Modular air-liquid interface aerosol exposure system (MALIES) to study toxicity of nanoparticle aerosols in 3D-cultured A549 cells in vitro.

We present a novel lung aerosol exposure system named MALIES (modular air-liquid interface exposure system), which allows three-dimensional cultivation of lung epithelial cells in alveolar-like scaffolds (MatriGrids®) and exposure to nanoparticle aerosols. MALIES consists of multiple modular units for aerosol generation, and can be rapidly assembled and commissioned. The MALIES system was proven for its ability to reliably produce a dose-dependent toxicity in A549 cells using CuSO4 aerosol. Cytotoxic effects of BaSO4- and TiO2-nanoparticles were investigated using MALIES with the human lung tumor cell line A549 cultured at the air-liquid interface. Experiments with concentrations of up to 5.93 × 105 (BaSO4) and 1.49 × 106 (TiO2) particles/cm3, resulting in deposited masses of up to 26.6 and 74.0 µg/cm2 were performed using two identical aerosol exposure systems in two different laboratories. LDH, resazurin reduction and total glutathione were measured. A549 cells grown on MatriGrids® form a ZO-1- and E-Cadherin-positive epithelial barrier and produce mucin and surfactant protein. BaSO4-NP in a deposited mass of up to 26.6 µg/cm2 resulted in mild, reversible damage (~ 10% decrease in viability) to lung epithelium 24 h after exposure. TiO2-NP in a deposited mass of up to 74.0 µg/cm2 did not induce any cytotoxicity in A549 cells 24 h and 72 h after exposure, with the exception of a 1.7 fold increase in the low exposure group in laboratory 1. These results are consistent with previous studies showing no significant damage to lung epithelium by short-term treatment with low concentrations of nanoscale BaSO4 and TiO2 in in vitro experiments.

Death in four RHDV2-vaccinated pet rabbits due to rabbit haemorrhagic disease virus 2 (RHDV2).

OBJECTIVES: To create awareness of single RHDV2 infections and cases of death despite immunisation with RHDV2-specific vaccine. MATERIALS AND METHODS: Retrospective case series of four companion rabbits. Patient signalment, vaccination history, pathology and molecular examinations were obtained from the medical records. RESULTS: The animals died peracutely or with nonspecific symptoms like apathy and inappetence. Pathological examination indicated and molecular biological findings confirmed RHDV2 infection in four animals. Several partner animals died at the same time under the same circumstances. CLINICAL SIGNIFICANCE: This is the first report of fatalities in RHDV2-vaccinated companion rabbits due to rabbit haemorrhagic disease virus 2 infection with documented case and vaccination history. Veterinarians should be aware of possible single fatal RHDV2 infections despite vaccination, should initiate the clarification of suspected cases and inform vaccine manufacturers and competent authorities.

Medical treatment of 145 cases of gastric dilatation in rabbits.

Gastric dilatation is an acute and life-threatening condition in pet rabbits commonly caused by an intestinal obstruction with pellets of compressed hair. Surgery is normally considered to be the treatment of choice to alleviate the obstruction. However, for various reasons such as restrictions by the owner, a high anaesthetic risk due to the critical condition of the patient or concurrent diseases, surgical treatment may be impossible. In a three-year period, 145 cases of gastric dilatation were treated medically with a combination of metoclopramide, metamizole, balanced fluid electrolyte solution with glucose and syringe feeding. No gender or breed predisposition could be noted. Four animals were euthanased, three of them directly after diagnosis. Eleven animals died, eight of them on the day of presentation. The medical treatment was successful in 130 cases (89 per cent) with a mean treatment time of three days. The animals were released from hospital when eating and defecating normally. Although the use of medical treatment of gastric dilatation has to be thoroughly considered, especially regarding the severity of obstruction, the painfulness and the animal's welfare, the good survival rate observed with these animals makes it a good option for all cases where surgical treatment is contraindicated.

[Atresa ani and rectocutaneous fistula in an adult guinea pig (Cavia porcellus)]. / Atresia ani und rektokutane Fistel bei einem adulten Meerschweinchen (Cavia porcellus).

A 4-year-old, male guinea pig in a good general condition was presented for a routine castration. Since its birth, the animal had been kept outdoors with a male sibling. At the initial examination the perineal sac was smaller compared to other intact male guinea pigs. At the caudal end of the perineal sac a small dimple was noted, that ended blindly after 3mm. The following examination under anaesthesia revealed a fistula opening 1cm caudodorsally to the anus. The skin around the opening was bulging but without any signs of inflammation. Slightly protruding and reddened mucosa was visible inside the opening. After instillation of a contrast agent into the fistula a radiograph showed a contrast-filled caudal region of the large intestine. The intestine appeared to be normal and no other abnormalities were present. The excretion of normal faeces through the fistula was visible and atresia ani with a rectocutaneous fistula was diagnosed. The examination of the male sibling showed a normally developed anus. As the guinea pig had no clinical signs or associated malformations apart from the smaller perineal sac and the passage of normal faeces was possible through the fistula opening, no therapy was scheduled. To the authors' knowledge, this is the second report of this congenital abnormality in a guinea pig. The characteristics of this case include the high age of the animal at diagnosis of the atresia ani, the gender and the formation of a single rectocutaneous fistula.

In vitro Sertoli cell bioassay of follicle-stimulating hormone (FSH)-serum from different animal species alters the morphology of rat Sertoli cells without affecting their response to FSH.

Follicle-stimulating hormone (FSH) stimulates aromatase activity of immature rat Sertoli cells cultured in vitro in the presence of an androgen substrate, a phenomenon that can be used to measure FSH bioactivity. This paper analyzes the effects of sera from various animal species on both aromatase activity and morphological aspects of cultured rat Sertoli cells. Treatment of Sertoli cells with increasing concentrations of serum from humans, monkeys, rats, hamsters, and mice gave a dose-dependent stimulation of estradiol production parallel to the response obtained with FSH standard. Serum from these species and from rabbits also had a characteristic morphological effect on the cells, to produce a fibroblast-like aspect and clumping. This effect was dose-dependent, was increased by the addition of FSH, and was eliminated by heating the sera at 56 degrees for 30 min prior to incubation with the cells. Clumping did not interfere with the aromatase activity of Sertoli cells and did not cause the cellular response to deviate from parallelism with the standard curve. Heat treatment of FSH standard and serum samples did not significantly change the aromatase activity. FSH bioactivity could be measured accurately in intact and castrated monkeys. It is concluded that the Sertoli cell aromatase bioassay can be applied directly to the measurement of serum bioactive FSH in several animal species without any preliminary treatment of the serum samples.

Time course of human sperm transformation in the cytoplasm of zona-free hamster ova.

The time required for nuclear transformation of human spermatozoa in the cytoplasm of zona-free hamster ova was determined using semen samples from four individuals. Zona-free ova were incubated with capacitated spermatozoa for either 3 h or for 4-8 h. After the first hour of insemination, up to 85 ova were fixed at 1-h intervals. Assessments of sperm transformation were carried out on Giemsa-stained preparations of ova. Considerable variation between individuals was found in the maximal time of sperm decondensation, ranging from 2 to 7 h. Decondensing sperm heads developed into pronuclei within 3-5 h. At and subsequent to pronuclear formation, the incidence of decondensing sperm heads was considerably reduced in both insemination times. This variability in sperm transformation is relevant in clinical evaluations of the functional capacity of spermatozoa.

Chemical modification of membrane proteins in relation to inhibition of anion exchange in human red blood cells.

Mono-, di-, and trisulfonic acids, including 4,4'-diacetamido stilbene-2,2'-disulfonic acid (DAS) and 2-(4'-amino phenyl)-6-methylbenzene thiazol-3',7-disulfonic acid (APMB) produce a reversible inhibition of sulfate equilibrium exchange in human red cells. A study of the sidedness of the action of a number of these sulfonic acids in red cell ghosts revealed that some, like DAS, inhibit only at the outer membrane surface while others, like APMB, inhibit at either surface. This finding suggests that at least two different types of membrane sites are involved in the control of anion permeability. The nature of the anion permeability controlling sites in the outer cell surface was investigated by studying the effects of DAS on the inhibition by dinitrofluorobenzene (DNFB) of anion equilibrium exchange and on the binding of DNFB to the proteins of the red blood cell membrane. After exposure to DNFB in the presence of DAS for a certain period of time, there was a reduction of both the inhibitory effect of DNFB on sulfate exchange and the binding of DNFB to the protein in band 3 of SDS polyacrylamide gel electropherograms (nomenclature of Steck, J. Cell. Biol., 62: 1, '74). Since binding to other membrane proteins was not affected, this observation supports the assumption that the protein in band 3 plays some role in anion transport. In accordance with the absence of an inhibitory effect at the inner membrane surface, internal DAS does not affect DNFB binding to the protein in band 3. DAS protected the anion exchange system not only against inhibition by DNFB but also by m-isothiocyanato benzene sulfonic acid. In contrast to DAS, the equally inhibitory phlorizin does not reduce the rate of dinitrophenylation of the protein in band 3. This suggests that either not all inhibitors of anion exchange exert their action by a combination with sites on the protein in band 3 or that in spite of the described evidence this protein is not involved in the control of anion movements.

Calcium-potassium-stimulated net potassium efflux from human erythrocyte ghosts.

In the presence of 8 mM external Ca++, the K+ permeability of human red cell ghosts increases provided K+ is also present in the medium. This increase does not represent K+/K+ exchange but a stimulation of net K+ efflux. The stimulation is half-maximal at 0.7 +/- 0.15 mM (n=5). At concentrations above 4.0 mM, external K+ inhibits net K+ efflux. Similar stimulatory and inhibitory effects of external K were also observed in intact cells after exposure to Pb++ or to Ca++ in the presence of fluoride, iodoacetate plus adenosine, or propranolol, suggesting that a common K+ -activated K+ -specific transfer system may be involved under all of these various circumstances. Internal K+ also stimulates net K+ efflux from ghosts, but it is uncertain whether internal K+ is an absolute requirement for the K+ permeability increase. In contrast to external Na+ which slightly stimulates K+ efflux, internal Na+ inhibits. The inhibition by internal Na+ is abolished by sufficiently high concentrations of external K+, showing that K+ binding to the outer membrane surface and Na+ binding to the internal surface are mutually interdependent. In red cell ghosts the Ca++ -K+ -stimulated net K+ efflux increases with increasing pH until a plateau is reached between pH 7.2 and 8.0. In fluoride-poisoned intact cells, the Ca++-K+ stimulated flux passes through a maximum around pH 6.8. Neither internal nor external Mg++ interferes with the combined effects of Ca++ and K+. Similarly, external EDTA has no influence at concentrations which are far lower than the Ca++ concentration required to produce a maximal response. In contrast, low concentrations of internal EDTA prevent the permeability change.