Rapid laboratory diagnosis of ulceroglandular tularemia with polymerase chain reaction.

Tularemia is a zoonotic disease which, in Scandinavia, is usually acquired through a mosquito bite. As the infecting organism, Francisella tularensis, is highly virulent the culturing of F. tularensis has generally been avoided. PCR offers a safe way to rapidly confirm diagnosis of tularemia. The case of a 9-y-old boy with ulceroglandular tularemia is presented. The diagnosis was made rapidly with DNA amplification from a pus specimen. The efficacy of ciprofloxacin treatment of tularemia in children is also discussed.

Characteristics of clodronate-induced apoptosis in osteoclasts and macrophages.

Bisphosphonates (BPs), such as clodronate and pamidronate, are inhibitors of bone resorption and are used on a widespread basis in the treatment of hyper-resorptive bone diseases. At the cellular level, BPs inhibit osteoclasts, but the precise molecular mechanisms are unclear. BPs have also been shown to affect the survival of macrophages, cells ontogenetically related to osteoclasts. We show that both clodronate and pamidronate induce apoptosis in isolated osteoclasts. Clodronate, when administered in liposomes, also induced apoptosis in rat peritoneal macrophages in vitro and in liver macrophages of mice in vivo but not in murine macrophage-like RAW-264 cells. The subcellular localization and staining intensity of Bcl-2, an anti-apoptotic protein known to protect several cell types against drug-induced apoptosis, were similar in RAW-264 and peritoneal macrophage cells, as revealed by immunofluorescence. The clodronate-induced apoptotic pathway was further characterized in isolated osteoclasts cultured on glass coverslips through the use of clodronate-containing liposomes and several inhibitors of the apoptotic cascade. None of the agents tested could totally prevent clodronate-induced osteoclast death. Partial protection was, however, obtained by the addition of staurosporine or homocysteine. The results suggest that primarily cytoplasmic, protein kinase C-activated mechanisms are involved in the execution of clodronate-induced apoptosis of osteoclasts.

A difference in the enzyme contents of resorption lacunae and secondary lysosomes of osteoclasts.

The distributions of tartrate sensitive lysosomal acid phosphatase (LAP) and cathepsin L in osteoclasts and the effect of parathyroid hormone (PTH) stimulation on them were investigated by using the protein A-gold method on ultracryosections of rat trabecular bone. LAP was located in association with the ruffled border membrane, in the resorption lacuna, on the mineral phase surrounding the lacuna, and in some primary lysosomes. After PTH treatment, the extracellular and ruffled border membrane associated LAP apparently increased. Heavy gold labelling for cathepsin L was confined exclusively to secondary lysosomes. No labelling was seen in the extracellular resorption lacuna or at the ruffled border. Acceleration of bone resorption by PTH-treatment did not change detectably the distribution or intensity of labelling. This study shows that the enzyme contents of secondary lysosomes and resorption lacunae are different and suggests that LAP is directly involved in extracellular bone degradation whereas the role of cathepsin L is restricted to lysosomes.

Mitochondrial carbonic anhydrase in osteoclasts and two different epithelial acid-secreting cells.

Acid secreting cells are rich in mitochondria and contain high levels of cytoplasmic carbonic anhydrase II. We have studied the ultrastructural distribution of a mitochondrial isoenzyme, carbonic anhydrase V, in two different acid-secreting epithelial cells, gastric parietal cells and kidney intercalated cells as well as in osteoclasts, which are the main bone resorbing cells. The mitochondria differ in carbonic anhydrase V content in these three acid-producing cells: gastric parietal cell mitochondria show strong immunolabelling for this isoenzyme, osteoclast mitochondria faint labelling and kidney intercalated cell mitochondria no labelling. The immunolabelling was located in the mitochondrial matrix, often in close contact with the inner mitochondrial membrane. These results show that mitochondrial carbonic anhydrase levels are not related to acid-transporting activity.

Vitronectin receptor has a role in bone resorption but does not mediate tight sealing zone attachment of osteoclasts to the bone surface.

During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption lacunae. Vitronectin receptor has previously been shown to be expressed in osteoclasts and it has been suggested that it mediates the tight attachment at the sealing zone. In this study we have shown that glycine-arginine-glycine-aspartic acid-serine pentapeptide inhibits bone resorption by isolated osteoclasts and drastically changes the morphology of the osteoclasts. When the vitronectin receptor was localized by immunofluorescence in rat and chicken osteoclasts cultured on bone slices, it was found to be distributed throughout the osteoclast cell membrane except in the sealing zone areas. Immunoperoxidase staining of rat bone sections at the light microscopical level also revealed intense staining of the cell membrane with occasional small unstained areas, probably corresponding to the sealing zones. Immunoelectron microscopy confirmed the results obtained by light microscopy showing specific labeling only at the ruffled borders and basolateral membranes (0.82 and 2.43 gold particles/microns of membrane, respectively), but not at the sealing zone areas (0.06 gold particles/microns of membrane). Both alpha v and beta 3 subunits of the vitronectin receptor were similarly localized. These results strongly suggest that, although the vitronectin receptor is important in the function of osteoclasts, it is not mediating the final sealing zone attachment of the osteoclasts to the mineralized bone surface.

Differential regulation of carbonic anhydrase II by androgen and estrogen in dorsal and lateral prostate of the rat.

Hormone regulation of carbonic anhydrase II (CA II) was studied in rat dorsal and lateral prostate. CA II is a major soluble protein in these accessory sex glands. The immunoelectronmicroscopy showed that CA II is expressed in their epithelial cells only. For studies on hormone regulation, adult male rats were castrated for 2 or 7 days. Groups of 7-day castrates and normal rats were treated daily either with testosterone or 17-beta-estradiol for 6 days and 2-day castrates for 1 day. CA II protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantified by RIA. The levels of CA II mRNA were studied by Northern blotting and hybridization of total RNA with a 32P-labeled mouse CA II cDNA clone. Castration of the rats decreased the concentration of CA II in lateral prostate but increased in dorsal prostate. These changes were reversed in both prostatic lobes by testosterone treatment. Estrogen treatment of castrated rats enhanced CA II concentration in lateral prostate but no effects were seen in the dorsal prostate of the same animals. In normal rats estrogen increased CA II concentration of dorsal prostate but there was no change in lateral prostate. Corresponding changes were observed in the levels of CA II mRNA in both tissues. The morphometric analyses showed that the castration- and hormone-induced changes of the mRNA and protein levels of the exclusively epithelial CA II could not be explained by any alterations in the proportions of epithelial and stromal components of the glands after hormone manipulations. The results demonstrate the differential steroid regulation of CAII in two prostatic lobes. Androgen regulates the expression of CAII at messenger RNA level, but the responses of CAII to testosterone are opposite in dorsal and lateral prostate. Estrogen increases CA II expression in lateral prostate but in dorsal prostate the castration-like effects of estrogen on CAII expression are probably indirect.

Carbonic anhydrase II in rat acid secreting cells: comparison of osteoclasts with gastric parietal cells and kidney intercalated cells.

Location of carbonic anhydrase II, an important enzyme involved in acid production, was studied using an immunogold method on ultracryosections. Its distribution in osteoclasts was compared with that in gastric parietal cells and kidney intercalated cells of the inner stripe of outer medulla. It is shown that the distribution of carbonic anhydrase II is much similar in all of these acid producing cells: most of the enzyme is cytoplasmic and nucleoplasmic and only a small fraction of the enzyme is associated with the apical plasma membrane. It seems likely that carbonic anhydrase II has a similar role in all of these acid producing cells.

Evidence for the presence of a proton pump of the vacuolar H(+)-ATPase type in the ruffled borders of osteoclasts.

Microsomal membrane vesicles prepared either from chicken medullary bone or isolated osteoclasts were shown to have ATP-dependent H(+)-transport activity. This activity was N-ethylmaleimide-sensitive but resistant to oligomycin and orthovanadate, suggesting a vacuolar-type ATPase. Furthermore, immunological cross-reactivity of 60- and 70-kD osteoclast membrane antigens with Neurospora crassa vacuolar ATPase was observed when analyzed by immunoblotting. Same antibodies labeled only osteoclasts in chicken and rat bone in immunohistochemistry. Immunoelectronmicroscopy localized these antigens in apical membranes of rat osteoclasts and kidney intercalated cells of inner stripe of outer medulla. Pretreatment of animals with parathyroid hormone enhanced the immunoreaction in the apical membranes of osteoclasts. No immunoreaction was seen in osteoclasts when antibodies against gastric H+,K(+)-ATPase were used. These results suggest that osteoclast resorbs bone by secreting protons through vacuolar H(+)-ATPase.