissue and the association between IL-8 mRNA levels and interstitial lung changes in an experimental model of warm lung ischemia­reperfusion. Material and methods: We studied 16 New Zealand rabbits divided into 3 groups: control, ischemia (tissue taken from right lower lobe after 1, 2, or 3 hours of ischemia), and reperfusion (tissue taken from right upper and middle lobes after 1 hour of ischemia and 1, 2, or 3 hours of reperfusion). Expression of IL-8 mRNA was determined by reverse transcription and polymerase chain reaction. Interstitial infiltration by polymorphonuclear neutrophils was determined. The Mann­Whitney U-test was used for statistical comparisons, with P<.05 considered to indicate a significant result. Results: During ischemia, IL-8 mRNA levels were elevated at the end of hour 1 (P=.009) with respect to the control group, but not thereafter. Interstitial changes were minimal. IL-8 mRNA levels during reperfusion were similar to those observed during ischemia, with a slight increase at the end of hour 2. There were no significant differences between hours 1, 2, and 3. Polymorphonuclear neutrophil recruitment occurred at the beginning of reperfusion (P=.014), but no significant differences were observed at hours 2 or 3. Progressive thickening of alveolar septa and edema was documented. Conclusions: Changes in IL-8 mRNA expression during ischemia precede interstitial infiltration by polymorphonuclear neutrophils during reperfusion, suggesting that the 2 processes are related. Quantification of IL-8 mRNA expression could facilitate early diagnosis of graft dysfunction

Targeting a marker of the tumour neovasculature using a novel anti-human CD105-immunotoxin containing the non-toxic type 2 ribosome-inactivating protein nigrin b.

Targeting tumour neovasculature using antibodies to the endothelial receptor CD105 (endoglin), is a potentially useful approach for anti-tumour therapy. We report on the preparation and the cytotoxicity of a novel immunotoxin consisting in the non-toxic type 2 ribosome-inactivating protein (RIP) nigrin b linked to the monoclonal anti-human CD105 (hCD105) antibody 44G4. The immunotoxin kills specifically mouse fibroblasts expressing the biomarker CD105 (L929-hCD105+ cells) with an IC(50) value of 6x10(-10)M while nigrin b does it at 2.4x10(-7)M. Immunofluorescence analysis indicated that the immunotoxin accumulates in a perinuclear region. In contrast, 44G4 showed a specific localization on the cell surface.

Intraretinal immunohistochemistry findings in proliferative vitreoretinopathy with retinal shortening.

UNLABELLED: To report the major intraretinal pathological changes in retinas with proliferative vitreoretinopathy (PVR) and retinal shortening, 13 human retinal samples from postoperative PVR after primary surgery for retinal detachment were immunostained for vimentin, glial fibrillary acidic protein (GFAP), cytokeratins, and CD68. One more sample was studied with electron microscopy. Retinal disorganization, neuronal loss, and gliosis were observed in 12 out of 13 samples, but all 13 were positive for GFAP. Muller cell processes showed different degrees of intermediate filament hyperplasia. CD68-positive cells were present in 11 of 13 retinal samples. CONCLUSION: A gliotic response plays a major role in retinal shortening in PVR. In addition, the presence of macrophage-like cells in retinal tissues suggests a possible role of these cells in the pathogenesis of this variety of PVR.

Histological changes in rat nasal epithelia after unilateral neonatal naris occlusion.

The authors studied the extent of the different epithelia lining the nasal fossae of the albino rat after neonatal closure of one naris. Newborn pups were anesthetized by hypothermia and the external opening of their right naris cauterized, while littermates served as controls. Animals were sacrificed at 3 months, and the occluded (OCF) and nonoccluded (NOF) fossae of experimental animals as well as both fossae of control animals (CTF) were histologically studied. In both control and experimental animals, nasal fossae were lined by five different types of epithelia: squamous stratified, transitional, metaplastic, respiratory, and olfactory epithelia. It was found that closure of one naris provokes reorganization of the epithelial lining in both the occluded and nonoccluded side. In CTF airflow, physical conditions as well as pollutants and biological agents irritate the epithelial lining, causing squamous metaplasia as well as metaplastic epithelium showing inflammation in rostral levels. In CTF caudal levels, the metaplastic epithelium appears to a lesser degree and the respiratory epithelium prevails, except for the most caudal level where the olfactory epithelium is prevalent. In OCF, the protected environment created prevents the occurrence of metaplastic epithelium, the transitional, respiratory, and olfactory epithelia developing in the corresponding area instead. In NOF, where the airflow is double, the same pattern occurs as in CTF, although metaplastic epithelium values are approximately double, suggesting a clear linear effect. An outstanding feature observed was the increased extent of the olfactory epithelium in OCF regarding NOF, although changes in its morphological structure were not found. Airflow properties, including pressure, coldness, velocity, and turbulence, as well as biological and chemical hazards present in inflow, cause histological reorganization of the nasal epithelium lining during postnatal development. Results prove the need to consider airflow changes in nasal fossae surgery and point to the protective value of naris closure in ENT clinics, supporting it as a treatment of atrophic rhinitis.