Beryllium sensitivity among workers at a Norwegian aluminum smelter.

BACKGROUND: Sensitivity to beryllium was investigated among workers at an aluminum smelter in Norway as a consequence of the findings in an occupational exposure survey. METHODS: Three hundred and sixty-two employees and 31 reference persons were tested for sensitization to beryllium with the beryllium lymphocyte proliferation test (BeLPT) based on specifications by the US Department of Energy in 2001. The results are reported as abnormal, borderline, or normal. RESULTS: One person (0.28%) from the aluminum smelter was found to have abnormal results in two separate blood samples and is sensitized to beryllium. Three other persons had one abnormal test that was not confirmed by a second test. One person in the reference group had one abnormal and one normal test result. No borderline samples were detected. None of the employees with one or more abnormal sample results had pot room asthma. The sensitized individual worked in a Soederberg line in 1972-1974. The beryllium concentration in the work atmosphere is estimated to have been similar as today (0.1-0.3 microg/m(3)), but work routines, etc. would cause higher total exposures. CONCLUSIONS: Only one sensitized person of 362 is in line with what is found in other studies in the aluminum industry. The low number, compared with the beryllium handling industry, may be attributable to lower work atmosphere concentrations, beryllium speciation effects, or use of respiratory protection equipment. Pot room asthma does not appear to be associated with beryllium sensitization.

Muscle wasting in chronic kidney disease: the role of the ubiquitin proteasome system and its clinical impact.

Muscle wasting in chronic kidney disease (CKD) and other catabolic diseases (e.g. sepsis, diabetes, cancer) can occur despite adequate nutritional intake. It is now known that complications of these various disorders, including acidosis, insulin resistance, inflammation, and increased glucocorticoid and angiotensin II production, all activate the ubiquitin-proteasome system (UPS) to degrade muscle proteins. The initial step in this process is activation of caspase-3 to cleave the myofibril into its components (actin, myosin, troponin, and tropomyosin). Caspase-3 is required because the UPS minimally degrades the myofibril but rapidly degrades its component proteins. Caspase-3 activity is easily detected because it leaves a characteristic 14kD actin fragment in muscle samples. Preliminary evidence from several experimental models of catabolic diseases, as well as from studies in patients, indicates that this fragment could be a useful biomarker because it correlates well with the degree of muscle degradation in dialysis patients and in other catabolic conditions.

Monocytes stimulated with group B streptococci or interferons release tumour necrosis factor-related apoptosis-inducing ligand.

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a cytotoxic member of the TNF family. Some reports have shown that TRAIL is released from cells in a soluble form. In this work, we have investigated the mechanism of release of TRAIL from monocytes. First, we show that whole gram-positive, gram-negative and mycoplasmal bacteria as well as lipopolysaccharide (LPS), interferon-alpha (IFN-alpha), -beta and -gamma all induced upregulation of TRAIL on the surface of human monocytes. Next, we show that IFN-alpha, -beta and -gamma all induced a dose-dependent release of TRAIL, giving significant amounts of soluble TRAIL after 2 days. Of the bacteria, only the Group B streptococcus COH-1 (GBS) induced release of TRAIL and concomittantly induced IFN-alpha. Monocytes stimulated with GBS or IFN-alpha also showed extensive cell death. When monocyte apoptosis was prevented by interleukin-1, GM-CSF, LPS or the caspase inhibitor zVADfmk, the IFN-alpha-induced release of TRAIL was reduced, whereas agents inducing necrosis caused increased release of TRAIL. LPS also prevented release of TRAIL from GBS-stimulated monocytes. The release of TRAIL from IFN-alpha-stimulated monocytes was reduced by inhibitors of both cysteine and metalloproteases. We conclude that bacteria and IFN induce upregulation of membrane TRAIL and that release of TRAIL is associated with cell death.

Lipopolysaccharide induces expression of APO2 ligand/TRAIL in human monocytes and macrophages.

Monocytes express cytotoxic factors of the tumour necrosis factor (TNF) ligand superfamily, including TNF and Fas ligand, both on the cell surface and in secreted form. In this report, we show that human monocytes and monocyte-derived macrophages stimulated with lipopolysaccharide (LPS) express APO2 ligand (APO2L, TRAIL), a recently discovered cytotoxic member of the TNF ligand superfamily. LPS increased the transcription of APO2L mRNA in monocytes and macrophages. Flow cytometric analysis showed low surface and high intracellular levels of APO2L, and LPS increased the expression of both. In addition, LPS increased the monocyte- and macrophage-mediated cytotoxicity against the APO2L-sensitive Jurkat and RPMI-8226 cells. Addition of the APO2L-binding decoy receptor 1 (DcR1)-Fc fusion protein inhibited the cytotoxicity by 30-70%. LPS also stimulated the release of soluble APO2L from the monocytes and macrophages. Monocytic phagocytosis of target cells was increased by LPS and partially inhibited by DcR1-Fc. Taken together, these data demonstrate a novel mechanism of cytotoxicity mediated by LPS-activated human monocytes and macrophages.

Induction of Fas ligand in murine bone marrow NK cells by bacterial polysaccharides.

Bacterial polysaccharides have a wide range of activities in mammals. We have studied the effect of LPS and poly-beta-(1-->4)-D-mannuronate (mannuronan, poly-M), an exopolysaccharide from Pseudomonas aeruginosa, on the cytotoxicity mediated by murine bone marrow cells (BMC). Addition of LPS or mannuronan to BMC induced a time- and dose-dependent cytotoxicity against Jurkat cells. The LPS- or mannuronan-induced cytotoxicity was due to increased Fas ligand (FasL) expression by BMC, since 1) Fas-transfected L1210-Fas target cells were more susceptible to lysis than the Fas(low)-expressing parent L1210 cells, 2) stimulated BMC from FasL-defective gld/gld mice were not cytolytic and, 3) the cytolytic activity of normal BMC was inhibited by a Fas-Fc fusion protein. Flow cytometry showed an increase in surface FasL in LPS-stimulated BMC. RT-PCR analysis of BMC revealed constitutive expression of FasL mRNA, which was increased after LPS stimulation. Immunomagnetic depletion of NK1.1-, CD2-, or CD32/16-expressing cells from BMC abrogated the LPS-induced BMC cytotoxicity against L1210-Fas cells, suggesting that NK cells were the cytotoxic effector cells. Depletion of CD45R/B220-, Gr-1-, or CD11b/Mac-1-expressing cells only partially decreased BMC-mediated cytotoxicity, and depletion of CD4- or CD8a-expressing cells had no effect. The results support the conclusion that LPS and mannuronan induce expression of cytotoxic FasL on bone marrow NK cells.

Mannuronan enhances survival of lethally irradiated mice and stimulates murine haematopoiesis in vitro.

Mannuronan (poly-beta-(1-->4)-D-mannuronate or poly-M), produced by Pseudomonas aeruginosa as a mucoid exopolysaccharide, has previously been shown to exhibit immunostimulating activity. The authors investigated the in vivo and in vitro effects of mannuronan on murine haematopoiesis. In vivo, prophylactic (-24 h, intraperitoneal) administration of mannuronan enhanced survival of lethally irradiated mice from zero day 40 survivors (NaCl) to 20, 80 and 70% survival at 0.5, 1 and 2 mg/kg bw mannuronan, respectively. In vitro, primary stromal cultures stimulated with mannuronan produced high levels of interleukin(IL)-1, IL-6 and colony stimulating activity. Mannuronan alone did not have any colony stimulating activity on GM-CFC, BFU-E, Mix-CFC or HPP-CFC progenitors in clonogenic assays, but acted synergistically with suboptimal amounts of growth factors on GM-CFC, Mix-CFC and HPP-CFC colony formation. Limiting dilution analysis showed that 1 of 423 bone marrow cells formed colonies in response to suboptimal GM-CSF plus mannuronan compared to 1 of 592 for suboptimal GM-CSF alone. The primitive Lin-Sca-1+ haematopoietic progenitors showed increased day 10 colony size in the presence of mannuronan in single cells assays. These stimulating effects of mannuronan on haematopoiesis may prove to have clinical importance.

Polysomy of chromosome 7 is associated with amplification and overexpression of the EGF-receptor gene in a human carcinoma cell line derived from a brain metastasis.

Overexpression of the EGF-receptor gene is associated with the malignant nature of some tumors. We have recently reported the establishment of a human carcinoma cell line (T-CAR1), derived from a brain metastasis, that had 7 million EGF receptors per cell and was growth inhibited by EGF. The present study was carried out in order to further characterize the EGF-receptor protein in T-CAR1 cells, and to see if the overexpression of the EGF-receptor gene in these cells was associated with abnormalities at the genomic level. We have compared the T-CAR1 cells with the human glioblastoma cell line T-MG1, which has 135,000 EGF-receptors and is growth stimulated by EGF. The MW of the EGF receptors in T-CAR1 cells and T-MG1 cells was estimated to be 170 kDa, equal to the normal EGF-receptor. However, in T-CAR1 cells an additional protein reacted with the monoclonal antibody directed against the internal domain of the EGF receptor. The levels of EGF receptor-related RNAs in T-CAR1 cells and T-MG1 cells reflected the number of EGF receptors in these cell lines. The EGF-receptor gene was amplified ten-fold in T-CAR1 cells, while it was not amplified in T-MG1 cells. No restriction fragment length polymorphism of DNA digested with various restriction enzymes was seen in either of the cell lines. Chromosomal analysis of T-CAR1 cells showed polysomy of chromosome 7 and marker chromosomes derived partly from chromosome 7. Thus, in the T-CAR1 cell line it was an association between polysomy of chromosome 7 and EGF-receptor gene amplification.

Overexpression of the epidermal growth factor receptor gene in a human carcinoma cell line, derived from a brain metastasis.

Abnormally high expression of epidermal growth factor receptors (EGF-receptors) may contribute to the unregulated growth of some tumors. We here report the EGF-receptor numbers and the effects of epidermal growth factor (EGF) on two human cell lines. The glioblastoma cell line T-MG1 had 135,000 EGF-receptors per cell, was slightly growth stimulated by EGF and showed no obvious change in morphology after exposure to EGF. The carcinoma cell line T-CAR1, derived from a brain metastasis of a carcinoma of the adrenal cortex, had approximately 7 million EGF-receptors per cell. EGF had a significant antiproliferative effect on these cells and caused rounding and detachment of cells in adherent cultures. The cell lines may become useful in future studies concerning the role of the EGF-receptors in malignant growth.

The effects of type beta transforming growth factor on proliferation and epidermal growth factor receptor expression in a human glioblastoma cell line.

Type beta transforming growth factor (B-TGF) is a potent growth inhibitor to many human tumor cell lines. Very little is known about the mechanism for this growth inhibitory action of B-TGF. We here report the effect of B-TGF on proliferation and epidermal growth factor receptor (R-EGF) expression in a human glioblastoma cell line named T-MG1. B-TGF inhibit the soft agar growth of T-MG1 cells. Maximum inhibition was 70%, achieved with 0.5 units B-TGF. B-TGF had no effect on monolayer growth of T-MG1 cells. T-MG1 cells contained abundant R-EGF, which could be divided into two subpopulations, one high affinity and one low affinity population of R-EGF. Treatment with B-TGF caused an initial decrease (0-6 h) in EGF-binding, followed by an increase in EGF-binding which reached maximum after 24 h exposure to B-TGF. Since addition of EGF to agar cultures gave no additional increase in inhibition by B-TGF and EGF alone had no inhibitory effect, we believe that binding of EGF to its receptor is not part of the pathway mediating the inhibitory effect of B-TGF. All neoplastic cells have lost some measure of growth control and the cellular elements involved are growth factors, growth factor receptors and oncogenes. T-MG1 cells contain abundant R-EGF and this may partly explain their malignant nature (malignant nature is here defined as ability to proliferate in agarose). Type alpha transforming growth factors, which in some cancer cells act as uncontrolled autocrine growth factors, were not found in protein extracts from T-MG1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Type beta transforming growth factor and epidermal growth factor suppress the plasminogen activator activity in a human glioblastoma cell line.

In the human glioblastoma cell line, T-MG1, plasminogen activator activity (PA-activity) was demonstrated by using the chromogenic substrate S-2251. Using monoclonal antibodies against human urokinase type PA (u-PA) and human tissue type PA (t-PA), only u-PA activity was found in T-MG1 cell extracts. The u-PA activity in T-MG1 cells was suppressed in a dose-dependent manner by B-TGF and EGF after 24 hours of exposure to these growth factors. Twenty units of B-TGF caused a decrease in PA-activity of 80%, while 10 ng/ml EGF gave a decrease in PA-activity of 60%. The suppressive effects of B-TGF and EGF were observed after 2 hours and 4 hours of incubation, respectively and sustained for at least 24 hours. The effects of B-TGF and EGF were not antienzymatic, but rather mediated through regulatory mechanisms. In view of the capacity of invasive growth of gliomas and the potential role of PA in invasive growth, the suppression of PA-activity in gliomas by B-TGF and EGF may be of importance.

The effect of type beta transforming growth factor on proliferation of clonogenic cells from human gliomas.

Type beta transforming growth factor (B-TGF) is a potent regulator of cell growth and differentiation. Growth of many human tumour cell lines are inhibited by B-TGF. The effect of B-TGF on proliferation of clonogenic cells from 12 human glioma biopsies was registrated in a thymidine incorporation assay. B-TGF appeared to be a potent growth inhibitor for some gliomas, while it had no effect on others. Maximum inhibition was about 60%. Though not significant, glioblastomas appeared to be less sensitive to inhibition by B-TGF than astrocytomas and oligodendrogliomas. Very little is known about the growth inhibitory action of B-TGF. The negative autocrine growth theory for cancer cells postulates that a reduced production, or production of a defective growth inhibitor normally found in the cell, may account for the autonomous nature of some cancer cells. In view of this theory, we searched for B-TGF in protein extracts from a glioblastoma cell line, T-MGl, whose growth was inhibited by B-TGF. Protein extract from T-MGl cells was analysed for B-TGF activity using a soft agar colony formation assay with normal rat kidney fibroblasts. B-TGF was not found in the extract. Since, according to the literature B-TGF has been found in all other cell lines examined, we believe that there may be a lack of B-TGF or an altered B-TGF in the gliomas whose growth are inhibited by B-TGF. This problem will be studied further.

Amplification of the epidermal growth factor receptor gene in biopsy specimens from human intracranial tumours.

Amplification and overexpression of proto-oncogenes are associated with the malignant nature of some human tumours. In this study we have determined the prevalence of amplification of the proto-oncogenes c-erb B1 (= epidermal growth factor receptor gene), c-erb B2 and c-myc in 44 human intracranial tumours (27 gliomas, six metastases to the brain and 11 meningiomas). None of the tumours had an amplified c-erb B2 gene and only two tumours had an amplified c-myc gene. Nineteen per cent (five out of 27) of the gliomas, 50% (three out of six) of the brain metastases and 0% (0 out of 11) meningiomas had an amplified EGF-receptor gene. Amplification of the EGF-receptor gene appeared to give a growth advantage when single-cell suspensions of the tumours were grown in agarose.

Effects of type beta transforming growth factor in combination with retinoic acid or tumor necrosis factor on proliferation of a human glioblastoma cell line and clonogenic cells from freshly resected human brain tumors.

Type beta transforming growth factor (beta-TGF) is a potent regulator of cell growth and differentiation. The human glioblastoma cell line, T-MGI, was growth inhibited by beta-TGF under anchorage independent conditions. The antiproliferative effect of beta-TGF was potentiated to nearly total arrest by low doses of retinoic acid (RA) or tumor necrosis factor (TNF), while epidermal growth factor, platelet-derived growth factor, interleukin-2, and gamma interferon did not have this potentiating effect. The potentiation of the beta-TGF effect by RA and TNF could not be explained by modulation of the epidermal growth factor receptor, the beta-TGF receptor, or the TNF receptor. beta-TGF alone and in combination with RA or TNF were further tested on primary cultures from freshly resected human glioma biopsies (n = 13). There was great individual variation in sensitivity to beta-TGF, RA, or TNF. The astrocytoma and oligodendroglioma cells were inhibited to various degrees by beta-TGF or TNF, while most of the glioblastomas were not sensitive to these agents. Most of the biopsies were stimulated by RA. RA or TNF did not potentiate the growth inhibitory effect of beta-TGF on biopsy cells. We therefore think it unlikely that beta-TGF in combination with RA or TNF will be effective agents in the treatment of gliomas.

An isotope agarose assay for rapid testing of the sensitivity of glioma biopsies to chemotherapeutic drugs and biological response modifiers. Effects of BCNU, vincristine, lymphokines and the recombinant agents interferon alpha 2c, interferon gamma and tumour necrosis factor.

A method based on growth of cells from glioma biopsies in a triple layer agarose system has been used to test the sensitivity of the tumour cells to different chemotherapeutic and immunotherapeutic agents. A special agarose that could be melted at 65 degrees C, enabled registration of proliferation by an easy and precise thymidine incorporation technique. The well known concept that agarose permits proliferation of malignant cells and inhibits growth of benign cells was confirmed in this assay by using the malignant glioma cell lines T-MG 1 and U 251 MG, and the benign glia cells T-BG 2 and T-BG 3. All of the 15 glioma biopsies grew exponentially during the first 7 days. The correlation between thymidine incorporation on day 7 and colony number on day 14 was very good for the glioma cell line (r = 0.96) and also for the glioma biopsies (r = 0.89), indicating that the sensitivity evaluation can be done as early as 7 days after the operation. Lymphokines, made by BCG-stimulated lymphocytes, had a strong inhibitory effect on the growth of some glioma biopsies, while others were only slightly inhibited or even stimulated. There was also a huge variation in the sensitivity of the biopsies to BCNU, but the sensitivity pattern was completely different for BCNU and lymphokines. rIF-A had a moderate inhibitory effect on growth of the biopsies, while rTNF had a weak inhibitory effect. The response to rIF-G varied from stimulation of some biopsies to strong inhibition of others. There was no similarity in the sensitivity pattern of the biopsies to rIF-A and rIF-G.(ABSTRACT TRUNCATED AT 250 WORDS)

A new technique to register proliferation of clonogenic cells from brain tumors.

The soft agar technique for culturing human clonogenic tumor cells has been usefully applied for predicting individual clinical responses to chemotherapy, for screening of new antineoplastic drugs, and in basic biological research. The counting of colonies formed by clonogenic cells is, however, a rather time consuming and inaccurate procedure. We here report a method to combine the easy and precise registration of DNA-synthesis by 3H-thymidine incorporation with the ability of soft agar to permit proliferation of clonogenic cells and inhibit proliferation of non-neoplastic cells. The glioma cell lines U 251 MG and T-MG 1, the benignant glia cells T-BG 1, T-BG 2, T-BG 3 and fibroblasts were cultured in Furcellaran gel. Twenty hours before harvesting 3H-thymidine was added. The Furcellaran gel was resolved by 50 mM LiI. The cells were trapped on glass fiber filters and incorporated radioactivity was measured. 3H-thymidine incorporation in malignant cells increased exponentially with time, while 3H-thymidine incorporation in the benignant glia cells and fibroblasts was inhibited. The correlation between number of colonies counted after 16 days and 3H-thymidine incorporation registered after different culture times was very good. The correlation was best when the cultures were harvested after 8 days (r = 0.95), indicating that it is possible to reduce the assay time. The five glioma biopsies tested grew well with a mean plating efficiency of 0.4% (range 0.02-1.8%). The most intense proliferation seemed to take place during the first week in culture. The good correlation between 3H-thymidine incorporation on day 7 and colony number on day 14 (r = 0.93), indicate that reduction of assay time is possible also for the glioma biopsies.