Description of a novel FR1 IgH PCR strategy and its comparison with three other strategies for the detection of clonality in B cell malignancies.

PCR amplification of IgH gene V-D-J junctional variability (IgH PCR) is increasingly replacing Southern analysis for the detection of clonal lymphoid populations in cases presenting diagnostic difficulties. In order to determine the most efficient strategy, we have compared three known methods, using consensus primers against the VH FR3 or FR2 (FR256) regions, or a mix of six primers against the FR1 region (FR1f), with a new approach using a consensus primer against FR1 (FR1c), never previously described for diagnostic purposes, on DNA from 89 monoclonal B-cell proliferations (16 ALL, 28 CLL/PLL, 15 myelomas, 30 NHL). We obtained a detection rate of 70% for FR3, 64% for FR1f and 77% and 78% for FR256 and FR1c, respectively.

Bone matrix proteins in osteogenesis and remodelling in the neonatal rat mandible as studied by immunolocalization of osteopontin, bone sialoprotein, alpha 2HS-glycoprotein and alkaline phosphatase.

The neonatal rat mandible was used as a model to study bone formation, mineralization, quiescence, and resorption, using immunolocalization and a variety of tissue-processing techniques. Monospecific antibodies for osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (AP) and alpha 2HS-glycoprotein (alpha 2HS-GP) were used on fixed paraffin-embedded tissue, fixed frozen tissue and unfixed frozen tissue. Immunostaining was correlated with mineral content by two procedures, the von Kossa and the morin techniques.

Dexamethasone alters the subpopulation make-up of rat bone marrow stromal cell cultures.

Bone marrow stromal cells comprise a heterogeneous population including fibroblastic, adipocytic, hemopoietic, and osteogenic cells. Although the conditions under which different lineages are regulated have not been fully elucidated, dexamethasone clearly stimulates osteogenic expression in stromal cultures. The purpose of this study was to begin to elucidate and quantify some of the subpopulations present when rat bone marrow stromal cells are grown with or without dexamethasone under conditions favoring bone formation.

Evaluation of a new non-isotopic sandwich hybridization for the detection of HIV1-specific PCR products.

A new non-isotopic sandwich hybridization assay was developed to detect human immunodeficiency virus type 1 (HIV1) provirus amplified by the polymerase chain reaction. The sensitivity and specificity of this new technique using 96-well microplates as the support for the sandwich hybridization procedure and stable enzyme-linked oligomer as the detection probe were compared with those of Southern hybridization using a 32P-labelled oligomer probe. Three laboratories studied 437 peripheral blood mononuclear cell samples from 294 different subjects including both HIV1-seropositive and -seronegative individuals.

Simultaneous detection of multiple bone-related mRNAs and protein expression during osteoblast differentiation: polymerase chain reaction and immunocytochemical studies at the single cell level.

Messenger RNA expression analyzed by in situ hybridization and Northern analysis and protein expression analyzed biochemically or immunocytochemically have been used to study the developmental expression of various osteoblast (OB)-associated molecules. These approaches have shown that over a time course of OB differentiation in vivo and in vitro, the expression of macromolecules associated with OB cells changes. However, ambiguities in data from different approaches and in populations representative of cells at different developmental stages are extant.

Identification of human herpesvirus 6 variants A and B by amplimer hybridization with variant-specific oligonucleotides and amplification with variant-specific primers.

Two distinct PCR-based procedures were evaluated for the detection and identification of human herpesvirus 6 (HHV-6) variants A and B in uncultured human samples. Variant-specific oligonucleotide hybridization (VSOH) is based on the amplification of two distinct regions of the HHV-6 genome, followed by hybridization of amplimers with variant-specific oligonucleotide probes. Variant-specific primer PCR (VSPP) is based on the amplification of each variant by using variant-specific primers.

Displacement and translocation of osteoblast-like cells by osteoclasts.

Rabbit osteoclasts and rabbit osteoblast-like stroma cells (OB cells) were placed onto plastic surfaces and the migration patterns of individual osteoclasts and osteoclast-OB interactions were analyzed with time-lapse recording. To induce directed migration, the cultures were exposed to an electrical field of 0.01 or 0.

Carbonic anhydrase II mRNA expression in individual osteoclasts under "resorbing" and "nonresorbing" conditions.

Rabbit osteoclasts can be transformed from a nonresorbing state to a resorbing state by transferring them from culture medium at pH 7.5 to one at pH 6.5.

1,25-dihydroxyvitamin D3 stimulates adipocyte differentiation in cultures of fetal rat calvaria cells: comparison with the effects of dexamethasone.

Progenitor cells for several mesenchymally derived cell types exist within freshly isolated fetal rat calvaria (RC) cell populations. We have characterized the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the differentiation of adipocytes from primary RC cells and compared these effects with those of dexamethasone (Dex). RC cells were plated at 3 x 10(4)/35-mm dish, and cultures were maintained for 14-19 days in alpha-Minimum Essential Medium containing 10% fetal bovine serum, 50 micrograms/ml ascorbic acid, 10 mM Na beta-glycerophosphate, and 0.

[A new virus: the human herpesvirus 6].

Human herpesvirus 6 (HHV-6) was discovered in 1986. This novel virus is genetically related to cytomegalovirus. HHV-6 mainly infects T lymphocytes but its tropism appears to be much wider and probably involves some epithelial cells.

Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures.

Rat bone marrow stromal cells comprise a heterogeneous mixture of cell lineages including osteoblastic cells. When grown in the presence of ascorbic acid, beta-glycerophosphate and 10(-8) M dexamethasone, osteoprogenitor cells within the population divide and differentiate to form bone nodules (Maniatopoulos et al., 1988, Cell Tissue Res.

Utilization of a specialized clinic following an ecological accident.

On August 23, 1988, a fire broke out in a warehouse storing PCBs, forcing the evacuation of 5,500 citizens. Three days later, a specialized clinic was opened to examine and reassure the population. Seventy percent of the evacuated people showed up at the clinic.

Differential effects of fluoride during initiation and progression of mineralization of osteoid nodules formed in vitro.

Osteoid nodules form in cultures of fetal rat calvarial (RC) cells grown in medium containing 10% FBS and 50 micrograms/ml of ascorbic acid. When 10 mM beta-glycerophosphate (beta-GP) is added, osteoid nodules mineralize in two phases: an initiation phase, which is dependent upon alkaline phosphatase activity for conversion of beta-GP to P(i), and a progression phase that proceeds independently of alkaline phosphatase activity and does not require exogenous phosphate. We have now used this system to investigate the effects of fluoride (F-) on mineralization.

Osteoclast differentiation in cocultures of a clonal chondrogenic cell line and mouse bone marrow cells.

Previous reports have demonstrated that hemopoietic progenitor cells derived from mouse bone marrow can form osteoclast-like cells when cultured in the presence of stromal cells and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. We show here that in cocultures of mouse bone marrow cells and a clonal chondrogenic cell line (C5.18), a stimulation of the number of tartrate-resistant acid phosphatase-positive (TRAP+) colonies is seen with or without the addition of 1,25-(OH)2D3 to the cultures.

Antigenic and genetic differentiation of the two putative types of human herpes virus 6.

Ten human herpes virus 6 (HHV-6) strains from different origins were studied using reactivity to monoclonal antibodies and polymerase chain reaction analysis. Using immunofluorescence and neutralization assays, two monoclonal antibodies gave a positive reaction with the ten strains while three others only reacted with a fraction of these strains. This differential reactivity permitted segregation of the ten strains into two non-overlapping antigenic groups, designated as I and II.

Characterization of the 1,25-(OH)2D3-induced inhibition of bone nodule formation in long-term cultures of fetal rat calvaria cells.

We investigated the effects of 1,25-dihydroxyvitamin D3[1,25-(OH)2D3], on osteoprogenitor cell differentiation and bone nodule formation at various stages of differentiation by evaluating the effects on long term cultures of fetal rat calvaria (RC) cells. RC cells were plated at 3 x 10(4) cells/35-mm dish in alpha-minimal essential medium containing 15% fetal bovine serum, ascorbic acid, and beta-glycerophosphate (beta-GP), conditions under which bone nodules form. 1,25-(OH)2D3 inhibited bone nodule formation in a dose-dependent manner with total inhibition occurring at 1-10 nM and half-maximal inhibition occurring at approximately 0.

The role of phospholipase A2 in interleukin-1 alpha-mediated inhibition of mineralization of the osteoid formed by fetal rat calvaria cells in vitro.

Interleukin-1 (IL-1) may be an important mediator of bone remodeling, since it is a potent stimulator of bone resorption and has biphasic effects on bone formation. Continuous exposure of fetal rat calvaria (RC) cells to IL-1 alpha or IL-1 beta results in a dose-dependent inhibition of both bone nodule formation and mineralization of the organic matrix. In this study, the effects of recombinant human IL-1 alpha on the mineralization process were examined by the addition of IL-1 alpha late in the culture period, after osteoid nodules had formed and when they were induced to mineralize by the addition of organic phosphate.

Differential regulation of phospholipase A2 by cytokines inhibiting bone formation and mineralization.

Treatment of fetal rat calvarial cells with interleukin-1 alpha, tumor necrosis factor-alpha, transforming growth factor-beta 1, or group II phospholipase A2 inhibits the number of bone nodules formed in long-term cultures. These same mediators also inhibit the mineralization of fully developed bone nodules in a time and dose-dependent fashion. The pro-inflammatory cytokines interleukin-1 alpha and tumor necrosis factor-alpha cause a dose-dependent induction of rat calvarial cell phospholipase A2-II mRNA levels, suggesting that their effects on bone formation may be mediated indirectly by activation of this enzyme.

beta-Glycerophosphate-induced mineralization of osteoid does not alter expression of extracellular matrix components in fetal rat calvarial cell cultures.

When fetal rat calvarial cells are cultured in medium containing vitamin C, osteoid nodules develop after approximately 15 days of culture. Upon addition of an organic phosphate (beta-glycerophosphate, beta GP), these nodules mineralize. We have now used this system to explore the suggestion made by others that a negative feedback may exist between matrix mineralization on the one hand and the synthesis of alkaline phosphatase and bone matrix collagen on the other by analyzing the synthesis of these proteins and the levels of their mRNAs in mineralizing and nonmineralizing cultures.

Differential chemotactic responses of different populations of fetal rat calvaria cells to platelet-derived growth factor and transforming growth factor beta.

We tested the chemotactic response to platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF beta) of cells released enzymatically from fetal rat calvaria (RC). Both factors were chemotactic for RC cells, but the magnitude of the chemotactic response differed markedly between different populations and varied with time in culture of the cell populations. Cells released earlier from the calvaria showed a greater response than osteoblast-enriched populations released later.

Isolation of monoclonal antibodies recognizing rat bone-associated molecules in vitro and in vivo.

Knowledge of the number and kinds of differentiation steps that characterize cells of the osteoblast lineage is inadequate. To further analyze osteoblast differentiation, we generated a series of monoclonal antibodies (MAb) to osteogenic cells. Spleen cells from mice immunized with whole-cell populations enriched for expression of osteoblast-associated properties or bone formation in vitro were fused with the SP2/0 myeloma cell line.

Inorganic phosphate added exogenously or released from beta-glycerophosphate initiates mineralization of osteoid nodules in vitro.

Rat calvaria (RC) cells grown in medium containing ascorbic acid form nodules of osteoid and cells. When 10 mM beta-Glycerophosphate (beta-GP) is added, the osteoid mineralizes in two phases: an initiation phase that is dependent upon alkaline phosphatase activity and a progression phase that proceeds independently of the activity of alkaline phosphatase and does not require added beta-GP (Bellows et al., Bone Miner 1991;14:27-40).