Can ultrastructural particle location predict aseptic loosening? A biopsy study of nonloose hip implants one year postoperative using three bearing material combinations.

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.

Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death.

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.

The transferrin receptor of Actinobacillus pleuropneumoniae: quantitation of expression and structural characterization using a peptide-specific monoclonal antibody.

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 60-70kDa 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.

Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor.

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.

Selective depletion of heat shock protein 70 (Hsp70) activates a tumor-specific death program that is independent of caspases and bypasses Bcl-2.

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.

Effect of mts1 (S100A4) expression on the progression of human breast cancer cells.

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.

A20 zinc finger protein inhibits TNF and IL-1 signaling.

A20 zinc finger protein is a product of a cytokine-induced primary response gene. In this report, we demonstrate that A20 specifically inhibits signal transduction pathways induced by TNF and IL-1, suggesting that it functions as a negative regulator of the cytokine response. Overexpression of A20 in MCF7 breast carcinoma cells or in WEHI-S fibrosarcoma cells inhibits apoptosis induced by TNF, whereas cytotoxicity induced by anti-Fas (anti-CD95); lymphokine-activated killer (LAK) cells, serum starvation, oxidative stress, or okadaic acid is not inhibited. Overexpression of A20 also inhibits TNF-induced activation of phospholipase A2 in a similar dose-dependent manner as it inhibits TNF-mediated apoptosis, whereas it does not affect the activation of phospholipase A2 by anti-Fas. Interestingly, A20 also blocks TNF-induced signal transduction pathways not directly related to the cytotoxicity, namely activation of NF-kappa B and AP-1 transcription factors. Activation of these transcription factors by a functionally related cytokine, IL-1, is also inhibited by A20, whereas activation induced by hydrogen peroxide or phorbol ester is unaffected. Overexpression of A20 does not affect the binding of TNF to its cell surface receptors. These data suggest that A20 functions as a negative regulator of TNF and IL-1, interfering with signal transduction pathways at an early point following receptor binding but before the activation of various second messengers, leading to the wide variety of effects induced by these cytokines.

Localization of NCAM on NCAM-B-expressing cells with inhibited migration in collagen.

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.

Epithelial transport and bioavailability of intranasally administered human growth hormone formulated with the absorption enhancers didecanoyl-L-alpha-phosphatidylcholine and alpha-cyclodextrin in rabbits.

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.

Immunoelectron microscopy of the receptor for urokinase plasminogen activator and cathepsin D in the human breast cancer cell line MDA-MB-231.

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.

Confocal fluorescence microscopy of urokinase plasminogen activator receptor and cathepsin D in human MDA-MB-231 breast cancer cells migrating in reconstituted basement membrane.

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.

Ultrastructural localization of the Pasteurella multocida toxin in a toxin-producing strain.

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.

Early effects of radiotherapy in small cell lung cancer xenografts monitored by 31P magnetic resonance spectroscopy and biochemical analysis.

31P magnetic resonance spectroscopy (31P MRS) and biochemical analysis of extracts were applied to study the metabolic response to X-irradiation of small cell lung cancer in nude mice. Two small cell lung cancer xenografts, CPH SCCL 54A and 54B, with different radiosensitivity, although derived from the same patient, were studied. A total of 126 individual tumors were examined. Following 5.0-Gy irradiation, a reversible increase in the ATP/Pi ratio, reaching twice the pretreatment level within 2 wk, was observed with 31P MRS, while 20 Gy induced a reversible decrease in the ATP/Pi ratio. The t1/2 of this decline was 2 to 3 h for 54A and about 6 h for the less radiosensitive 54B. The 31P MRS data were compared with biochemical analysis of tumors freeze-clamped and extracted at similar intervals after 20 Gy. It appeared that an acute reversible increase in Pi concentration was the major cause of the ATP/Pi decrease induced by 20 Gy. A linear correlation between ATP/Pi estimated by 31P MRS and by analytical biochemistry was found. The ATP/Pi ratio may be valuable for early assessment of radiosensitivity of small cell lung cancer tumors.

Characterization of the dermal lesions induced by a purified protein from toxigenic Pasteurella multocida.

The dermonecrotic effect of purified Pasteurella multocida toxin (PMT) was studied sequentially in guinea pigs and rats. The skin reaction was initially an acute inflammatory reaction, with edema and emigration of neutrophils and a few eosinophils and diapedesis of some erythrocytes. Four hours after intracutaneous injection the vessels were congested and thrombocytes were focally attached to the endothelial wall. Twenty-four h after the injection the inflammatory reaction appeared more severe and venules and arterioles were thrombosed. Necrotic changes were seen in hair follicles and in striated muscle fibers. Crude extracts from P. multocida and Clostridium perfringens injected intracutaneously into guinea pigs induced skin lesions qualitatively similar to the lesions induced by the purified PMT, indicating that dermonecrotic bacterial toxins may share similar biochemical properties.

Ductus arteriosus in pilot whales.

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.

Adult rhabdomyoma of the oropharynx recurring three times within thirty-five years.

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.

Monoclonal antibodies to K88ab, K88ac and K88ad fimbriae from enterotoxigenic Escherichia coli.

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 effect of bromhexine on the kidney lesions in NZB-NZW-F1 mice.

The NZB-NZW-F1 mice develop a clinical picture resembling SLE and an exocrinopathy resembling Sjögren's syndrome. Three groups of hybrids were treated from their 20th week of age for 10, 17 and 20 weeks (groups 1, 2, 3) with Bromhexine in two different concentrations--6 & 60 mg/kg and placebo. NMRJ mice treated with placebo acted as healthy controls. After the treatment the kidneys were examined by light microscopy. The kidneys exhibited lupus-like lesions. Animals treated with 60 mg/kg Bromhexine for 17 weeks had a significantly lower degree of changes than had the other hybrids.