Intravenously injected Newcastle disease virus in non-human primates is safe to use for oncolytic virotherapy.

Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic potential. Detailed preclinical information regarding the safety of oncolytic NDV is scarce. In this study, we evaluated the toxicity, biodistribution and shedding of intravenously injected oncolytic NDVs in non-human primates (Macaca fascicularis). Two animals were injected with escalating doses of a non-recombinant vaccine strain, a recombinant lentogenic strain or a recombinant mesogenic strain. To study transmission, naive animals were co-housed with the injected animals. Injection with NDV did not lead to severe illness in the animals or abnormalities in hematologic or biochemistry measurements. Injected animals shed low amounts of virus, but this did not lead to seroconversion of the contact animals. Postmortem evaluation demonstrated no pathological changes or evidence of virus replication. This study demonstrates that NDV generated in embryonated chicken eggs is safe for intravenous administration to non-human primates. In addition, our study confirmed results from a previous report that naïve primate and human sera are able to neutralize egg-generated NDV. We discuss the implications of these results for our study and the use of NDV for virotherapy.

Is dolphin morbillivirus virulent for white-beaked dolphins (Lagenorhynchus albirostris)?

The virulence of morbilliviruses for toothed whales (odontocetes) appears to differ according to host species. In 4 species of odontocetes, morbilliviruses are highly virulent, causing large-scale epizootics with high mortality. In 8 other species of odontocetes, including white-beaked dolphins (Lagenorhynchus albirostris), morbilliviruses have been found as an incidental infection. In these species, the virulence of morbilliviruses is not clear. Therefore, the admission of 2 white-beaked dolphins with morbillivirus infection into a rehabilitation center provided a unique opportunity to investigate the virulence of morbillivirus in this species. By phylogenetic analysis, the morbilliviruses in both animals were identified as a dolphin morbillivirus (DMV) most closely related to that detected in a white-beaked dolphin in Germany in 2007. Both animals were examined clinically and pathologically. Case No. 1 had a chronic neural DMV infection, characterized by polioencephalitis in the cerebrum and morbillivirus antigen expression limited to neurons and glial cells. Surprisingly, no nervous signs were observed in this animal during the 6 months before death. Case No. 2 had a subacute systemic DMV infection, characterized by interstitial pneumonia, leucopenia, lymphoid depletion, and DMV antigen expression in mononuclear cells and syncytia in the lung and in mononuclear cells in multiple lymphoid organs. Cause of death was not attributed to DMV infection in either animal. DMV was not detected in 2 contemporaneously stranded white-beaked dolphins. Stranding rate did not increase in the region. These results suggest that DMV is not highly virulent for white-beaked dolphins.

Low pathogenic avian influenza A(H7N9) virus causes high mortality in ferrets upon intratracheal challenge: a model to study intervention strategies.

Infections with low pathogenic avian influenza (LPAI) A(H7N9) viruses have caused more than 100 hospitalized human cases of severe influenza in China since February 2013 with a case fatality rate exceeding 25%. Most of these human infections presented with severe viral pneumonia, while limited information is available currently on the occurrence of mild and subclinical cases. In the present study, a ferret model for this virus infection in humans is presented to evaluate the pathogenesis of the infection in a mammalian host, as ferrets have been shown to mimic the pathogenesis of human infection with influenza viruses most closely. Ferrets were inoculated intratracheally with increasing doses (>10 e5 TCID50) of H7N9 influenza virus A/Anhui/1/2013 and were monitored for clinical and virological parameters up to four days post infection. Virus replication was detected in the upper and lower respiratory tracts while animals developed fatal viral pneumonia. This study illustrates the high pathogenicity of LPAI-H7N9 virus for mammals. Furthermore, the intratracheal inoculation route in ferrets proofs to offer a solid model for LPAI-H7N9 virus induced pneumonia in humans. This model will facilitate the development and assessment of clinical intervention strategies for LPAI-H7N9 virus infection in humans, such as preventive vaccination and the use of antivirals.

Spatial variability of genomic aberrations in a large glioblastoma resection specimen.

In the present study, the distribution of genetic aberrations in a glioblastoma resection specimen of unusually large size (9x8x2 cm) was investigated using comparative genomic hybridization (CGH). CGH was performed on 20 samples taken from the specimen, and the genetic aberrations found were compared with the regional histology. The samples were histopathologically graded according to WHO criteria, and a division in high- and low-grade areas and infiltration rims was made. In high-grade areas, low-grade areas as well as infiltration rims, gains on 10p11.2-pter (14/20), 11q12-q22 (6/20) and losses on 4q13-qter (9/20), 10q22-qter (8/20), 11p14-pter (5/20), 13q12-qter (7/20) were revealed. Gains on 1q21-32 (2/4) and losses on 7p21-pter (3/4) were exclusively found in the high-grade areas. In the low-grade tumor samples and in the infiltration rim, gains on 16p11.2-pter (6/16), 17p11.2-pter (6/16), 17q11.2-qter (5/16), 20q11.2-q13 (3/16) and deletions on 5q31-qter (4/16) were detected. Gains on 7q21-qter (8/11) and 8q11.2-qter (6/11), and loss of chromosome 9 (4/11) and the Y-chromosome (4/11) were found in the high-grade and low-grade samples, not in the infiltration rims. The finding of a set of identical chromosomal aberrations throughout the resection specimen, most of which have been previously reported in gliomas, confirms a mechanism of clonal tumor proliferation operative in gliomas. The previously unreported genetic alterations which were predominantly traced in the tumor rims, might reflect either selection for properties related to infiltrating behavior, or genomic instability of subclones. The findings illustrate the importance of searching for high-grade genetic aberrations in low-grade tumor samples taken from cases in which sampling error is suspected.

Accumulation and release of iron in polarly and non-polarly cultured trophoblast cells isolated from human term placentas.

We investigated the usefulness of membrane grown human term trophoblast cells in transferrin-mediated iron transfer studies. We showed that diferric transferrin is taken up both at the microvillous and at the basal membrane by means of receptor-mediated endocytosis. Uptake from the microvillous side is predominant. This corresponded with a much higher expression of transferrin receptors at the microvillous membrane as compared to the basal one. Iron appeared to accumulate in the cell. Accumulation was higher when transferrin was supplied at the microvillous side. Transfer of iron could not be assessed because uptake of transferrin by the cells was much less than passive diffusion of transferrin through the cell-free filter. The observation of iron accumulation was unexpected for a transfer epithelium. Could it be that part of the iron taken up by the cells is rapidly released whereas the remaining part accumulates? In this case the rate of iron uptake should be higher than the rate of iron accumulation. This question was assessed with non-polarly cultured trophoblast cells. We showed that like in polar cells iron accumulated in ferritin. A new experimental design enabled us to demonstrate that indeed the rate of transferrin-mediated iron is in excess over iron accumulation. We thus provide evidence for a mechanism that enables rapid transfer of iron across the syncytiotrophoblast cell layer.

Genetic aberrations in oligodendroglial tumours: an analysis using comparative genomic hybridization (CGH).

Four low-grade oligodendrogliomas, nine anaplastic oligodendrogliomas and two mixed oligoastrocytomas were investigated for chromosomal aberrations by comparative genomic hybridization on formalin-fixed, paraffin-embedded tissue samples. The most frequent losses observed involved 1p, 9p, 10pq, 14q, 16p, 19q, while the most frequent gains were seen on 7pq, 11pq, 17p, 19pq, and Xp. In one oligodendroglioma, a highly specific amplification of 1q32.1 was seen. The frequent losses of 14q have not been reported previously. In the two cases of mixed oligoastrocytomas multiple gains and losses were found that did not show a clear overlap with the alterations found in the pure oligodendrogliomas.

Exposure of laboratory animal workers to airborne rat and mouse urinary allergens.

BACKGROUND: Laboratory animal workers are at high risk of developing occupational allergy. Little is known about the relationship between levels of exposure and the risk of developing laboratory animal allergy. Since laboratory animal work comprises a large number of different-often short lasting-tasks, it is of interest to assess which activities are associated with high, low or intermediate levels of allergen exposure. OBJECTIVE: To develop and evaluate highly sensitive immunoassays in order to quantify rat and mouse urinary allergens in airborne dust sampled during short-lasting tasks. METHODS: Personal air dust samples were taken during full-shift periods as well as during specific tasks in seven laboratory animal facilities. Two sandwich enzyme immunoassays were developed, using rabbit antisera against rat and mouse urinary proteins. The rabbit antibodies were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting and compared with IgE antibodies from sensitized laboratory animal workers. RESULTS: The rabbit antibodies were highly specific for rat and mouse urinary proteins and reacted with all IgE-binding allergens in either urinary protein preparation. The assays for rat and mouse urine were very sensitive, with detection limits of 0.075 ng/mL. The coefficient of variation of the analysis was 12.9% for both assays. Animal caretakers appeared to experience the highest exposure to aeroallergens. A large variation in exposure within jobs was found, due to differences between tasks performed during the sampling day and the facility worked at. The highest exposure levels were found during removal of contaminated bedding from the cages. However, rat and mouse allergen exposure levels during this task varied enormously between facilities, 1.1-158 ng eq/m3 and 0.63-2000 ng eq/m3, respectively. CONCLUSION: Both sandwich immunoassays are highly specific and sensitive and are able to identity tasks of relatively short duration with high, medium and low exposure to airborne rat and mouse urinary allergens.

Airborne levels of alpha-amylase allergens in bakeries.

BACKGROUND: In the baking industry the use of enzymes has increased throughout the 1980s. Several studies have reported sensitization and respiratory disorders among bakery workers caused by enzymes in dough improvers. Fungal alpha-amylase is the most frequently reported cause of allergy. alpha-Amylase allergen exposure levels in the bakery industry, however, have not yet been reported. OBJECTIVE: The main objective of this study was to quantify personal alpha-amylase exposure levels of bakery workers. METHODS: alpha-Amylase allergens were measured in 507 personal samples of airborne dust taken in bakeries by using a newly developed sandwich enzyme immunoassay with affinity-purified polyclonal rabbit IgG antibodies. A cascade impactor was used to estimate the size of dust particles carrying alpha-amylase allergens. RESULTS: The rabbit IgG antibodies used in the assay showed, in immunoblotting with commercially available alpha-amylase, a reaction profile very similar to that of IgE from sensitized bakers. The enzyme immunoassay appeared to be highly specific for fungal amylase. Allergen exposure levels varied considerably among bakery workers, depending on the type of bakery and job category (range, 0 to 40 ng/m3). In confectioneries no alpha-amylase allergens were detected. In other bakeries alpha-amylase exposure was only found for workers directly involved in dough making. Measurements of the particle size distribution in these bakeries showed that alpha-amylase allergens are most likely to be deposited in the nose and ciliated airways. CONCLUSION: This study shows that personal monitoring of fungal amylase allergen exposure in bakeries is possible. This permits the identification of high-risk tasks and allergen sources, as well as the study of exposure-response relationships.

Transcytosis of 6.6-nm gold-labeled transferrin: an ultrastructural study in cultured porcine blood-brain barrier endothelial cells.

The mechanism and regulation of iron transport to the brain are largely unknown. The large surface area of the blood-brain barrier capillaries and the presence of transferrin receptors on the luminal plasma membranes of the blood-brain barrier endothelial cells (BBB-ECs) suggest that these cells actively participate in the transport of iron into the brain. In this paper, we describe the ultrastructural morphology of primary and first-passage cultures of BBB-ECs grown on different types of porous membranes. To investigate the mechanism of iron transport into and across the BBB-ECs, porous membrane grown first-passage cells were incubated with 6.6-nm gold-labeled transferrin and studied with electron microscopy. Results are suggestive for a transcytosis of transferrin through the BBB-ECs.

Ultrastructural osteopontin localization in papillary stones induced in rats.

OBJECTIVES: To detect in situ the precise osteopontin (OPN) localization in papillary stones. METHODS: Immunocytochemical labelling procedures are applied to detect OPN localizations in crystalline material of renal papillary stones. The tissue-processing procedure for electron microscopy, which includes OsO4 postfixation, preserves both immunocytochemical OPN reactivity and cellular membrane contrast up to the ultrathin section. Reflection-contrast light microscopical images are correlated with high resolution transmission-electron microscopical observations from consecutive ultrathin epon sections. RESULTS: Preserved crystalline material in interstitial and peripheral papillary stones is recognized as calcium oxalate monohydrate. After section incubation with markers conjugated to an antibody against OPN (alpha OPN) the crystals are converted into ghosts. In the ghosts, alpha OPN markers are present around microcrystals. The size of these microcrystals ranges from several nanometers to micrometers. It is observed (due to the OsO4-preserved membranes) that interstitial cells are separated from the stone surfaces by unidentified extracellular material, also present in the center as a stone matrix. CONCLUSION: The microcrystal-growth inhibitor OPN is detected in situ in interstitial stones induced in the rat's papilla and at the surface of the papilla.

Exposure-sensitization relationship for alpha-amylase allergens in the baking industry.

Fungal alpha-amylase is an important occupational allergen in the bakery industry. Epidemiologic studies focusing on the relationship between alpha-amylase allergen exposure and work-related respiratory allergy, however, have not been reported yet. In this cross-sectional study, sensitization to occupational allergens and work-related symptoms were studied in 178 bakery workers and related to allergen exposure. Alpha-amylase allergen concentrations were measured in personal dust samples, using a sandwich enzyme immunoassay. All workers were categorized into groups on the basis of their job histories and the alpha-amylase exposure levels of their job titles. Of all workers 25% had one or more work-related symptoms. As much as 9% of the bakery workers showed a positive skin prick test reaction to fungal amylase, and in 8% amylase-specific IgE was demonstrated. Alpha-amylase exposure and atopy appeared to be the most important determinants of skin sensitization, with prevalence ratios for atopy of 20.8 (95% CI, 2.74 to 158) and for medium and high alpha-amylase exposure groups of 8.6 (95% CI, 1.01 to 74) and 15.9 (95% CI, 1.95 to 129), respectively. Furthermore, a positive association was found between positive skin prick tests to alpha-amylase and work-related respiratory symptoms. In conclusion, this study has shown that there is a strong and positive relationship between alpha-amylase allergen exposure levels in bakeries and specific sensitization in bakery workers.

Wheat antigen exposure assessment for epidemiological studies in bakeries using personal dust sampling and inhibition ELISA.

BACKGROUND: Asthma in bakery workers caused by exposure to wheat flour proteins is an important occupational health problem. Until recently, gravimetric dust measurements were the only available technique for quantitative exposure assessment in bakeries. However, it is questionable whether dust levels are a good exposure parameter or only give a crude approximation of the actual flour allergen concentration. OBJECTIVE: In the present study we have investigated a method to measure wheat flour antigens with immunochemical methods. METHODS: Wheat flour antigens were measured in 449 personal dust samples taken in bakeries, using enzyme-linked immunosorbent assay (ELISA) inhibition and an anti-wheat IgG4 serum pool. Western-blotting was performed to compare the wheat flour proteins detected by IgE and IgG4. RESULTS: Electrophoresis and immunoblotting showed that many wheat flour proteins can bind IgG4 and IgE, but also a reasonable similarity in major allergens detected by our IgG4-serum pool and IgE-positive sera. Inhibition tests showed some cross-reactivity with some cereal species, but not with other ingredients used in bakeries. In bakeries, large differences in personal airborne flour levels were found between occupational titles. For several groups clear differences in wheat antigen exposure levels existed, where no differences in dust exposure levels could be found. The relationship between dust and wheat antigen exposure varied considerably, depending on the specific bakery occupation, the size of the bakery, and the type of product produced by the bakery. This study also shows that personal sampling of wheat antigens is possible on a large scale and can be used for epidemiological field studies. CONCLUSION: Measurement of airborne wheat antigens in bakeries is a more specific and sensitive measurement tool than measuring dust samples, and will probably be essential for epidemiologic field studies focusing on exposure-response relationships.

Lectin-cytochemistry of experimental rat nephrolithiasis.

Lectin reactivity in epithelial apical cell coats of normal rat kidneys was compared to that from animals subjected to crystal inducing diets (CID). The aim was to see whether the absence of lectin reactivity in cell coats is related to intratubular calcium oxalate crystal retention. In normal rat kidneys, after a pre-embedding procedure, it was observed that at the ultrastructural level, reactivity was present but that the lectin specificity for the various parts of the nephron might have to be reconsidered. There was heterogeneity between the epithelial cells with respect to the presence of coat material in the tubular cell apices. Tubular epithelial cell apices from CID rats showed no obvious changes in lectin reactivity pattern. Lectin reactivity was present at the periphery of intratubular crystals but undetectable at true crystal attachment sites or reduced at cell apices in the vicinity of recently attached crystals or agglomerates. After a post-embedding reaction procedure, wheat-germ agglutinin (WGA)-lectin reactivity confirmed the presence of coat material in the cleft between cell apex and retained crystal at crystal-attachment sites. The WGA/Au-10 nm reaction products were also seen inside epithelial cells. WGA/Au-10 nm reaction products mark a crystal matrix component inside intratubular and retained crystals. A similar matrix was also marked by an alpha-osteopontin (alpha OPN/Au-10 nm) reaction product.

Pathological and immunocytochemical changes in chronic calcium oxalate nephrolithiasis in the rat.

In the present study, we exposed rats to a crystal-inducing diet (CID) consisting of vitamin D3 and 0.5% ethylene glycol (EG), and we investigated histologically the kidney damage induced by the deposition of calcium oxalate (CaOx) crystals. After 28 days, 50% of the animals had renal CaOx crystals, of which 60% also had small papillary stones. Most crystals were present in the cortex. The occurrence of these crystals coincided with morphological and cytochemical changes: glomerular damage, tubular dilatation and necrosis, and an enlargement of the interstitium. The number of epithelial and interstitial cells positive for the proliferating cell nuclear antigen (PCNA) was increased. Tamm-Horsfall protein (THP) was not only demonstrable in the thick ascending limb of the loop of Henle (TAL), but also frequently in glomeruli, in the proximal tubular epithelium, and in the papilla. In the lumen of the tubular system, it was associated with urinary casts. Reflection contrast microscopy (RCM) showed that the crystals were coated with a thin layer of THP. In spite of the high urinary oxalate concentrations, the above described cellular changes were not observed in CID-fed rats without renal crystals. We conclude, therefore, that in the kidney, the retained CaOx crystals rather than the urinary oxalate ions are responsible for the observed morphological and immunocytochemical changes.

Etiology of calcium oxalate nephrolithiasis in rats. I. Can this be a model for human stone formation?

Crystal retention is studied in a rat-model system as a possible mechanism for the etiology of human nephrolithiasis. A crystal-inducing diet (CID) of ethylene glycol plus NH4Cl in their drinking-water is offered to healthy rats to generate intratubular crystals. Subsequently, the fate of retained crystals is investigated by allowing the rats a tissue recovery/crystalluria phase for three, five and ten days, respectively, on normal drinking water. The process of exotubulosis is observed in cortex and medulla of aldehyde-fixed kidneys after three days recovery. After five days, crystals are predominantly seen there in the interstitium. After ten days, cortex and medulla are virtually free of crystals. However, in the papillary regions after five and ten days recovery, three types of calcium oxalate monohydrate (COM) crystals are present: (1) free in the calycine space, (2) sub-epithelially located surrounded by interstitial cells within, and (3) covered by macrophage-like cells, outside the original papillary surface. After a CID plus three days recovery, a further thirty-seven days extra oxalate challenge with solely 0.3 vol% ethylene glycol induced intratubular and interstitial oxalate crystals. In the papillary region, large sub-epithelial crystals are seen. However, no crystals are seen in kidneys from rats given solely (0.5 or 0.8 vol.%) ethylene glycol for thirty days. An oxalate re-challenge retards crystal removal.

Etiology of calcium oxalate nephrolithiasis in rats. II. The role of the papilla in stone formation.

In kidneys of healthy rats submitted to a crystal-inducing diet (CID) with ethylene glycol (EG) and NH4Cl, the fate of retained crystals in the papillar region is studied during a recovery period of one, five or ten days, as model system for human nephrolithiasis. Scanning electron microscopy (SEM) shows, at papillary tips bulging into the calycine space, crystal masses covered either by the epithelium or a thin fibrous veil, or by unidentified mobile cuboidal cells. After CID plus one or five days recovery, small sub-epithelial swellings are seen of large sub-epithelial crystals at or around the papillary tip. After CID plus ten days, massive sub-surface crystal-containing micrometer-sized stones are seen in which the presence of calcium is confirmed by X-ray microanalysis. The papillary tip of rats after a re-challenge with an oxalate load from 0.1 vol% EG for twelve or forty-two days shows minor lesions. But a re-challenge with 0.3 vol% EG for thirty-seven days induces large sub-epithelial papillary millimeter-sized stones. The Von Kossa section staining converts the crystals into a black precipitate, but large peri-tubular or peri-vascular calcium deposits are absent. A new hypothesis about the etiology of an inductive calcium oxalate monohydrate nephrolithiasis is formulated which differs from the one proposed by Randall based on his deductive human kidney studies.

Etiology of experimental calcium oxalate monohydrate nephrolithiasis in rats.

In a rat-model system, tubular crystal retention as a possible mechanism for the etiology of nephrolithiasis in man, was studied by conventional transmission electron microscopy. The animals were supplied for nine days with a crystal-inducing diet, with ethylene glycol plus NH4Cl in their drinking-water. After this induction period, a two day regime with fresh drinking-water was included, to allow crystals to be removed by spontaneous crystalluria. After aldehyde fixation of the rat kidneys, large crystals were seen inside the tubular lumen. The crystals were attached to cell surfaces and covered by neighboring epithelial cells. Some crystals were overgrown by several epithelial cells and underwent a process of so-called exotubulosis, resulting in free or cell-surrounded crystals in the interstitium, and possibly in crystals in Giant cells. To investigate the fate of the retained crystals, some animals were additionally exposed to a low-oxalate challenge from drinking water containing 0.1 volume per cent of ethylene glycol for 12 or 30 days, respectively. It was assumed that this would interfere with the retained intratubular or interstitial crystals, and allow the crystals to grow into mini-stones. This was not observed. After the oxalate challenge, no crystals were found to be retained in the tubules (free or covered by cells). Interstitial crystals were observed, but it remains to be demonstrated whether such crystals actually grow into mini-stones or that they are removed by the sterile inflammation process observed.

Effects of limited reduction on disulfide bonds in human IgA1 and IgA1 fragments.

Human IgA occurs in body fluids as monomers, dimers and secretory IgA (sIgA). Besides the cysteine residues in intra-domain, inter-chain and inter-subunit disulfide bonds IgA molecules contain several cysteine residues with unknown function and reactivity. Limited reductions on serum IgA1 and secretory IgA1 with glutathione revealed that four cysteine residues per monomer or subunit were part of labile bonds. Six cysteine residues were reduced in F(ab')2 fragments and about three in Fc fragments, but none in Fab fragments, indicating that the labile bonds occur in the Fc fragment. By SDS-PAGE analyses of reduced proteins labile inter-alpha chain bond(s) were detected in F(ab')2 and F(abc)2 fragments but not in Fc fragments and intact IgA1, thus showing the importance of the CH3 domains for the structural stability of the hinge region. Nine cysteine residues per IgA1 were reduced with 0.01 M DTT and a large proportion of the IgA1 myeloma proteins formed half-molecules consisting of an alpha- and a light chain, but sIgA1 remained intact. This indicates a relative stability of heavy to light chain and inter-subunit bonds. Reductions in the presence of 2% SDS disrupted several intra-chain bonds. Binding studies with (CH2)2-specific monoclonal antibodies, which detect an epitope expressed only on IgA molecules with disulfide linked alpha chains, were in accordance with the SDS-PAGE results. A new model for the location of labile and more stable disulfide bonds is discussed.

Neural induction is mediated by cross-talk between the protein kinase C and cyclic AMP pathways.

Embryonic inductions appear to be mediated by the concerted action of different inducing factors that modulate one another's activity. Such modulation is likely to reflect interactions between the signal transduction pathways through which the inducing factors act. We tested this idea for the induction of neural tissue. We report that both adenylate cyclase activity and cAMP concentration increase substantially in induced neuroectoderm during neural induction. The enhancement of adenylate cyclase activity requires protein kinase C (PKC) activation, indicating cross-talk between these two signal transduction pathways. This cross-talk appears to be essential for neural induction. Whereas cAMP analogs alone were not neural inducers, they had a synergistic inducing effect if ectoderm was first incubated with TPA (12-O-tetradecanoylphorbol 13-acetate), a PKC activator. These results strongly suggest that at least two signals mediate neural induction. The first signal activates PKC and the second signal then activates the cAMP pathway effectively.