Search

Ochratoxin A was localized in the kidneys of pigs and rats by means of immunofluorescence microscopy after short-time exposure. Antibody against ochratoxin A was obtained from rabbits after repeated injections of bovine serum albumin-ochratoxin A conjugate. Ochratoxin A was localized exclusively in the proximal tubule. Light microscopically, necrosis and desquamation of epithelial cells in the pig kidneys were restricted to the proximal tubule where the toxin was found. The investigation has demonstrated conclusively that the proximal tubule is the target part of the nephron in ochratoxin A-induced acute mycotoxic nephropathy.

A case of recurrent rhabdomyoma in the oropharynx of a 72-year-old man is presented. Diagnosis was based on routine histology, and in the third and last recurrence it was further established by electron microscopy, immune peroxidase staining for myoglobin, actin and fibronectin, and special strains. All recurrences were histologically identical and metastases were never observed. The adult rhabdomyoma is almost exclusively located in the head/neck region, often adjacent to vital organs, complicating radical surgery. The present case indicates that recurrences can be expected often at extraordinarily long intervals.

A florid necrotizing glomerulonephritis was found in all 48 pigs that were fed a waste product from the industrial production of the proteolytic enzyme Alcalase® NOVO. In addition, three of the animals developed a lesion identical to polyarteritis nodosa. Focal necrosis of the glomeruli was observed in all animals. Electron microscopy showed electron dense deposits at the subendothelial and subepithelial side of the basement membrane of the glomerular capillary wall and in the mesangium. Immunofluorescence microscopy showed IgM in a fine granular pattern in the glomeruli of all 48 pigs. This appears to be the first report on nutritionally induced glomerulonephritis and polyarteritis nodosa in pigs.

Structural changes in kidney biopsies were investigated from five patients with primary oliguric extracapillary glomerulonephritis in whom the renal function was adequately maintained during extended combined immunosuppressive treatment. The most important structural change was a pronounced decrease in the number of crescents. Reduction in numbers of crescents in the late biopsies was significantly greater than the increase in the number of hyalinized glomeruli. Tubular parenchyma showed only slight diffuse atrophy, and a moderate interstitial fibrosis was always present during the latter stages of treatment. Disappearance of crescents in the glomeruli was not accompanied by disappearance of immunoglobulins. Successfull immunosuppressive treatment of extracapillary glomerulonephritis causes the disappearance of structural characteristics of the kidney that are diagnostic for this disease.

To find predisposing parameters for aseptic loosening the present study determined the ultrastructural morphology in the pseudosynovial membrane from non-loose hip arthroplasties to compare the intra- and extracellular distribution of wear particles one year after total hip replacement using three different bearing material combinations.A total of 37 patients from a larger prospective randomised trial of 225 patients had biopsies taken arthroscopically from the pseudosynovial membrane one year after insertion of identical endoprostheses except for the bearing materials, polyethylene-on-zirconia (n=15), alumina-on-alumina (n=13), and CoCr-CoCr (n=9), respectively. The granulomatous inflammation seen in biopsies from these well-fixated implants was qualitatively comparable to the pattern seen in aseptic loose implants. Wear particles were seen in the extracellular matrix and intracellularily in macrophages, fibroblasts, and in endothelial cells. It was not possible systematically to distinguish the morphology between the three groups, though in one patient with CoCr-CoCr bearing material necrotic tissue was seen and exclusively extracellular location was not found in this group. The transport mechanism may vary with these materials and particle number. At this initial stage after hip surgery, the present study did not provide evidence for different types of bearing materials having significant impact on ultrastructural morphology adjacent to hip arthroplasties within the first year after surgery.

Wear-resistant bearing materials may hypothetically reduce chronic inflammation in the pseudosynovial membrane as compared to less wear-resistant bearing materials such as polyethylene. We assessed the foreign body response in the pseudosynovial membrane in vivo after total hip replacement.37 patients from a larger prospective randomized trial of 225 patients had biopsies taken arthroscopically from the artificial hip joint (i.e. the pseudosynovial membrane) 1 year after insertion of the implant. All patients had an identical hip prosthesis (Bimetric-RingLoc) except for the bearing materials, which consisted of polyethylene on zirconia, CoCr on CoCr, or alumina on alumina. Histological quantification was performed on 2-mum-thick semi-thin plastic sections or paraffin sections by point counting technique to compare the volume fraction of macrophages, granulomas and endothelial cells in biopsies of the pseudosynovial membrane.The median macrophage volume fractions for polyethylene-on-zirconia bearing material (n = 15), CoCr-on-CoCr (n = 9), and alumina-on-alumina (n = 11) were 0.02, 0.04, and 0.004, respectively. The median granuloma volume fractions for polyethylene-on-zirconia (n = 13), CoCr-on-CoCr (n = 9), and alumina-on-alumina (n = 13) were 0.02, 0.04, and 0.02, respectively. The median endothelial cell volume fractions for polyethylene-on-zirconia (n = 15), CoCr-on-CoCr (n = 9), and alumina-on-alumina (n = 11) were 0.03, 0.02, and 0.05, respectively. Statistical analysis showed no significant differences between the three groups with the different bearings with respect to volume fraction of macrophages, granulomas and endothelial cells.Our study demonstrated that a granulomatous inflammation is a common finding in non-loose implants as early as 1 year after the operation not demonstrating a difference in macrophages and granuloma formation with the various bearing materials. Thus a high volume fraction of macrophages was found in the osteoarthritis control group compared to the operated group.

A chemotherapeutic vitamin D analogue, EB1089, kills tumor cells via a caspase-independent pathway that results in chromatin condensation and DNA fragmentation. Employing transmission- and immunoelectronmicroscopy as well as detection of autophagosome-associated LC3-beta protein in the vacuolar structures, we show here that EB1089 also induces massive autophagy in MCF-7 cells. Interestingly, inhibition of autophagy effectively hindered apoptosis-like nuclear changes and cell death in response to EB1089. Furthermore, restoration of normal levels of beclin 1, an autophagy-inducing tumor suppressor gene that is monoallelically deleted in MCF-7 cells, greatly enhanced the EB1089-induced nuclear changes and cell death. Thus, EB1089 triggers nuclear apoptosis via a pathway involving Beclin 1-dependent autophagy. Surprisingly, tumor cells depleted for Beclin 1 failed to proliferate suggesting that even though the monoallelic depletion of beclin 1 in human cancer cells suppresses EB1089-induced autophagic death, one intact beclin 1 allele is essential for tumor cell proliferation.

When Actinobacillus pleuropneumoniae ( A. pp ) is grown under iron-restricted conditions in vitro, transferrin binding proteins (Tbps) are induced. The functional transferrin receptor of A. pp is composed of two outer membrane proteins (Tbp1 and Tbp2) and shows an exquisite specificity for porcine transferrin. This complex was studied using a monoclonal antibody (Mab 1.48) raised against a synthetic peptide corresponding to a hydrophilic domain of Tbp2 common to several A. pp serotypes. The antibody reacted specifically with a 60–70 kDa Tbp2-antigen found in all serotypes of A. pp obtained from iron-restricted culture. It was found that Tbp2 was not expressed in iron replete medium by any serotype except serotypes 5a, 5b and 6 where a weak expression was seen. There was a weak expression of related antigens in Actinobacillus indolicus and Actinobacillus suis under iron-depleted conditions while no similar antigens were detected with the Mab in iron-starved Actinobacillus lignieresii, Actinobacillus porcinus, Actinobacillus minor, Haemophilus influenzae , and Haemophilus parasuis . Using an enzyme-linked immunosorbent assay (ELISA) based on the Mab 1.48, Tbp2 could be detected in both recombinant E. coli expressing Tbp2 and in wild type A. pp grown under iron restricted conditions . The subcellular location of Tbp2 in A. pp was studied by immunoelectron microscopy using the Mab 1.48. Interestingly, all antibody binding was found inside the A. pp cells, while Tbp2 expressed in recombinant E. coli was found both in the cytosol and on the outer membrane. These results indicate that the Mab 1.48-reactive epitope of Tbp2 is surface exposed when it is expressed without Tbp1 in E. coli while the inaccessibility of this epitope of Tbp2 in A. pp could be due to shading by the association between Tbp2 and Tbp1 .

Death receptors can trigger cell demise dependent or independent of caspases. In WEHI-S fibrosarcoma cells, tumor necrosis factor (TNF) induced an increase in cytosolic cathepsin B activity followed by death with apoptotic features. Surprisingly, this process was enhanced by low, but effectively inhibiting, concentrations of pan-caspase inhibitors. Contrary to caspase inhibitors, a panel of pharmacological cathepsin B inhibitors, the endogenous cathepsin inhibitor cystatin A as well as antisense-mediated depletion of cathepsin B rescued WEHI-S cells from apoptosis triggered by TNF or TNF-related apoptosis-inducing ligand. Thus, cathepsin B can take over the role of the dominant execution protease in death receptor-induced apoptosis. The conservation of this alternative execution pathway was further examined in other tumor cell lines. Here, cathepsin B acted as an essential downstream mediator of TNF-triggered and caspase-initiated apoptosis cascade, whereas apoptosis of primary cells was only minimally dependent on cathepsin B. These data imply that cathepsin B, which is commonly overexpressed in human primary tumors, may have two opposing roles in malignancy, reducing it by its proapoptotic features and enhancing it by its known facilitation of invasion.

Heat shock protein 70 is an antiapoptotic chaperone protein highly expressed in human breast tumors and tumor cell lines. Here, we demonstrate that the mere inhibition of its synthesis by adenoviral transfer or classical transfection of antisense Hsp70 cDNA (asHsp70) results in massive death of human breast cancer cells (MDA-MB-468, MCF-7, BT-549, and SK-BR-3), whereas the survival of nontumorigenic breast epithelial cells (HBL-100) or fibroblasts (WI-38) is not affected. Despite the apoptotic morphology as judged by electron microscopy, the asHsp70-induced death was independent of known caspases and the p53 tumor suppressor protein. Furthermore, Bcl-2 and Bcl-X L , which protect tumor cells from most forms of apoptosis, failed to rescue breast cancer cells from asHsp70-induced death. These results show that tumorigenic breast cancer cells depend on the constitutive high expression of Hsp70 to suppress a transformation-associated death program. Neutralization of Hsp70 may open new possibilities for treatment of cancers that have acquired resistance to therapies activating the classical apoptosis pathway.

The mts1 (S100A4) gene, encoding a Ca(2+)-binding protein of the S-100 subfamily, is involved in the control of tumor metastasis in some murine tumor cell lines. To further analyze its role, we transfected hormone-responsive human breast cancer MCF-7 cells with the mts1 gene under the control of a strong constitutive promoter. All of the 3 tested clones (MCF-7/mts1) producing Mts1 protein acquired an ability for hormone-independent growth in nude mice. Tumors derived from mts1 transfectants revealed local invasiveness into surrounding muscle and adipose tissues and metastasized to regional lymph nodes and lungs, characteristics which are rarely observed with parental MCF-7 cells. Electron-microscopic analysis of MCF-7/mts1 cells demonstrated structural changes in anchoring junctions, particularly in intermediate filament attachment site (desmosomes). The mts1-transfected clones expressed estrogen receptor, and their growth in tissue culture was both estrogen- and anti-estrogen responsive. Changes in regulation of the estrogen-dependent proteins progesterone receptor and cathepsin D were observed in some of the transfected clones. Our results indicate that mts1 expression in human breast cancer cells induces several changes characteristic of malignant phenotype and tumor progression.

The extracellular matrix is a key element in neuronal development and tumour invasion, providing a substratum which sustains the adhesion and migration of cells. In order to study interactions between the neural cell adhesion molecule (NCAM) and collagen, we transfected mouse L cells with cDNA encoding the human transmembrane NCAM isoform of 140 kDa (NCAM-B). An L-cell/collagen type I system was used to study the influence of NCAM expression on in vitro invasion. We here report that migration of NCAM-expressing cells in collagen was inhibited compared to that of NCAM-negative cells transfected with the empty vector. Immunofluorescence confocal laser scanning microscopy (CLSM) and immunogold electron microscopy using anti-human NCAM antibodies demonstrated a heterogeneous distribution of NCAM on the plasma membrane of transfected L cells grown on collagen. NCAM was preferentially located at the surface of broad cytoplasmic protrusions and slender extensions, some of which were facing the collagen. This was in contrast to the homogeneous surface distribution of NCAM on cells grown on plastic. These data suggest that NCAM and collagen type I interact, and that this might lead to the migration inhibition of NCAM-expressing cells.

Using confocal fluorescence microscopy with a monoclonal antibody, we have localized the receptor for urokinase plasminogen activator (uPAR) in MDA-MB-231 human breast cancer cells migrating into a reconstituted basement membrane. Patchy and polarized uPAR immunoreactivity was found at the cell membrane, and strong staining was found both in the ruffled border or leading edge of the cells and at pseudopodia penetrating into the membrane. Intracellular uPAR staining was localized in the paranuclear region and in rounded granule-like structures; some of these were identified as lysosomes by double staining for uPAR and the lysosomal enzyme cathepsin D. Urokinase plasminogen activator (uPA) activity has previously been shown to play a role in migration of cells into basement membranes, and it has been proposed that uPAR also is involved in this process. uPA is known to be internalized and degraded after complex formation with the inhibitor PAI-1. Lysosomal uPAR immunoreactivity may result from concomitant internalization of the receptor.

Receptors for urokinase-type plasminogen activator (uPAR) are present on the surface of many cell types and appear to be the key determinant controlling extracellular proteolysis catalyzed by the urokinase-type plasminogen activator (uPA). Receptor-bound uPA may be inhibited by the specific inhibitors PAI-1 and PAI-2, and the complex thus formed may subsequently be internalized and degraded in lysosomes. Biochemical evidence has recently indicated that also uPAR is internalized with the uPA/uPAI complex. We report here the subcellular localization of uPAR and cathepsin D in the MDA-MB-231 human breast cancer cell line studied by immuno-electron microscopy of ultrathin cryosections using single or double immunostaining techniques. Cell surface uPAR was preferentially localized at cell-cell junctions; cytoplasmic uPAR was inside large vesicles of different morphology and in flat Golgi saccules. A number of vesicles also contained cathepsin D. The uPAR was exclusively membrane-bound at the cell surface and in cytoplasmic vesicles without cathepsin D. In lysosomal vesicles with both cathepsin D and u-PAR, uPAR was probably degraded as it was observed in the luminal contents.

Macroautophagy is an evolutionary conserved lysosomal pathway involved in the turnover of cellular macromolecules and organelles. In spite of its essential role in tissue homeostasis, the molecular mechanisms regulating mammalian macroautophagy are poorly understood. Here, we demonstrate that a rise in the free cytosolic calcium ([Ca(2+)](c)) is a potent inducer of macroautophagy. Various Ca(2+) mobilizing agents (vitamin D(3) compounds, ionomycin, ATP, and thapsigargin) inhibit the activity of mammalian target of rapamycin, a negative regulator of macroautophagy, and induce massive accumulation of autophagosomes in a Beclin 1- and Atg7-dependent manner. This process is mediated by Ca(2+)/calmodulin-dependent kinase kinase-beta and AMP-activated protein kinase and inhibited by ectopic Bcl-2 located in the endoplasmatic reticulum (ER), where it lowers the [Ca(2+)](ER) and attenuates agonist-induced Ca(2+) fluxes. Thus, an increase in the [Ca(2+)](c) serves as a potent inducer of macroautophagy and as a target for the antiautophagy action of ER-located Bcl-2.

The apoptosome, a heptameric complex of Apaf-1, cytochrome c, and caspase-9, has been considered indispensable for the activation of caspase-9 during apoptosis. By using a large panel of genetically modified murine embryonic fibroblasts, we show here that, in response to tumor necrosis factor (TNF), caspase-8 cleaves and activates caspase-9 in an apoptosome-independent manner. Interestingly, caspase-8-cleaved caspase-9 induced lysosomal membrane permeabilization but failed to activate the effector caspases whereas apoptosome-dependent activation of caspase-9 could trigger both events. Consistent with the ability of TNF to activate the intrinsic apoptosis pathway and the caspase-9-dependent lysosomal cell death pathway in parallel, their individual inhibition conferred only a modest delay in TNF-induced cell death whereas simultaneous inhibition of both pathways was required to achieve protection comparable to that observed in caspase-9-deficient cells. Taken together, the findings indicate that caspase-9 plays a dual role in cell death signaling, as an activator of effector caspases and lysosomal membrane permeabilization.

Morphological evidence of apoptosis in transient forebrain ischemia is controversial. We therefore investigated the time sequence of apoptosis-related antigens by immunohistochemistry and correlated it with emerging nuclear patterns of cell death in a model of transient forebrain ischemia in CA1 pyramidal cells of the rat hippocampus. The earliest ischemic changes were found on day 2 and 3, reflected by an upregulation of phospho-c-Jun in a proportion of morphologically intact CA1 neurons, which matched the number of neurons that succumbed to ischemia at later time points. At day 3 and later 3 ischemic cell death morphologies became apparent: pyknosis, apoptosis-like cell death and necrosis-like cell death, which were confirmed by electron microscopy. Activated caspase-3 was present in the vast majority of cells with apoptosis-like morphology as well as in a small subset of cells undergoing necrosis; its expression peaked on days 3 to 4. Silver staining for nucleoli, which are a substrate for caspase-3, revealed a profound loss of nucleoli in cells with apoptosis-like morphology, whereas cells with necrosis-like morphology showed intact nucleoli. Overall, cells with apoptosis-like morphology and/or caspase-3 expression represented a minor fraction (

The active form of vitamin D(3) (1,25(OH)(2)D(3)) induces an increase in the intracellular free calcium ([Ca(2+)](i)) and caspase-independent cell death in human breast cancer cells. Here we show that the treatment of MCF-7 breast cancer cells with 1,25(OH)(2)D(3) or its chemotherapeutic analog, EB 1089, releases Ca(2+) from the endoplasmic reticulum. The increase in [Ca(2+)](i) was associated with the activation of a calcium-dependent cysteine protease, mu-calpain. Interestingly, ectopic expression of a calcium-binding protein, calbindin-D(28k), in MCF-7 cells not only attenuated the elevation in [Ca(2+)](i) and calpain activation, but also reduced death triggered by vitamin D compounds. Similarly, the inhibition of calpain activity by structurally unrelated chemical inhibitors increased the survival of the cells and reduces the amount of annexin V-positive cells. Despite the complete absence of effector caspase activation, transmission electron microscopy of MCF-7 cells treated with 1,25(OH)(2)D(3) or EB 1089 revealed apoptosis-like morphology characterized by the condensed cytoplasm, nuclei, and chromatin. Overall, these results suggest that calpain may take over the role of the major execution protease in apoptosis-like death induced by vitamin D compounds. Thus, these compounds may prove useful in the treatment of tumors resistant to therapeutic agents dependent on the classical caspase cascade.

The transepithelial transport of biosynthetic human growth hormone (hGH) formulated with the absorption enhancers didecanoyl-L-alpha-phosphatidylcholine (DDPC) and alpha-cyclodextrin (alpha-CD) was studied after intranasal administration to rabbits. Plasma concentrations of the hormone were determined until 240 min post administration by ELISA, and the absolute bioavailability was estimated to be in the vicinity of 20%. The localization of hGH was studied 15 min after application of the powder formulation in the initial absorptive phase. To visualize the hormone, a two-step indirect immuno-gold technique was used on semithin and ultrathin cryosections and Epon sections. Polyclonal rabbit anti-hGH was used as primary antibody and gold-conjugated goat anti-rabbit IgG as secondary antibody, succeeded by silver enhancement. Growth hormone was mainly found in the cytoplasm and nuclei of ciliated cells, showing distinct morphological signs of early necrosis, and in lamina propria, including the venules. Minute amounts of hGH were found in endocytotic vesicles in morphologically normal epithelial cells and in the intercellular compartment. We conclude that the major transport route of hGH formulated with absorption enhancers DDPC and alpha-CD was transcellular through lethally damaged ciliated cells.

Toxigenic strains of Pasteurella multocida produce the 147 kDa protein Pasteurella multocida toxin (PMT) which is responsible for the osteoclastic bone resorption in progressive atrophic rhinitis in pigs and induces such resorption in all experimental animals tested so far. In the present study we have carried out immunocytochemistry on formaldehyde- and glutaraldehyde-fixed ultracryocut P. multocida using a pool of monoclonal antibodies against different epitopes on PMT as the first layer and affinity purified rabbit anti-mouse IgG as the second layer. Goat anti-rabbit IgG conjugated with 5 nm gold particles was used as marker. The gold particles were silver-enhanced prior to examination in the transmission electron microscope. Whole bacteria were also immunostained after fixation and critical point drying and examined by scanning transmission electron microscopy. The results showed that PMT was located in the cytoplasm of P. multocida. PMT could not be detected on intact, undamaged P. multocida by scanning electron microscopy. Neither pili nor flagella could be detected on the surface of the negatively stained P. multocida strains investigated. PMT has a series of characteristics encompassed in the definition of an exotoxin. However, that PMT was not secreted by living intact P. multocida is unexpected for an exotoxin.

Four pigs were treated with ochratoxin A (800 micrograms/kg) for five consecutive days. Subsequently, urine and bile were collected and kidneys were perfusion fixed unilaterally. Liver and kidney samples were examined for the distribution of ochratoxin A and metabolites in subcellular fractions and the effects of the toxin on protein synthesis and enzyme activities. Ochratoxin A and the hydrolytic product, ochratoxin alpha, were found in urine. Elevated levels of toxin accumulation in kidney (283 ng/g) compared with liver (189 ng/g) and toxin-mediated reductions in protein synthesis and enzyme activities in kidney identified it as a target organ of ochratoxin toxicity. Ultrastructural investigations of kidney in toxin-exposed animals identified a process of condensation of cellular material with disappearance of membranes and continuous desquamation in the lower part of the proximal convoluted tubules. In target cells peroxisomes appeared to have lost membrane integrity and the organelles were leaking materials into the cytosol. Reduction of structural integrity was associated with an increase in the presence of catalase and cyanide insensitive fatty acid oxidase activity in the soluble kidney fractions.

(1983). Environmental Conditions That Facilitate Ochratoxin Contamination of Agricultural Commodities. Acta Agriculturae Scandinavica: Vol. 33, No. 2, pp. 113-128.

K88ab, K88ac and K88ad fimbriae derived from enterotoxigenic Escherichia coli strains involved in porcine colibacillosis were used to immunize BALB/c mice. Several hybridomas secreting monoclonal antibodies (MAbs) against the three intact K88 fimbriae subtypes were produced by fusion of spleen cells from these mice with P3-X63-Ag8.653 myeloma cells. Hybridomas producing MAbs with affinity for all 3 E. coli K88 subtypes proved to be the most frequent ( 248 303 ), but subtype-specific monoclonals ( 39 303 ) as well as MAbs reacting with two but not with the third subtype ( 16 303 ) were also produced. The antibody-containing culture supernatants from 71 selected hybridomas were characterized by enzyme-linked immunosorbent assay (ELISA) titrations, ELISA inhibition experiments and further examined by immunoblotting. Derivation of several MAbs specific for one of the E. coli fimbrial antigens, K88ab, K88ac or K88ad, was of interest in view of the extensive sequence homology in their primary structures. Specific binding of the MAbs to fimbriae on the surface of K88-positive E. coli strains was indicated by agglutination tests and visualized by immuno gold labeling and electron microscopy. The present MAbs against K88 fimbriae have potential veterinary applications for diagnosis and treatment of porcine colibacillosis. Preliminary results indicate the therapeutic value of oral administration of murine ascitic fluid containing anti-K88 MAbs to piglets experimentally infected with E coli K88.

The ductus arteriosus (DA), connecting the aorta with the pulmonary artery in the fetus, which normally closes up just after birth in terrestrial mammals, has been claimed not to close, but to remain open in normal, adult cetaceans, just as in the adult lungfish. We have examined the hearts from two Pilot Whales. In those we found no persisting DA, but an almost totally obliterated lumen. Blood flow through the ductus of these two whales could be excluded. Instead of an anatomical shunt mammals may use a functional pulmonary shunt. To the extent diving mammals can empty their alveoli for air at depth through reinforced bronchioli, and their very compliant thorax, they block alveolar gas exchange, and thus avoid decompression sickness, nitrogen narcosis and pulmonary squeeze.