Comparison of the developmental effects of two mercury compounds on glial cells and neurons in aggregate cultures of rat telencephalon

A three-dimensional cell culture system was used as a model to study the influence of low levels of mercury in the developing brain. Aggregating cell cultures of fetal rat telencephalon were treated for 10 days either during an early developmental period (i.e., between days 5 and 15 in vitro) or during a phase of advanced maturation (i.e., between days 25 and 35) with mercury. An inorganic (HgCl2) and an organic mercury compound (monomethylmercury chloride, MeHgCl) were examined. By monitoring changes in cell type-specific enzymes activities, the concentration-dependent toxicity of the compounds was determined. In immature cultures, a general cytotoxicity was observed at 10(-6) M for both mercury compounds. In these cultures, HgCl2 appeared somewhat more toxic than MeHgCl. However, no appreciable demethylation of MeHgCl could be detected, indicating similar toxic potencies for both mercury compounds. In highly differentiated cultures, by contrast, MeHgCl exhibited a higher toxic potency than HgCl2. In addition, at 10(-6) M, MeHgCl showed pronounced neuron-specific toxicity. Below the cytotoxic concentrations, distinct glia-specific reactions could be observed with both mercury compounds. An increase in the immunoreactivity for glial fibrillary acidic protein, typical for gliosis, could be observed at concentrations between 10(-9) M and 10(-7) M in immature cultures, and between 10(-8) M and 3 x 10(-5) M in highly differentiated cultures. A conspicuous increase in the number and clustering of GSI-B4 lectin-binding cells, indicating a microglial response, was found at concentrations between 10(-10) M and 10(-7) M. These development-dependent and cell type-specific effects may reflect the pathogenic potential of long-term exposure to subclinical doses of mercury.     

Immunotoxic effects of mercuric compounds on human lymphocytes and monocytes. IV. Alterations in cellular glutathione content

The major goal of this investigation was to determine if the sensitivity of lymphocytes and monocytes to mercury (Hg++) was related to intracellular glutathione (GSH) levels and the thiol redox status [GSH/glutathione disulfide (GSSG)]. To isolate cells based upon their GSH content, T and B-cells were stained with monochlorobimane (MCB) and separated into high and low fluorescent groups by FACS analysis. Cells with high GSH fluorescence were found to be resistant to both the cytotoxic and immunotoxic effects of HgCl2 as evidenced by cell viability and their responsiveness to mitogen, respectively. In contrast, cells with low levels of GSH were extremely sensitive to mercury. To further examine the relationship between GSH level and mercury exposure, T-cells, B-cells and monocytes were treated with different doses of HgCl2 for 12 hrs. All cells exhibited a dose-dependent decrease in GSH content with a concomitant reduction in GSSG levels. However, the GSH/GSSG ratio in these cells remained constant, or increased following exposure to mercury. GSH levels were also reduced in monocytes following exposure to HgCl2; in this case, GSSG levels remained constant and a decline in the GSH/GSSG ratio was observed. For all cell types, mercury did not inhibit the activities of GSH reductase and GSH peroxidase, enzymes responsible for oxidation/reduction of GSH and GSSG, respectively. Results of the study clearly show that susceptibility to the immunotoxic effects of HgCl2 is, in part, dependent upon GSH levels and further that mercury inhibits GSH generation by lymphocytes and monocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na(+)-H+ exchanger in isolated epithelial tracheal cells from sheep. Involvement in tracheal proton secretion

Heavy metals have been shown to exert immunotoxic effects on humoral immunity. To ascertain the mechanisms by which these immunotoxic effects are exerted, the effects of CdCl2 and HgCl2 on the biology of murine B-lymphocytes were studied. It was shown that CdCl2 and HgCl2 inhibited B-cell RNA and DNA synthesis. The IC50 (the concentration required to inhibit a specific B-cell function by 50%) for CdCl2 was 30 microM for RNA synthesis and DNA synthesis. The IC50 for HgCl2 was 50 and 120 nM for RNA and DNA synthesis, respectively. Cell cycle analysis revealed that B-cells were arrested throughout the cell cycle with CdCl2 and HgCl2. The inhibitory effects exerted by CdCl2 and HgCl2 were rapid, inhibiting RNA synthesis within 2 h of activation. Differentiation to Ig secretion was inhibited by CdCl2 and HgCl2 in culture and there appeared to be selective effects on specific Ig isotypes. IgG3 production was most sensitive to inhibition by CdCl2 and HgCl2 followed by IgG1 and IgG2b and then IgM and IgG2a. Changes in the expression of B-cell surface antigens induced by LPS were also influenced by CdCl2. LPS-induced increases in class II MHC expression was inhibited by CdCl2, as was the constitutive expression of class I MHC antigen. A summary of the IC50 for CdCl2 and HgCl2 are presented. In summary, both CdCl2 and HgCl2 exert early, inhibitory effects on B-cell activation. This is manifested by the inhibition of RNA, DNA and antibody synthesis. However, selective effects on the production of specific Ig isotypes by these metals may influence the ability of B-cells to mount effective immune responses to pathogens.     

The repetitive activation of extracellular signal-regulated kinase is required for renal regeneration in rat

In this study, we investigated the activation of p42 extracellular signal-regulated kinase (ERK2) during renal regeneration after HgCl2-induced acute renal failure (ARF) in rat. ERK2 activation was observed at 5 and 29 hr after HgCl2 injection, respectively. The tyrosine phosphorylation of hepatocyte growth factor receptor (c-MET) occurred between 2.5 and 5 hr after the treatment. On the other hand, the phosphorylation of epidermal growth factor receptor (EGFR) was transiently observed at 29 hr after the injection. The peak of ornithine decarboxylase activity as a marker of G1 phase was at 10 hr, and subsequently the labeling index of proliferating cell nuclear antigen as a marker of S phase increased at 53 hr. These results indicate that the repetitive activation of ERK2 related to the phosphorylation of c-MET and EGFR is required for the renal regeneration in HgCl2-induced ARF of rat.     

Methyl mercury-induced autoimmunity in mice

Female SJL/N, A.SW, B10.S (H-2s), BALB/C, DBA/2 (H-2d), A.TL and B10. TL (H-2t1) mice were treated with sc injections of 1.0 mg CH3HgCl/kg body weight every third day for 4 weeks. Controls were given sterile, isotonic NaCl. CH3HgCl (MeHg) induced in SJL, A.SW and B10.S mice antinucleolar antibodies (ANoA) targeting the nucleolar 34-kDa protein fibrillarin. The susceptibility to develop ANoA in response to MeHg was linked to the mouse major histocompatibility complex (H-2), since H-2s but not H-2t1 mice sharing background (non-H-2) genes developed ANoA. However, the background genes decided the strength of the ANoA response in the susceptible H-2s mice, and the ANoA titer was in the order: A.SW > SJL > B10.S. Although MeHg as well as inorganic mercury induced ANoA, the two forms of mercury differed both quantitatively and qualitatively in their effect on the immune system. MeHg induced in H-2s mice a weaker general (polyclonal) and specific (ANoA) B-cell response than HgCl2, probably due to weaker activation of Th2 cells with lower IL-4 production, as indicated by the minimal increase in serum IgE. The A. TL strain with a susceptible genetic background, but a H-2 haplotype resistant to HgCl2, responded to MeHg with a modest polyclonal B-cell response dominated by Th1-associated Ig isotypes. H-2s mice treated with MeHg showed in contrast to HgCl2-treated mice no systemic immune-complex (IC) deposits, which may be due to the weaker immune activation after MeHg treatment. The increase in serum IgE concentration and ANoA titer 2-6 weeks after stopping treatment with MeHg is identical to reactions during the first 2-3 weeks of HgCl2 treatment. Therefore, demethylation of MeHg probably increased the concentration of inorganic mercury in the body sufficiently to reactivate the immune system. This reactivation indicated that genetically susceptible mice are not resistant to challenge with mercury, making them distinctly different from rats.     

Exposure to mercury alters early activation events in fish leukocytes

Although fish in natural populations may carry high body burdens of both organic and inorganic mercury, the effects of this divalent metal on such lower vertebrates is poorly understood. In this report, inorganic mercury in the form of mercuric chloride (HgCl2) is shown to produce both high-dose inhibition and low-dose activation of leukocytes in a marine teleost fish, Sciaenops ocellatus. Concentrations of inorganic mercury > or = 10 microM suppressed DNA synthesis and induced rapid influx of radiolabeled calcium, as well as tyrosine phosphorylation of numerous cellular proteins. Lower concentrations (0.1-1 microM) of HgCl2 that activated cell growth also induced a slow sustained rise in intracellular calcium in cells loaded with the calcium indicator dye fura-2, but did not produce detectable tyrosine phosphorylation of leukocyte proteins. These studies support the possibility that subtoxic doses of HgCl2 may inappropriately activate teleost leukocytes, potentially altering the processes that regulate the magnitude and specificity of the fish immune response to environmental pathogens.     

Toxicity of methylmercury chloride in rats. III. Long-term toxicity study

In the range-finding test, 6 groups of 4 male and 4 female weanling rats were given dietary levels of 0, 0.1,0.5, 2.5, 12.5 and 250 ppm methylmercury chloride (MeHgCl) for 2 weeks. Signs of central nervous system toxicity, weight loss and high mortality appeared at 250 ppm but not at lower levels. No haematological changes were observed at 0.1-12.5 ppm. The relative weights of the liver in females on 2.5 and 12.5 ppm and of the kidneys in females on 12.5 ppm were significantly increased; the effects in males were less marked. Total mercury concentration in the kidneys increased proportionally with increasing dietary levels of MeHgCl. In the short-term test, 5 groups of 15 male and 10 female weanling rats were given dietary levels of 0, 0.1, 0.5, 2.5 and 25 ppm MeHgCl for 12 weeks. Toxic signs, weight loss and restricted food intake were observed at 25 ppm starting from week 9 onwards. Haematological, serum enzyme and urinalysis changes were seen at 25 ppm. Liver microsomal enzyme activity was increased non-significantly and liver glycogen was depressed at 25 ppm. Organ weight changes were evident at 25 ppm and histological changes seen in the spleen, kidneys, brain, spinal cord and peripheral nerves were confined to the 25 ppm level. Histochemical changes in kidney enzymes occured at 2.5 and 25 ppm. Hg concentrations in blood, hair, kidneys, liver and brain were higher at 12 weeks than 6 weeks and generally increased with increasing MeHgCl level in the diet.     

Thiamine, riboflavin, folate, and vitamin B12 status of infants with low birth Weights receiving enteral nutrition

The purpose of the present study was to monitor the vitamin status of 14 low-birth-weight (LBW) infants (< 1,750 g birth weight) at 2 weeks and an additional four infants at 3 weeks who were receiving an enteral formula providing 247 micrograms/100 kcal thiamine, 617 micrograms/100 kcal riboflavin, 37 micrograms/100 kcal folate, and 0.55 micrograms/100 kcal vitamin B12. The mean birth weight of the 18 infants was 1,100 +/- 259 g, and mean gestational age was 29 +/- 2 weeks. Weekly blood, 24-h urine collections, and dietary intake data were obtained. For thiamine, red blood cell (RBC) transketolase activity was within the normal range for all infants. For riboflavin, RBC glutathione reductase activity was normal for all infants except one. We calculated from intake and urinary excretion data that these infants require 225 micrograms/100 kcal thiamine and 370 micrograms/100 kcal riboflavin, respectively. Mean plasma folate levels were 21 +/- 11 ng/ml at 2 weeks and 18 +/- 5 ng/ml at 3 weeks. RBC folate levels were 455 +/- 280 ng/ml at 2 weeks and 391 +/- 168 ng/ml at 3 weeks. All folate blood values were normal, except for one subject with an elevated level (59 ng/ml). Vitamin B12 plasma values were 737 +/- 394 pg/ml at 2 weeks and 768 +/- 350 pg/ml at 3 weeks, and all values were normal except for three infants with elevated values. In conclusion, appropriate vitamin status was maintained during this short observational period, during administration of this enteral formula; however, riboflavin concentrations in the enteral feed may be excessive.     

Purification, physicochemical characterization and biological properties of a lectin from Erythrina velutina forma aurantiaca seeds

A lectin was purified from seeds of Erythrina velutina forma aurantiaca by affinity chromatography on cross-linked guar gum. The lectin is a potent agglutinin for human (minimal concentration of protein able to cause visible agglutination of a 2% erythrocyte suspension varying from 1 to 4 micrograms/ml), rabbit (4 micrograms/ml) and chicken erythrocytes (8 micrograms/ml) but presented low activity against cow (250 micrograms/ml) or sheep (333 micrograms/ml) blood cells. Hemagglutination of human O+ erythrocytes was inhibited by D-lactose (0.2 mM) > D-galactose (0.8 mM) > D-raffinose (2.1 mM). At pH 7.5, chromatography on a Superose 12 HR 10/30 column showed that the lectin was primarily a dimer (56.0 kDa) composed of two identical subunits (31.6 kDa each). A small amount of a tetrameric form was also apparently present. The lectin is a glycoprotein (7.3% carbohydrate), has a pI of 4.5, contains high levels of acidic (Asp and Glu, 64.2 and 51.6 residues/mol, respectively) and hydroxy amino acids (Ser and Thr, 42.9 and 38.5 residues/mol, respectively) but relatively low amounts of sulfur amino acids (Cys and Met, 1.0 and 5.0 residues/mol, respectively) and has an N-terminal sequence of Val-Glu-Thr-Ile/Leu-Pro-Phe-Ser. Its hemagglutinating activity was abolished by heating at 70 degrees C for 10 min. The activation energy (delta G') required for denaturation measured by loss of hemagglutination activity was 24.87 kcal/mol. In rats, the purified lectin (100 micrograms) induced neutrophil migration into the peritoneal cavity (3.7 +/- 0.6 x 10(6) neutrophils/ml) or into the air pouch (2.75 +/- 0.25 x 10(6) neutrophils/ml), 8 and 10 times greater than the negative control, respectively.     

Hypothermia as an indicator of the acute effects of lipopolysaccharides: comparison with serum levels of IL1 beta, IL6 and TNF alpha

1. Hypothermia was investigated as a parameter indicating the severity of the acute effects of lipopolysaccharides (LPS) in BALB/c mice, and was compared with the induction of serum levels of IL1 beta, TNF alpha and IL6. 2. Hypothermia induced by low doses of LPS (10-50 micrograms/mouse IP LPS E. coli 0111:B4) peaked at 2 hr after LPS and then either plateaued (50 micrograms) or declined. LPS, 100 and 300 mu, induced greater degrees of hypothermia that plateaued or continued to increase with time for 8 hr. Higher doses of LPS induced similar levels of hypothermia until 4 hr but then continued to increase markedly until 8 hr. 3. TNF alpha levels peaked early (1-2 hr) and declined rapidly, IL6 levels peaked at 3 hr and then declined slowly, and IL1 beta levels peaked at 4 hr, declined at lower doses of LPS, plateaued at higher doses and continued to slowly increase at highest doses. 4. The peak levels of the cytokines (IL1 beta up to 4 hr) and hypothermia (4 hr) increased in relation to the dose of LPS and maximum responses were apparently achieved in all cases at 300-1000 micrograms LPS. 5. A similar parallel between hypothermia and induction of cytokines was observed in C57BL6 and OF1 mice, which were good and poor responders to LPS, respectively, and with the more potent Shigella dysenteria LPS in BALB/c mice. 6. In conclusion, hypothermia is a useful parameter for indicating the strength of the acute effects of LPS. Further studies are necessary to determine whether or not the cytokines studied here play a causative role in hypothermia.     

Effect of sublethal doses of cadmium, inorganic mercury and methylmercury on the cell morphology of an insect cell line (Aedes albopictus, C6/36)

The effect of CdCl2 (44 microM), HgCl2 (3.7 microM), and MeHgCl (2 microM) on the morphology of Aedes albopictus C6/36 cells was studied at the light microscopical level. Treatment times and metal concentrations were in the sublethal range as determined by a fluorometric dye exclusion test. The three metal species had profound effects on the cell morphology. MeHgCl treatment induced the development of a large number of short, actin-supported, tangled filopodia. Both CdCl2 and HgCl2 induced long extensions. Pretreatment with colchicine but not with cytochalasin B prevented formation of these extensions which suggests that they were supported by microtubules. This was confirmed by immunostaining for microtubules. The extensions were relatively stable towards colchicine post-treatment. To authors' knowledge, this effect has not yet been described for heavy metals. The similarity with 20-hydroxyecdysone-treated cells and the occurrence of cytoplasmic feet in insect cells is discussed.     

Effects of cadmium on survival and morphology of cultured rat Sertoli cells

The toxicity of different metals on isolated Sertoli cells grown in culture has been investigated. Methyl mercury (CH3HgCl) and mercury chloride (HgCl2) were more toxic than cadmium (CdCl2) which was slightly more toxic than arsenic (As2O3). Isolated peritubular cells and Sertoli cells were equally sensitive to cadmium. Cadmium reduced the Sertoli cell survival over the concentration range of 1--10 microM. Freeze-etch electron microscopy of cadmium-exposed Sertoli cells revealed circular areas of average diameter 500 nm devoid of intramembrane particles in the nuclear membrane, and general signs of degeneration such as vesiculation of the plasma membrane and intramembrane particle aggregation. However, cadmium did not dissolve junctional complexes between Sertoli cells. Isolated Sertoli cells were protected against cadmium-induced damage when the cells were preincubated for 48 h with selenium, zinc or low doses of cadmium. Preincubation with cobalt, FSH, testosterone or oestradiol did not protect against cadmium-induced damage. Cadmium bound to metallothionein had no toxic effects on isolated Sertoli cells.     

Systemic autoimmunity due to mercury vapor exposure in genetically susceptible mice: dose-response studies

Six groups of genetically mercury-susceptible female SJL/N (H-2s) mice were exposed to mercury vapor at a concentration of 0.3-1.0 mg Hg/m3 air for 0.5-19 hr/day 5 days a week for 10 weeks. The absorbed doses were calculated to be between 75 and 2365 micrograms Hg/week/kg body wt (micrograms Hg/week/kg). The correlation between the dose and the concentration of Hg in kidney, spleen, and thymus was highly significant (p < 0.0001; Spearman's rank correlation test). The lowest observed adverse effect level (LOAEL) for serum IgG antinucleolar antibodies (ANoA) was 170 micrograms Hg/week/kg, corresponding to a renal mercury concentration of 4.0 +/- 0.76 micrograms Hg/g wet wt. The correlation between the absorbed dose and the ANoA titer was highly significant (p < 0.0001; Spearman's rank correlation test), and all mice were ANoA-positive at a dose of 480 micrograms Hg/week/kg. High-titer ANoA targeted the nucleolar 34-kDa protein fibrillarin. The LOAEL for B-cell stimulation, measured as an increase in serum IgG2a and IgG1 concentrations, was 360 micrograms Hg/week/kg, but the increase was fivefold higher and also included IgE at a dose of 690 and 2365 micrograms Hg/week/kg. The serum Ig concentrations peaked after 2-4 weeks and then slowly declined but, except for IgE, remained significantly increased during the entire exposure time. Glomerular, mesangial IgG immune complex (IC) deposits, accompanied by systemic vessel wall IC deposits, were first detected at a dose of 480 micrograms Hg/week/kg. The mesangium also showed increased titers of IgM IC deposits and complement factor C3c. The correlation between the absorbed dose, and the individual titer of IgG, IgM, and C3c, was highly significant (p < 0.0001; Spearman's rank correlation test). In conclusion, mercury vapor efficiently induced an autoimmune syndrome in genetically susceptible mice, and the LOAEL for the adverse effects varied in the order ANoA < B-cell stimulation < IC deposits. Comparing the body burden of mercury in mice at the LOAEL for autoantibodies with the body burden in populations of occupationally exposed humans suggests that the safety margin may be narrow for genetically susceptible individuals.     

Naltrexone: disposition, metabolism, and effects after acute and chronic dosing

The disposition of naltrexone during acute and chronic administration of 100-mg oral dose was studied in 4 subjects. Following an acute dose the mean (X) peak naltrexone plasma level was 43.6 +/- 29.9 ng/ml at 1 hr and for the major biotransformation product, beta-naltrexol, was 87.2 +/- 25.0 ng/ml at 2 hr. Twenty-four hours after the dose the X levels of naltrexone and beta-naltrexol declined to 2.1 +/- 0.47 and 17.6 +/- 5.0 ng/ml, respectively. Following chronic administration and X peak plasma levels of naltrexone and beta-naltrexol rose to 46.4 +/- 18.5 and 158.4 +/- 89.9 ng/ml at 1 hr, but by 24 hr both compounds declined to levels of the same order as in the acute state at 24 hr. Plasma levels of naltrexone and beta-naltrexol measured 24 hr after the daily doses of naltrexone throughout the study indicated that steady-state equilibrium was rapidly attained and that there was no accumulation of naltrexone and beta naltrexol in the plasma after chronic treatment on 100 mg oral doses. Biexponential kinetics were observed for naltrexone and beta-naltrexol in the first 24 hr. The half-life of naltrexone and beta-naltrexol decreased slightly from the acute to thechronic study from 10.3 +/- 3.3 to 9.7 +/- 1.1 hr and from 12.7 +/- 2.6 to 11.4 +/- 2.0 hr. The plasma levels of naltrexone declined slowly from 24 through 72 hr from 2.4 to 1.7 ng/ml, with an apparent half-life of 96 hr. The renal clearance data indicate that naltrexone is partially reabsorbed while beta naltrexol is actively secreted by the kidney. During acute and chronic naltrexone administration the mean fecal excretion was 2.1% and 3.6% while urinary excretion was 38% and 70% of the dose in a 24-hr period. Opiate antagonism to 25 mg heroin challenges was nearly complete through 48 hr after naltrexone. At 72 hr the objective responses reappeared to a greater extent than the subjective ones. Correlation coefficient (r) between naltrexone plasma levels and opiate antagonism was 0.91 and between individual half-life of naltrexone and opiate antagonism it was 0.99.     

Involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist in LPS-induced rabbit uveitis

The objective of the study was to investigate involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist (IL-1Ra) in lipopolysaccharide (LPS)-induced uveitis. Intravitreal injection of LPS (100 ng) to rabbits induced a massive leukocyte infiltration and protein leakage into the aqueous humor. Aqueous leukocyte counts and protein levels reached a peak 24 hr after this injection. The peak concentrations of aqueous TNF alpha (230 +/- 37 pg ml-1, at 9 hr) and IL-1 beta (185 +/- 80 pg ml-1, at 18 hr) preceded peak levels of aqueous leukocyte counts and protein levels. In contrast, the levels of aqueous IL-1Ra peaked at 48 hr (12,239 +/- 1964 pg ml-1) and a fairly high concentration of IL-1Ra remained when the inflammatory reactions subsided. Immunohistochemistry and leukocyte-depletion studies showed that infiltrating leukocytes were the major cellular sources of aqueous TNF alpha, IL-1 beta and IL-1Ra. Intravitreal injection of homologous TNF alpha (0.1-1.5 micrograms) or IL-1 beta (0.5-5 ng) reproduced a rapid leukocyte infiltration and protein leakage. Administration of anti-TNF alpha mAb (10 micrograms) suppressed the number of LPS-induced infiltrating neutrophils by 50%, mononuclear cells by 58%, and protein leakage by 42%. Administration of rabbit IL-1Ra (10 micrograms) also suppressed neutrophil influx by 78%, however, neither mononuclear cell influx nor protein leakage was inhibited by rabbit IL-1Ra. Co-administration of the two inhibitors enhanced inhibition of neutrophil infiltration to 88%, and protein leakage to 64%. We conclude that TNF alpha and IL-1 beta are the principal mediators of LPS-induced uveitis. Our observations also suggest that endogenous IL-1Ra may down-regulate inflammatory reactions.     

The induction of polyclonal B-cell activation and interleukin-1 production by the 75-kDa cell surface protein from Porphyromonas gingivalis in mice

The immunobiological activities of 75-kDa protein, fimbrial protein and lipopolysaccharide lipopolysaccharide prepared from whole cells of Porphyromonas gingivalis 381 were compared. The 75-kDa protein was mitogenic for BALB/c nu/nu, BALB/c and lipopolysaccharide-responsive C3H/HeN mouse spleen cells and for lipopolysaccharide-non-responsive C3H/HeJ mouse spleen cells. The response was significant in BALB/c mouse spleen cells incubated with 1-100 micrograms/ml of the 75-kDa protein. Furthermore, the 75-kDa protein exhibited polyclonal B-cell activation in murine spleen cells, which was similar to the lipopolysaccharide of P. gingivalis. In contrast, fimbriae from P. gingivalis did not, or only weakly, activated murine spleen cells. C3H/HeN mouse macrophages exposed to 10 micrograms/ml of the 75-kDa protein released large amounts of interleukin-1 (IL-1), which were maximal for 48 h, whereas IL-6 activity in macrophage supernatants was not detected throughout the culture period. These results suggest that the 75-kDa protein is a potent polyclonal B-cell activator and that it stimulates IL-1 production from murine peritoneal macrophages as well as lipopolysaccharide, which may play an important part in the inflammatory response during the development of periodontal diseases.     

Establishment of primary cell culture from stria vascularis explants. Morphological and functional characterization

To provide the prerequisite for long-term study of the inner ear related to structural and functional integrity, tissue of stria vascularis with spiral ligament was isolated from Wistar rat cochleas and cultured using the explant-culture technique. The following culture media were used: EMEM with Hepes buffer, hydrocortisone (400 ng/ml), transferrin (5 micrograms/ml). triiodothyronine (10(-9) M), cholera toxin (10(-10) M), insulin (5 micrograms/ml), and epidermal growth factor (10 ng/ml). To characterize the cells growing out from the explant, immunofluorescence with cytokeratin (cytokeratin 18) and ultrastructural examination with SEM and TEM were performed. The marginal cell function was investigated by expression of Na+, K(+)-ATPase antisera against beta 2 subunit of rat Na+, K(+)-ATPase and P-NPPase. We were able to maintain the cultured cells for 3 weeks or more. Monolayered marginal cells were observed beyond 14 days in vitro and the expression of cytokeratin 18 was especially enhanced. The cultured marginal cells were almost identical to in vivo cells both as regards ultrastructural features and Na+, K(+)-ATPase activity. The present results suggest that the primary explant culture technique is a reliable in vitro model of strial marginal cells. However, establishment of the cell line is needed for long-term study.