Large differences in cost of illness and wellbeing between patients with fibromyalgia, chronic low back pain, or ankylosing spondylitis.

OBJECTIVE: To compare the cost of illness of three musculoskeletal conditions in relation to general wellbeing. METHODS: Patients with fibromyalgia, chronic low back pain (CLBP), and ankylosing spondylitis who were referred to a specialist and participated in three randomised trials completed a cost diary for the duration of the study, comprising direct medical and non-medical resource utilisation and inability to perform paid and unpaid work. Patients rated perceived wellbeing (0-100) at baseline. Univariate differences in costs between the groups were estimated by bootstrapping. Regression analyses assessed which variables, in addition to the condition, contributed to costs and wellbeing. RESULTS: 70 patients with fibromyalgia, 110 with chronic low back pain, and 111 with ankylosing spondylitis provided data for the cost analyses. Average annual disease related total societal costs per patient were 7813 euro for fibromyalgia, 8533 euro for CLBP, and 3205 euro for ankylosing spondylitis. Total costs were higher for fibromyalgia and CLBP than for ankylosing spondylitis, mainly because of cost of formal and informal care, aids and adaptations, and work days lost. Wellbeing was lower in fibromyalgia (mean, 48) and low back pain (mean, 42) than in ankylosing spondylitis (mean, 67). No variables other than diagnostic group contributed to differences in costs or wellbeing. CONCLUSIONS: In patients under the care of a specialist, there were marked differences in costs and wellbeing between those with fibromyalgia or CLBP and those with ankylosing spondylitis. In particular, direct non-medical costs and productivity costs were higher in fibromyalgia and CLBP.

Application of the CFU-GM assay to predict acute drug-induced neutropenia: an international blind trial to validate a prediction model for the maximum tolerated dose (MTD) of myelosuppressive xenobiotics.

In a previous study of prevalidation, a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed, to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up, by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper, we describe an international blind trial designed to apply this model to the clinical neutropenia, by testing 20 drugs, including 14 antineoplastics (Cytosar-U, 5-Fluorouracil, Myleran, Thioguanine, Fludarabine, Bleomycin, Methotrexate, Gemcitabine, Carmustine, Etoposide, Teniposide, Cytoxan, Taxol, Adriamycin); two antivirals (Retrovir, Zovirax,); three drugs for other therapeutic indications (Cyclosporin, Thorazine, Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin, Bleomycin, Etoposide, Fludarabine, 5-Fluorouracil, Myleran, Taxol, Teniposide, Thioguanine, and Thorazine), IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine, Cyclosporin, Cytosar-U, Cytoxan, Gemcitabine, Indocin, Lindane, Methotrexate, Retrovir, and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had "actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD, instead of the 4-fold range required by the model, whereas the prediction with Cytosar-U was approximately 10-fold in error. A possible explanation for the failure in the prediction of these three drugs, which are pyrimidine analogs, is discussed. We concluded that our model correctly predicted the human MTD for 20 drugs out of 23, since the other three drugs (Topotecan, PZA, and Flavopiridol) were tested in the prevalidation study. The high percentage of predicitivity (87%), as well as the reproducibility of the SOP testing, confirm that the model can be considered scientifically validated in this study, suggesting promising applications to other areas of research in developing validated hematotoxicological in vitro methods.

Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay.

This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement), two SOPs were assessed, by using two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer), the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance), dedicated to a training set of six anticancer drugs (adriamycin, flavopindol, morpholino-doxorubicin, pyrazoloacridine, taxol and topotecan), a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics. As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy. On the basis of these results, we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional xenobiotics that represent the spectrum of haematotoxic potential.

Modulation of phenotype, cytokine production and stimulatory function of CD34+-derived DC by NiCl(2) and SDS.

One of the most promising alternatives to identify the sensitizing potency of new products is the in vitro culture of human dendritic cells (DC). In vivo, dendritic cells present in the skin are highly specialized antigen-presenting cells (APC) of the immune system, which play a crucial role in the induction of allergic reactions. The DC produce specific cytokines and upregulate specific co-stimulatory molecules in addition to the antigen-MHC complex in order to promote an optimal T-cell activation. The aim of our study is to assess the phenotype, cytokine production and autologous T-cell stimulatory capacity of the in vitro CD34+-derived dendritic cells after 24 hours of incubation with the metal allergen NiCl(2) (100-300 microM) and the irritant sodium dodecyl sulfate (SDS; 0.01%), in order to find a sensitive endpoint to discriminate between sensitizers and irritants. After exposure to Ni, a significant increase in the number of CD83+ and CD86+ cells was noticed. The intensity of CD86 as well as the intensity of the HLA-DR molecule on the DC also showed a significant increase. The expression of the co-stimulatory molecule CD80 was not changed after exposure. SDS was not able to increase the expression of any of the analysed markers. The production of IL-6 increased significantly after exposure of dendritic cells to Ni, but not after SDS exposure. Results on tumor necrosis factor-alpha (TNF-alpha) production are somewhat equivocal. Although not statistically significant, TNF-alpha was upregulated in one out of three experiments after 48 hours of exposure to the Ni allergen, but increases were also noticed after exposure to SDS (two out of three experiments). Both exposure to Ni and SDS caused an upregulation (not significant) in the IL-12 production by DC, but the production was higher in Ni-exposed DC compared to SDS-exposed cells. In none of the exposed DC cultures was it possible to detect IL-1 beta. The antigen-presenting capacity of the DC in autologous MLR could not be demonstrated. Nevertheless, T-cell proliferation after DC stimulation was noticed in allogenic MLR.

Haemopoietic cell proliferation in murine bone marrow cells exposed to extreme low frequency (ELF) electromagnetic fields.

As leukemia is one of the health hazards that is sometimes associated with exposure to extreme low frequency fields, we studied the in vitro effects of ELF fields on haemopoietic cell proliferation. First, the cytotoxic effect of 80 microT, 50 Hz magnetic fields on 3T3 cell proliferation was investigated using the neutral red test. Many chemicals are believed to cause damage because they interfere with basal or "housekeeping" cell functions. The basal cell functions are present in every cell. Non-specialized, actively dividing cells are suitable for measuring cytotoxic effects. Cytotoxic doses can be identified by exposing actively dividing cells in vitro and measuring growth inhibition caused by interference with these basal cell functions. 80 microT, 50 Hz magnetic fields caused no cytotoxicity: we were not able to demonstrate any interference with essential cell functions in the non-differentiated 3T3 cell line. Furthermore, the in vitro effects of ELF fields on murine haemopoietic and stromal stem cell proliferation were studied. Haemopoiesis is a continuous process, where mature blood cells are replaced by the proliferation and differentiation of more primitive progenitor and stem cells. Blood formation is tightly regulated by the stromal micro-environment. Exposure of murine bone marrow cells, from male and female mice, to 80 microT (50 Hz) magnetic fields showed a reduction in the proliferation and differentiation of the granulocyte-macrophage progenitor (CFU-GM) compared to non-exposed bone marrow cells. The results on the effect of the ELF-field on stromal stem cell proliferation (CFU-f) are somewhat equivocal at the moment. CFU-f from female mice showed a reduction while CFU-f from male mice were not decreased.

Use of in vitro assays to assess hematotoxic effects of environmental compounds.

The number of chemicals being introduced into the environment increases and many of these substances may pose a health risk to exposed individuals. Many environmental toxicants with a potential toxicity to the hematopoietic system have been identified by animal experiments. Owing to the risks of severe chronic hematopoietic disorders, it is important to screen chemicals for their hematotoxicity. The aim of this work was to identify, through the use of in vitro techniques, targets for hematotoxic effects. Our study focused on myeloid and erythroid hematopoietic progenitors and stromal stem cells as possible targets. The in vitro assays showed that various hematotoxic compounds exert different effects on these cell populations. In vitro exposure of murine bone marrow cells to various inorganic (cadmium, lead) and organic (benzene metabolites, lindane. benzo-[a]-pyrene (BaP), PCB (polychlorinated biphenyl) congeners) environmental chemicals indicated that hematopoietic or stromal bone marrow cells were targets for most of the chemicals. Stromal cells were more affected by lead, cadmium, and BaP compared to myeloid cells. Benzene and phenol gave no response, but the metabolites catechol and hydroquinone were equally toxic to the stromal and the myeloid progenitor cells. Among the PCBs tested, PCB126 was most toxic. Human progenitor cells derived from cord blood were exposed in vitro to catechol, hydroquinone, lead nitrate, and PCBs. Human hematopoietic cells were sensitive to the tested compounds. Human erythroid progenitors are more susceptible to lead exposure than are myeloid progenitors. Based on the in vitro tests, humans are more sensitive to lead, catechol, and PCB126 than are mice. In contrast to the murine data, humans responded with individual differences to lead and PCB126.

Structural analysis of flavinylation in vanillyl-alcohol oxidase.

Vanillyl-alcohol oxidase (VAO) is member of a newly recognized flavoprotein family of structurally related oxidoreductases. The enzyme contains a covalently linked FAD cofactor. To study the mechanism of flavinylation we have created a design point mutation (His-61 --> Thr). In the mutant enzyme the covalent His-C8alpha-flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The H61T mutant displays a similar affinity for FAD and ADP (K(d) = 1.8 and 2.1 microm, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (k(cat) = 0.24 s(-)(1), K(m) = 40 microm) than the wild-type enzyme. The crystal structures of both the holo and apo form of H61T are highly similar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These results show that covalent flavinylation is an autocatalytical process in which His-61 plays a crucial role by activating His-422. Furthermore, our studies clearly demonstrate that in VAO, the FAD binds via a typical lock-and-key approach to a preorganized binding site.

The monoclonal antibodies 18d7/91f2 recognize a receptor regulatory protein on mouse bone marrow stromal cells.

Two monoclonal antibodies 18D7 and 91F2 were developed by immunizing rats with the mouse bone marrow-derived osteogenic cell line MN7. Hybridomas secreting rat antibodies against MN7 cell surface markers were selected by flow cytometry analysis. Both the monoclonal antibody 18D7 and the monoclonal antibody 91F2 are directed against the same cell surface antigen present on MN7 cells. Here, we report on the immunopurification of the 18D7/91F2 antigen and its identification as the prostaglandin F2 alpha receptor regulatory protein (FPRP). FPRP is expressed as a single messenger RNA (mRNA) of approximately 6 kilobases (kb) in MN7 cells and is differentially expressed in developing osteogenic cultures of bone marrow cells of the mouse. However, addition of the monoclonal antibodies 18D7 and 91F2 to these cultures did not inhibit bone formation in vitro. Both monoclonal antibodies reacted with mouse stromal cell lines established from bone marrow, thymus, spleen, and mandibular condyles. Immunohistochemical analysis of mature tibia of mice using the monoclonal antibody 18D7 revealed the presence of a distinct population of bone marrow cells close to trabecular and endosteal bone surfaces. In the central bone marrow, hardly any positive cells were found. In 17-day-old fetal mouse radius 18D7 immunoreactivity was restricted to cells in the periosteum in close vicinity to the bone collar. Mature osteoblasts, osteoclasts, osteocytes, growth plate chondrocytes, and mature macrophages were all negative. Taken together, these results suggest that FPRP plays a role in the osteogenic differentiation process.

Lead and catechol hematotoxicity in vitro using human and murine hematopoietic progenitor cells.

In vitro cloning assays for hematopoietic myeloid and erythroid precursor cells have been used as screening systems to investigate the hematotoxic potential of environmental chemicals in humans and mice. Granulocyte-monocyte progenitors (CFU-GM) from human umbilical cord blood and from mouse bone marrow (Balb/c and B6C3F1) were cultured in the presence of lead and the benzene metabolite catechol. Erythroid precursors (BFU-E) from human umbilical cord blood were cultured in the presence of lead. The in vitro exposure of the human and murine cells resulted in a dose-dependent depression of the colony numbers. The concentration effect relationship was studied. Results showed that: (1) Based on calculated IC50 values, human progenitors are more sensitive to lead and catechol than are murine progenitors. The dose that caused a 50% decrease in colony formation after catechol exposure was 6 times higher for murine cells (IC50 = 24 micromol/L) than for human cord blood cells (IC50 = 4 micromol/L). Lead was 10-15 times more toxic to human hematopoietic cells (IC50 = 61 micromol/L) than to murine bone marrow cells from both mice strains tested (Balb/c, IC50 = 1060 micromol/L; B6C3F1, IC50 = 536 micromol/L). (2) A lineage specificity was observed after exposure to lead. Human erythroid progenitors (hBFU-E) (IC50 = 3.31 micromol/L) were found to be 20 times more sensitive to the inhibitory effect of lead than were myeloid precursors (hCFU-GM) (IC50 = 63.58 micromol/L). (3) Individual differences in the susceptibility to the harmful effect of lead were seen among cord blood samples. (4) Toxicity of lead to progenitor cells occurred at environmentally relevant concentrations.

Long-term effects on tumour incidence and survival from 241Am exposure of the BALB/c mouse in utero and during adulthood.

BALB/c mice were given 100, 500 or 1500 Bq/g 241Am at day 14 of pregnancy. The offspring were separated from the mothers at birth and followed until death. In addition, adult females and one group of males were also studied for the effects of 241Am following treatment with 45-213 Bq/g. Adults treated with 241Am showed significantly shortened survival and increased incidence of osteosarcoma (to 40 - 50%). The data also suggest that the female mouse is more susceptible to induction of osteosarcoma than the male. There was also a significant increase in osteosarcoma, all bone tumours, all sarcomas, and all leukaemias in the offspring from the contaminated mothers, although this appeared to occur independently of dose. Calculations of the number of osteosarcomas induced per Gy varied for contamination of adult mice between 0.2 and 0.01 and for the offspring between 6 and 0.6. Thus, offspring seemed to be about 10 times more at risk if osteosarcomas induced per mouse Gy are compared. Surprisingly, offspring from mothers treated with 241Am displayed a longer survival time than controls, possibly due to fewer deterministic lung diseases appearing early in life.

Interleukin-10 stimulates hematopoiesis in murine osteogenic stroma.

Bone marrow from 5-fluorouracil-treated mice support osteogenesis when cultured in the presence of beta-glycerophosphate and vitamin C. These cultures are unable to support the growth of granulocyte/macrophage colony-forming units for longer than 2 weeks. In contrast, granulocyte/macrophage colony-forming units were detected for more than 6 weeks in interleukin-10 (IL-10)-treated cultures. In addition, IL-10-treated cultures contain long-term culture initiating cells, suggesting the presence of pluripotent hematopoietic cells. Apparently, IL-10 does not directly stimulate the proliferation of granulocyte/macrophage colony-forming units. Interleukin-10 is unable to stimulate [3H]-thymidine incorporation or to increase the number of granulocyte/macrophage colony-forming units in cell suspensions harvested from untreated or interleukin-10-treated bone marrow cultures. Interleukin-10 acts via an indirect pathway. Because exogenous transforming growth factor-beta (TGF-beta) reverses IL-10's stimulatory activity on myeloid progenitors, IL-10 most likely works by blocking TGF-beta synthesis, which acts as an endogenous suppressor of hematopoiesis in osteogenic marrow cultures. This is shown further by the increased numbers of granulocyte/macrophage colony-forming units in cultures treated with neutralizing anti TGF-beta antibodies (1D11.16). Interleukin-10 and 1D11.16 change the cultured bone marrow stroma from an osteogenic into a hematopoietic morphology. It may be that by blocking endogenous TGF-beta production, IL-10 drives marrow mesenchymal cells away from osteogenic differentiation toward hematopoietic support.

Haemopoietic and osteogenic toxicity testing in vitro using murine bone marrow cultures.

Bone marrow and the surrounding bone with its high storage capacity for inorganic compounds may accumulate various lipophilic and electrophilic substances that enter the bloodstream. In bone marrow a few stem cells are responsible for the continuous production of blood cells and bone cells during the entire life of the organism. Damage to these cells may result in haemopoietic failure, blood disorders or bone diseases. Therefore bone marrow needs to be considered as one of the major targets of chemicals that enter the circulation. A battery of different in vitro bone marrow assays is established in which interference of chemicals with proliferation and differentiation of marrow cells with haemopoietic, stromal or bone forming marrow commitment may be screened routinely. Stromal cells form the network of extracellular matrix and growth factors that is needed by the haemopoietic cells to proliferate and differentiate. If stromal marrow cells are cultured in the presence of ascorbic acid and beta-glycerophosphate, bone-specific proteins and an extracellular matrix are produced, which calcifies within 3 wk. To evaluate the specificity of the effects on marrow cells, a general cytotoxicity assay is included using 3T3-fibroblasts. Various concentrations of xenobiotics were added over the course of 3 days to the different asssays. Lead nitrate inhibited proliferation of stromal stem cells and their calcification in the bone-forming assay at much lower concentrations than those which were inhibitory to the proliferation of 3T3 cells. The benzene metabolite hydroquinone was equally inhibitory in all the marrow assays, but 3T3 cells needed 10 times more hydroquinone to reach the same degree of inhibition. Catechol, which is another benzene metabolite, was highly toxic but was equally effective in all the assays and showed no specific effects on the marrow cells. As in vivo, benzene itself and phenol showed hardly any effect in the in vitro assays. Not only pollutants but also cytokines may be screened with these assays. Differential effects on marrow cells could be demonstrated for interleukin 10.

Effects of in vitro alpha-particle irradiation on osteogenic bone marrow cultures.

Murine bone marrow contains osteogenic precursor cells that undergo differentiation during in vitro cultivation. In vitro these cells are potential target cells for alpha-irradiation-induced bone tumour formation. Under defined tissue culture conditions these differentiating cells were directly exposed to alpha-particle irradiation from the radon daughter 210Po. Po deposits in soft tissue and it was shown to be associated with marrow cells and with the extracellular marrow tissue formed in vitro. These differentiating marrow cultures showed high sensitivity to alpha-irradiation. Cell death was observed at 210Po concentrations in tissue culture medium (TCM) > 7 Bq 210Po/ml. At lower concentrations (between 1 and 5 Bq 210Po per ml TCM) proliferation was enhanced as measured by uptake of 3H-thymidine, also differentiation was stimulated as measured by alkaline phosphatase activity and incorporation of 3H-proline in newly synthesized collagen. At several times of culture, the association of 210Po with the extracellular matrix and cells was measured. These retention data enabled us to calculate the daily alpha-particle fluence. At 1 Bq 210Po present per ml tissue culture, a daily alpha-particle fluence as low as 3-6 per 1000 cells seemed very efficient in changing the expression of osteogenic differentiation of marrow cells.

Pharmacokinetics of alfentanil after epidural administration. Investigation of systemic absorption kinetics with a stable isotope method.

BACKGROUND: The effects of epidurally administered alfentanil may be due in part to its uptake into the systemic circulation. Therefore we examined the systemic absorption kinetics after epidural injection of alfentanil. METHODS: Pharmacokinetics were determined using a stable isotope method in ten patients, undergoing lower abdominal surgery under general anesthesia. After epidural injection of 0.68 mg deuterium-labeled alfentanil (alfentanil-d5), 1 mg unlabeled alfentanil was administered over 1 h by an intravenous infusion. Blood samples were collected for 12 h. Concentrations of alfentanil and alfentanil-d5 were measured by a combination of gas chromatography and mass fragmentography. The systemic absorption profiles of alfentanil-d5 were determined by deconvolution of the plasma alfentanil-d5 concentrations with the biexponential unit disposition functions, derived from the intravenous data. In addition, data were analyzed by moment analysis. RESULTS: The mean (+/- SD) steady-state volume of distribution, total plasma clearance, elimination half-life and mean residence time, derived from the unlabeled alfentanil concentration-time data, were 43.2 +/- 19.5 1,418 +/- 129 ml/min, 119 +/- 34 min, and 103 +/- 26 min, respectively. The absorption of alfentanil-d5 was monophasic in most patients. The mean systemic availability and mean absorption time derived from the deconvolution data were 100 +/- 17% and 114 +/- 24 min. The values determined by moment analysis were 107 +/- 18% and 112 +/- 36 min, respectively. CONCLUSIONS: After epidural administration alfentanil is slowly absorbed into the general circulation. Resulting plasma concentrations are very low and do not contribute appreciably to the systemic opioid effect.

Interleukin-10 inhibits the osteogenic activity of mouse bone marrow.

Murine bone marrow cells synthesize bone proteins, including alkaline phosphatase (ALP), collagen type I, and osteocalcin, and form a mineralized extracellular matrix when cultured in the presence of beta-glycerophosphate and vitamin C. Interleukin-10 (IL-10) suppressed the synthesis of these bone proteins and mineralization without affecting cell proliferation. In addition, mRNA levels for the latter proteins were reduced in IL-10-treated cultures. This inhibitory effect was most outspoken when IL-10 was added before ALP activity peaked, eg, day 15 of culture. No significant effect was observed when IL-10 was added at later time points. This finding suggests that IL-10 acts at osteogenic differentiation stages that precede ALP expression but is ineffective on cells that progressed beyond this maturation stage. Likewise, IL-10 appeared to be unable to block both ALP activity and collagen synthesis in the preosteosteoblastic cell lines MN7 and MC3T3 that constitutively synthesize these proteins. Whereas IL-10 did not alter the number of fibroblast colony-forming cells of the marrow, it significantly reduced their osteogenic differentiation potential. In contrast to control cultures, IL-10-treated stroma was unable to either synthesize osteocalcin or to mineralize when subcultured over a 25-day period in the absence of IL-10. The inhibitory activity of IL-10 coincided with significant changes in stroma morphology. Whereas control cultures contained mainly flat adherent polygonal cells, significant numbers of rounded semiadherent to nonadherent cells were observed in the presence of IL-10. Scanning and transmission electron microscopy showed that, in contrast to control cultures, IL-10-treated stromas completely lacked a mineralized extracellular matrix. Collectively, these data suggest that IL-10 may have important regulatory effects on bone biology because of its capacity to downregulate early steps of osteogenic differentiation.

Response of murine stromal bone marrow cultures to alpha-particle irradiation in vivo and in vitro at osteosarcomogenic alpha-radiation doses.

Stromal cells belonging to the bone marrow microenvironment are altered in mice at risk of bone tumour development after (241)Am injection. This can be observed with selective cell culture techniques long before the tumours become manifest. Colony forming assays in vitro showed increases in the number of stromal stem cells at lower dose levels but a decrease in cell number at higher dose levels. The in vitro osteogenic capacity of marrow, which is attributed to stromal cells of the osteogenic lineage, was significantly reduced. The changes were persistent until at least 1 yr after (241)Am injection. In vitro alpha-irradiation of bone marrow also reduced the osteogenic capacity of the bone marrow at dose levels that were comparable with those producing an effect in vivo. This suggests a direct effect of alpha-particle irradiation on stromal bone marrow cells. These data and previous results on the regulatory role of the stroma in haemopoiesis show that stromal bone marrow cells should be considered as sensitive targets for chronic low level alpha-particle irradiation.

Haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3-dimensional collagen sponges.

Adult murine bone marrow cells, cultured under conditions for long-term haemopoietic marrow cultures, produce bone matrix proteins and mineralized tissue in vitro, but only after the adherent stromal cells were loaded on a 3-dimensional collagen sponge. Provided more than 8 x 10(6) cells are loaded, mineralization as measured by 85Sr uptake from the culture medium, occurred in this 3-dimensional configuration (3-D) within 6 days. In contrast if undisrupted marrow fragments (containing more than 10(7) cells) are placed directly on a collagen sponge, then it requires more than 10 days before significant mineralization can similarly be detected. The 2-dimensional (2-D) long-term marrow culture system allows prior expansion of the stromal cells and some differentiation in an osteogenic direction within the adherent stromal layer. This is suggested by the presence of type I collagen and alkaline phosphatase positive cells. However; synthesis of osteonectin and a bone specific protein, osteocalcin, as well as calcification are only observed in 3-D cultures. Electron microscopy demonstrated hydroxyapatite mineral on collagen fibres, osteoblast-like cells, fibroblasts, cells which accumulated lipids, and macrophages which were retained on the collagen matrices. Irradiation of confluent long-term bone marrow cultures, prior to their loading on the collagen sponge showed that haemopoietic stem cells are not necessary for the mineralization.

High radiosensitivity of the mineralization capacity of adult murine bone marrow in vitro to continuous alpha-irradiation compared to acute X-irradiation.

Adult BALB/c mice, injected with osteosarcomogenic amounts of 241Am (between 40 and 500 Bq/g mouse) showed an impaired mineralization capacity of their femoral bone marrow. This effect persisted until at least 1 year after 241Am injection and was expressed after incubation of bone marrow cells in vitro in conditions allowing osteogenic differentiation. The mineralization capacity of marrow in vitro was evaluated by measurement of 85Sr uptake from the tissue culture medium. Two osteogenic assays were used: in marrow cultured as an intact organ (marrow organ cultures), reduced mineralization was observed in mice given 149 Bq 241Am/g mouse or more (skeletal dose rate of 25 mGy/day), in stromal marrow cells cultured from adherent cell layers and subsequently brought into a three-dimensional (3D) mineralizing condition (stromal 3D cultures), reduced 85Sr uptake was observed from the lowest dose level tested (42 Bq 241Am/g mouse, skeletal dose rate of 7 mGy/day). Taking into account that only a fraction of the skeletal alpha-dose reached the marrow of the femoral diaphyses, marrow organ cultures and stromal 3D cultures exhibited high radiosensitivity to alpha-irradiation in vivo. However, after acute X-irradiation of marrow in vivo or in vitro prior to initiation of the marrow organ cultures, X-ray doses of 4 Gy or higher were needed to significantly impair the mineralization capacity of marrow organ cultures in vitro. Our data demonstrated that the osteogenic cells from the bone marrow are subjected to long-term damage after low doses of continuous alpha-irradiation in vivo.

Culture of hematopoietic stem cells purified from murine bone marrow.

The results of the Y-chromosome in situ hybridization experiments, the MRA assessment, and the long-term production of CFU-GM in vitro indicate that our protocol to sort low density WGA+, 15/1.1-, Rh123 dull cells enriches about 200-fold for PHSC. Assays for spleen colony formation (CFU-S) and radioprotection (30-day survival) were shown to be unspecific for PHSC, and, therefore, we lack a quantitative PHSC assay. The absolute number of PHSC in the bone marrow is not known any more, the purity of our sorted population likewise is unknown. Long-term repopulating cells (PHSC) could be separated from short-term repopulating ones by using Rh123 staining. The short-term repopulating cells (Rh123 bright) provided sufficient offspring to protect lethally irradiated mice until endogenous PHSC could reconstitute hematopoiesis. These cells are therefore of interest for bone marrow transplantation, because they provide radioprotection without long-term repopulation and graft-versus-host disease. For gene therapy these cells are of limited use, and PHSC with extensive replication are needed. The PHSC were not cultured successfully. Less than 15% of the sorted Rh123 dull cells responded in semisolid or liquid cultures in the presence of growth factors. Proliferation without differentiation was not observed. This may indicate that the right growth factor has not been found yet. On the other hand, about 30% of the cells responded in stromal layers of long-term bone marrow cultures and prolonged CFU-GM production and cobblestone area formation were observed there, suggesting that cell-cell contact and adherence molecules play a regulatory role in PHSC replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Stromal cells from murine developing hemopoietic organs: comparison of colony-forming unit of fibroblasts and long-term cultures.

The adherent stromal layer in long-term marrow cultures is essential to the proliferation and differentiation of hemopoietic cells. Adhering cells are heterogeneous and morphologically not adequately characterized. Comparative morphological studies were conducted on adherent cells in short-term clonal assays and long-term cultures derived from liver and bone marrow. Liver and bone marrow at different developmental ages have different hemopoietic activities in vivo and in vitro, as tested via CFU-GM recovery in long-term cultures. Adherent cells from each organ were recovered at an age with high hemopoietic activity (fetal liver and adult bone marrow) and at an age with low hemopoietic activity (neonatal liver and bone marrow). The presence of macrophages, alkaline phosphatase, acid phosphatase, myeloperoxidase, sulfated and non-sulfated glycosaminoglycans (GAGs) and fibronectin was compared. For a given organ, CFU-f colonies showed characteristics similar to those of the confluent adherent stromal layer in long-term cultures. The presence of macrophages and GAGs (sulfated and non-sulfated) in the adherent layer were directly related to the hemopoietic activity. The amount of alkaline phosphatase-positive cells and the amount of fibronectin showed no correlation with the hemopoietic activity of the cultures.