A high proportion of novel mutations in BRCA1 with strong founder effects among Dutch and Belgian hereditary breast and ovarian cancer families.

We have identified 79 mutations in BRCA1 in a set of 643 Dutch and 23 Belgian hereditary breast and ovarian cancer families collected either for research or for clinical diagnostic purposes. Twenty-eight distinct mutations have been observed, 18 of them not previously reported and 12 of them occurring more than once. Most conspicuously, a 2804delAA mutation has been found 19 times and has never been reported outside the Netherlands. A common haplotype spanning > or = 375 kb could be identified for each of the nine examined recurrent mutations, indicating the presence of multiple BRCA1 founder mutations in the Dutch population. The 2804delAA mutation has been estimated to have originated approximately 32 generations ago. No specific breast or ovarian cancer phenotype could be assigned to any of the common mutations, and the ovarian cancer incidence among 18 families with the 2804delAA mutation was heterogeneous.

Changes in proteoglycans of ageing and osteoarthritic human articular cartilage: an electron microscopic study with polyethyleneimine.

BACKGROUND: Ageing and osteoarthritic (OA) cartilage show characteristic alterations in chondrocyte morphology and in the composition and content of matrix proteoglycans (PGs). Data concerning matrix components are mostly of biochemical nature. Ultrastructural histochemistry is needed to gain more information about distribution of these altered matrix components. METHODS: We used the cationic dye polyethyleneimine (PEI) to visualize at the EM level alterations in the distribution and dimensions of PGs of human healthy young, healthy aged, and OA articular cartilage. RESULTS: Young cartilage contained PEI-positive granules in the superficial layer and big winding PEI-positive structures in the deeper layers. In the healthy aged tissue, PEI-positive granules were observed throughout the matrix and smaller winding structures were present in the deeper layer. In OA cartilage both types of PEI-positive structures were absent in the superficial layer. Deeper in the matrix PEI-positive granules could be demonstrated. Moreover, PEI-positive angular structures were observed in the deeper zones. CONCLUSIONS: The differences in PEI-positive structures are a good reflection of the differences in PGs between young, ageing, and OA cartilage as demonstrated in biochemical studies. PEI, used at the EM level, gave more precise information concerning the localized changes in quality, quantity, and location of PGs in articular cartilage during ageing and disease.

Murine macrophage precursor cell lines are unable to differentiate into osteoclasts: a possible implication for osteoclast ontogeny.

Six murine macrophage precursor cell lines, thought to be arrested around the CFU-GM stage of the myeloid differentiation and shown to be negative for acid phosphatase, F4/80 antigen expression and phagocytosis capacity, were tested for their ability to differentiate into osteoclasts. Their differentiation potential was compared with that of the haemopoietic stem cell line FDCP-mix C2GM. None of the macrophage precursor cell lines could be induced to differentiate into osteoclasts when the cells were cocultured with either periosteum-free metatarsal bones of fetal mice, or monolayers of osteoblast-like cells. In contrast, when the haemopoietic stem cell line FDCP-mix C2GM, murine fetal liver cells or murine spleen cells were used as a source of haemopoietic precursor cells, numerous osteoclasts were formed in both culture systems. During cell culture a small percentage of the macrophage precursor cells attached to the bottom of the culture well. These firmly attached cells acquired acid phosphatase activity, F4/80 antigen expression and phagocytosis capacity. Furthermore, when the cell lines were cultured for 2 or 4 days with 1% DMSO, up to 30% of the precursor cells differentiated into metamyelocytes. These results suggest that the macrophage precursor cell lines are able to acquire macrophage and granulocyte characteristics, but are unable to differentiate into osteoclasts. In contrast, the haemopoietic stem cell line FDCP-mix C2GM is able to differentiate into both macrophages and osteoclasts. We therefore suggest that the osteoclast lineage branches off at an early stage of the myeloid differentiation pathway.

An electron microscopic study on the presence of proteoglycans in the mineralized matrix of rat and human compact lamellar bone.

The presence of proteoglycans (PGs) was studied in compact lamellar rat and human bone at the electron microscopic level. With the cationic dye cuprolinic blue (CB1), PGs could be demonstrated in the mineralized bone matrix. The amounts of PGs appeared to be equal in the different lamellae and osteons. More CBl-positive material was found in the outermost lamella of the cortex, in the perilacunar matrix around the osteocyte lacunae, and around the canaliculi. Enzyme digestion with chondroitinase ABC demonstrated that the CBl-positive rods consisted of PGs. These observations amplify biochemical studies in which PGs have been isolated from the mineralized bone matrix. The presence of CBl-positive rods in the mineralized matrix suggest that PGs do not have to be removed completely to make the matrix calcifiable.

Polyethyleneimine as a contrast agent for ultrastructural localization and characterization of proteoglycans in the matrix of cartilage and bone.

We examined the presence of proteoglycans in the extracellular matrix of cartilage and bone in fetal mouse radii at the ultrastructural level, using the cationic dye polyethyleneimine (PEI). After staining with this dye, the proteoglycans appeared as granules in the uncalcified bone matrix and as extended winding structures in the cartilage matrix. PEI-positive material was removed after treatment of the tissue with chondroitinase ABC. Inhibition of the proteoglycan synthesis by beta-D-xyloside resulted in smaller PEI-positive windings in the cartilage matrix. These observations suggest that the winding, PEI-positive structures represent proteoglycan aggregates. No loss of PEI-positive material in the calcified cartilage matrix was seen, suggesting that proteoglycans do not need to be removed to make the matrix calcifiable.

Morphologic features of bone in human osteopetrosis.

Trabecular bone biopsies obtained from six patients with malignant osteopetrosis, one patient with benign osteopetrosis, and two controls were examined by light and electron microscopy. Osteopetrotic osteoclasts showed little to no signs of active involvement in bone resorption. Ruffled borders and clear zones were absent in most cells. In all patients there were large numbers of osteoclasts. Numbers of osteoblasts, bone lining cells, and bone marrow stromal cells were extremely low in all patients with malignant osteopetrosis. In six of the patients a mineralized layer of amorphous organic material lacking collagen fibrils was seen covering large areas of the bone or cartilage matrix. We suggest that this layer represents a pathological calcification on which subsequently organic material has accumulated. The abnormalities in osteopetrotic bone are discussed in the light of the pathogenesis of this disease.

Osteoclast formation in vitro from progenitor cells present in the adult mouse circulation.

The development of multinucleated cells with tartrate-resistant acid phosphatase (TRAP) activity was studied in coverslip cultures of murine blood leukocytes and in cocultures of blood leukocytes with murine fetal bone rudiments. Cells with TRAP activity were not present among the leukocytes before culture and were absent in the bone rudiments at the time of explanation. After 14 days, macrophages with only tartrate-sensitive acid phosphatase activity developed in cultures of leukocytes without long bones. Multinucleated cells were not seen. In cocultures of leukocytes with bone rudiments, however, multinucleated cells with a strong TRAP activity had formed after 10-14 days of coculture. These TRAP-positive cells had invaded the bones and resorbed part of the calcified matrix. Electron microscopy revealed ruffled borders on the resorbing cells. In cocultures, TRAP-positive cells also formed from leukocyte fractions depleted of strongly adherent cells. Also on the cellophane supports of the cocultures, mononuclear cells with a stellate appearance and a strong TRAP activity were seen. We suggest that, in the cocultures, osteoclasts developed from a TRAP-negative, circulating progenitor cell. The presence of osteoclast progenitor cells in the circulation is discussed in light of the descent of osteoclasts from hematopoietic stem cells. That appearance of TRAP activity was always seen in resorbing cells and was not acquired in monocytes present in the leukocyte fraction by mere culture means that in the mouse TRAP is a useful marker for osteoclasts.

An electron microscopical study on the presence of proteoglycans in the calcified bone matrix by use of cuprolinic blue.

With Cuprolinic Blue (CBl) as contrasting agent, PGs could be demonstrated in mouse fetal bone matrix. Large CBl-positive rod-like structures proved to be present in and outside the calcification nodules in regions of beginning mineralization. In further developed bone also smaller rods were present in the mineralized matrix. The CBl-positive rods were sensitive to chondroitinase ABC and hyaluronidase. Under the circumstances we chose, this indicates that these structures are PGs containing chondroitin and/or dermatan sulphate. The fine filamentous and granular material in the nodules was still present after digestion with these enzymes, but disappeared after treatment with pronase. This is an indication that this material mainly contains proteins.

A morphologic study of osteoclasts isolated from osteopetrotic microphthalmic (mi/mi) mouse and human fetal long bones using an instrument permitting combination of light and scanning electron microscopy.

Cell surface structures of isolated osteopetrotic (mi/mi) and normal murine (+/+ and +/mi) and human osteoclasts were examined in a microscope combining light and scanning electron microscopy (LM/SEM). Tartrate-resistant acid phosphatase (TrAP) was used as an histochemical osteoclast marker. In osteopetrotic bone, as in normal murine bone, TrAP activity was exclusively seen in osteoclasts and preosteoclasts and was therefore judged a suitable marker for identification of isolated osteoclasts. A method was developed for preparation of LM/SEM specimens from osteoclast-enriched cell suspensions. In the LM/SEM isolated osteoclasts were easily recognized in the LM mode by TrAP contents. In specimens prepared from murine cells, but not human cells, LM identification of osteoclasts by TrAP was essential. This was in particular true for small, mononuclear, mi/mi osteoclasts. All osteoclasts examined had a villous appearance and were well spread over the glass substrate. There were no differences in cell surface morphology and in adherence to glass between osteopetrotic and normal osteoclasts.

Osteoclast generation from human fetal bone marrow in cocultures with murine fetal long bones. A model for in vitro study of human osteoclast formation and function.

Osteoclast formation in vitro from human progenitor cells was studied in cocultures of periosteum-free murine long-bone rudiments and human fetal tissues. No osteoclasts were generated from chorionic villi or from fetal liver, but bone marrow and purified bone-marrow fractions gave rise to multinucleated cells that resorbed calcified cartilage matrix. These polykarya react very strongly for tartrate-resistant acid phosphatase (TrAP) and upon ultrastructural examination show large ruffled borders in areas of resorption. Resorption of murine calcified cartilage matrix by human osteoclasts was less than resorption. by osteoclasts formed from murine fetal bone-marrow cells. Our results show that the murine long-bone rudiment can be used to generate osteoclasts from human sources of progenitor cells and to assess the biological activity of the formed osteoclasts. This coculture system thereby offers possibilities to study human osteoclast pathology in vitro. The use of TrAP as marker for osteoclasts in cell cultures is discussed.