Chronic lithium administration triggers an over-expression of GRP94 stress protein isoforms in mouse liver.

Moderate doses of lithium were chronically administered to mice in order to verify whether the cytoprotective effects of lithium could be in part attributed to a molecular protection conferred by stress proteins/chaperones accumulation. In order to reach serum lithium levels within the common therapeutic values, mice were fed for 6 months on food pellets contained 1 g (L1 group) or 2 g (L2 group) lithium carbonate/kg, resulting in serum concentrations of 0.5 and 0.9 mM Li, respectively. Under these experimental conditions, no clinical side-effects were observed. Urea and creatinine concentrations in serum, lipids peroxidation level and activities of catalase, superoxide-dismutase and glutathione-peroxidase in liver and kidney were not significantly different from control values. Although the expression level of the constitutive HSP73 was not significantly modified, HSP72 was found to be down-regulated in kidney after 1 month. In liver, three protein bands were immunodetected by the anti-GRP94 antibody: 98 kDa and 96 kDa proteins corresponding to more or less glycosylated forms and/or phosphorylated forms of GRP94 and a 80 kDa protein probably being a cleavage product of GRP94. The 96 kDa and 80 kDa proteins were significantly up-regulated in liver of lithium-treated mice as compared to controls.

The effects of subchronic lithium administration in male Wistar mice on some biochemical parameters.

Lithium salts are efficiently used for treatment of psychiatric disorders. However, prolonged treatment frequently involves adverse side effects. In this study, effects of lithium carbonate administration on some biochemical parameters were studied in male mice. Lithium carbonate (20, 40, or 80 mg/kg body weight corresponding to 3.77, 7.54, or 15.08 mg Li element/kg body weight, respectively) was injected daily for 14 or 28 days. The following parameters were recorded: drinking water consumption, body weight, lithium and testosterone serum concentrations, activities of catalase (CAT), superoxide-dismutase (SOD), and glutathione-peroxidase (GPX), and level of lipid peroxidation (expressed as TBARS) in liver was performed. Lithium treatment, especially at the highest dose for 28 days, was found to induce weight gain and polydipsia and a significant decrease of plasma testosterone level. Lipid peroxidation level and activities of SOD and GPX were increased in liver, which suggests a perturbation of the antioxidative status. Our results indicate that subchronic exposure to lithium, which induces weight gain and polydipsia under our experimental conditions, also damages the male reproductive system and triggers an oxidative stress in the liver.

The effects of subchronic lithium administration in male Wistar mice on some biochemical parameters.

Lithium salts are efficiently used for treatment of psychiatric disorders. However, prolonged treatment frequently involves adverse side-effects. In the present work, effects of lithium carbonate administration on some biochemical parameters were studied in male mice. Lithium carbonate (20, 40 or 80 mg/kg body weight, corresponding to 3.77, 7.54 or 15.08 mg Li element/kg body weight, respectively) was injected daily for 14 or 28 days. The following parameters were recorded: water consumption, body weight, lithium and testosterone serum concentrations, activities of catalase (CAT), superoxide-dismutase (SOD) and glutathione-peroxidase (GPX) and level of lipid peroxidation (expressed as TBARS) in liver was performed. Lithium treatment, especially at the highest dose for 28 days, was found to induce weight gain, polydipsia and a significant decrease of plasma testosterone level. Lipid peroxidation level and activities of SOD and GPX were increased in liver which suggests a perturbation of the antioxidative status. Our results indicate that subchronic exposure to lithium, which induces weight gain and polydipsia under our experimental conditions, also damages the male reproductive system and triggers an oxidative stress in the liver.

Long-term exposure to low lithium concentrations stimulates proliferation, modifies stress protein expression pattern and enhances resistance to oxidative stress in SH-SY5Y cells.

SH-SY5Y cells, derived from a human neuroblastoma, were submitted to short- or long-term exposures to lithium carbonate concentrations ranging from 0.5 to 8 mM. Short-term exposures (4 days) to concentrations higher than 6 mM were found to reduce cell growth rate while exposure to 8 mM resulted in significant cell mortality. These ranges of concentrations induced an overexpression of (1) the HSP27 stress protein, (2) a 108 kDa protein (P108) recognized by an anti-phospho-HSP27(Ser78) antibody, and probably corresponding to a phosphorylated HSP27 tetramer, (3) a 105 kDa protein (P105), possible glycosylated or phosphorylated form of the GRP94 stress protein and (4) a phosphorylated (inactivated) form of glycogen synthase kinase (GSK3alpha/beta) SH-SY5Y cells, when cultured in the presence of 0.5 mM lithium for 25 weeks, displayed interesting features as compared to controls: (1) higher cell growth rate, (2) increased resistance toward the inhibitory effects of high lithium concentrations on cell proliferation, (3) lower basal level of lipid peroxidation (TBARS) and improved tolerance to oxidative stress induced by high lithium concentrations, (5) reduced expression of monomeric HSP27 versus an increase of corresponding tetrameric protein (P108) and (6) overexpression of a 105 kDa protein (P105). In conclusion, our study suggests that chronic treatment (over several months) by therapeutic relevant lithium concentrations could favour neurogenesis, decrease the vulnerability of neuronal cells to oxidative stress and induce posttranslational changes of molecular chaperones.

Impact of dietary restriction on peroxidative effects of nickel chloride in wistar rats.

ABSTRACT The purpose of this study, carried out in Wistar rats, was to evaluate the protective effect of dietary restriction (performed by intermittent fasting) against oxidative stress induced by a low concentration of nickel chloride in kidney, liver, uterus, and ovary. Lipid peroxidation (TBARS), catalase activity, and the levels of vitamins E and A in the blood were investigated in rats feed for 1 month either daily (N) or 1 day over two (intermittent fasting, IF) and then injected (NNi, IFNi) or not with nickel chloride (30 mumoles/kg body weight/day) for 10 days. Ni induced a significant increase of TBARS in organs of N rats. Intermittent fasting alone or associated to nickel treatment did not result in TBARS change in IF and IFNi rats. Catalase activity levels were found to be similar in N and IF rats. In Ni-treated rats a transient increase of catalase activity appeared at day 1 in the kidney and days 1 and 3 in the liver. Then, catalase activity was found to be inhibited until day 10. In the uterus and ovary, catalase activity was always found to be inhibited. In IFNi rats, no significant increase of catalase activity was observed as compared to IF rats. Vitamin E was inhibited from the 1st to the 10th day in Ni rats, whereas no significant changes were noted in IFNi rats. A moderate decrease of vitamin A was only found at days 1 and 3 in Ni rats. In conclusion, intermittent fasting is able to protect from oxidative stress induced by low concentration of Ni, but catalase and Vitamins E and A do not seem to be involved.

Lithium toxicity and expression of stress-related genes or proteins in A549 cells.

To unveil some molecular mechanisms underlying lithium toxicity, expression changes of stress-related genes or proteins were analysed in A549 cells, cultured for 3 days in presence of lithium. A dose-dependent cell-growth inhibition was found for concentrations ranging from 2 (toxicity threshold) to 12 mM (lethality threshold). cDNA arrays technology was used to analyse effects of 5 and 10 mM lithium. Among genes involved in cell cycle regulation, proliferating cell nuclear antigen (PCNA) was down-regulated and cyclin kinase inhibitor p21 (CDKN1A), up-regulated. Genes of paraoxonase 2, known to prevent LDL lipid peroxidation, and of catalase and SOD were found to be down-regulated whereas genes of cytochrome P450 (CYP2F1, CYP2E1) were up-regulated. This probably results in higher intracellular levels of reactive oxygen species and account for increased levels of lipid peroxidation commonly associated with lithium exposure. Moreover, lithium was found to down-regulate genes coding for anti-apoptotic gene BAG-1 and for most of the molecular chaperones (HSP, GRP). This might account for lithium toxicity since these proteins are critical for cell survival. At translational level, a 105 kDa protein was found to be over-expressed. This protein was recognized by the anti-GRP94, anti-KDEL and anti-phosphoserine monoclonal antibodies suggesting that, lithium could induce post-translational modifications of GRP94 phosphorylation. Using tunicamycin and thapsigargin, it was concluded that lithium effects are not related to defect in N-linked glycosylation and/or to changes in calcium homeostasis.

Effect of selected insecticides on growth rate and stress protein expression in cultured human A549 and SH-SY5Y cells.

Two organochlorines (dienochlor, endosulfan) and one neonicotinoid (imidacloprid) insecticides were investigated as putative cellular aggressors, both as pure chemicals and as commercial formulations, in order to evaluate the additional toxicity due to additives present in the commercial formulations. Toxicity was evaluated on human cells in vitro, by culturing neuronal SH-SY5Y and pulmonary A549 cell lines for 3 days in the presence of increasing concentrations of the selected pesticides. LOEC (lowest observed effect concentration), IC50 (concentration leading to a 50% decrease of cell growth) and expression changes of molecular chaperones involved in cellular protein quality control were determined. The investigated molecular chaperones were the cytosolic resident heat shock proteins (HSP27, HSP72/73, and HSP90) and the glucose regulated proteins (GRP78, GRP94) located in the endoplasmic reticulum (ER). Organochlorines were found to be the most toxic in both A549 and SH-SY5Y cells, IC50 being respectively 0.95 and 0.36 microM for dienochlor, 34 and 20 microM for endosulfan, 1.8 and 1.5 mM for imidacloprid. This shows that neuronal cells were more sensitive than pulmonary cells. LOEC and IC50 appeared at lower concentrations of active molecule when using the commercial formulations Techn'ufan (endosulfan) and Confidor (imidacloprid), indicating an additional adverse effect of additives. Insecticide concentrations higher than IC50 were found to induce an underexpression of all cytosolic HSPs, probably resulting from a general inhibition of protein synthesis. HSP27 was found to be underexpressed at concentrations of imidacloprid or endosulfan (as Techn'ufan) lower than IC50. This underexpression of the anti-apoptotic HSP27 could contribute to the increase of cell mortality. GRP78 was up-regulated by endosulfan in A549, but not in SH-SY5Y cells, suggesting a damaging effect on proteins specific to pulmonary cells. Conversely, HSP72/73 was found to be down-regulated, resulting probably from the ER unfolded protein response (UPR) as previously reported [Skandrani, D., Gaubin, Y., Vincent, C., Beau, B., Murat, J.C., Soleilhavoup, J.P., Croute, F., 2006. Relationship between toxicity of selected insecticides and expression of stress protein (HSP, GRP) in cultured human cells: effects of commercial formulations versus pure active molecules. Biochim. Biophys. Acta 1760 (1), 95-103].

Changes in growth rate and thyroid- and sex-hormones blood levels in rats under sub-chronic lithium treatment.

Lithium therapy, mainly used in curing some psychiatric diseases, is responsible for numerous undesirable side effects. The present study is a contribution to the understanding of the pathophysiological mechanisms underlying lithium toxicity. Male and female mature rats were divided into three batches and fed commercial pellets: one batch was the control and the second and third batches were given 2 g (Li1) and 4 g (Li2) of lithium carbonate/kg of food/day, respectively. After 7, 14, 21 and 28 days, serum levels of free tri-iodothyronine (FT3), thyroxine (FT4), testosterone and estradiol were measured. Attention was also paid to growth rate and a histological examination of testes or vaginal mucosa was carried out. In treated rats, a dose-dependent loss of appetite and a decrease in growth rate were observed, together with symptoms of polydypsia, polyuria and diarrhea. Lithium serum concentrations increased from 0.44 mM (day 7) to 1.34 mM (day 28) in Li1 rats and from 0.66 to 1.45 mM (day 14) in Li2 rats. Li2 treatment induced a high mortality after 14 days, reaching 50-60% in female and male animals. From these data, the LD50 (14 days Li2 chronic treatment) was calculated to be about 0.3 g/day per kilogram of animal, leading to Li serum concentrations of about 1.4 mM. A significant decrease of FT3 and FT4 was observed in treated rats. This effect appeared immediately for the highest dose and was more pronounced for FT3, resulting in an increase of the FT4/FT3 ratio. In males, testosterone decreased and spermatogenesis was stopped. Conversely, in females, estradiol increased in a dose-dependent manner as the animals were blocked in the diestrus phase at day 28. This finding supports a possible antagonistic effect of lithium on the estradiol receptors.

Stress proteins (Hsp72/73, Grp94) expression pattern in rat organs following metavanadate administration. Effect of green tea drinking.

Expression pattern of heat shock proteins (Hsp) 72/73 and glucose regulated protein (Grp) 94 was studied in liver, kidney and testis of rats injected with sublethal doses of ammonium metavanadate (5 mg/kg/day). In addition, some batches of animals were given green tea decoction, known to be rich in anti-oxidative compounds, as sole beverage in order to evaluate its protective properties. In control animals, the stress proteins expression was found to be organ-dependent: anti-Grp94 antibody revealed two bands at 96 and 98 kDa in kidney and liver whereas the 98 kDa band only was found in testis; anti-Hsp72/73 antibody revealed that the constitutive Hsp73 was present in all organs whereas the inducible Hsp72 was only present in kidney and testis. In kidney of vanadium-treated rats, Hsp73 was over-expressed by about 50% whereas Hsp72 was down-regulated by 50-80%. No such effects were observed in liver and testis. In liver and kidney of vanadium-treated rats, Grp94 was over-expressed by 50% and 150% respectively whereas no change was found in testis. In rats given green tea as sole beverage, the 96 kDa protein expression level in liver was reduced both in controls and in vanadium-treated animals. However, green tea drinking failed to prevent the vanadium-induced Hsp72 under-expression in kidney of vanadium-treated rats.

Effects of nickel poisoning on expression pattern of the 72/73 and 94 kDa stress proteins in rat organs and in the COS-7, HepG2, and A549 cell lines.

The present study deals with the effects of Ni on the expression level of three stress proteins, namely, the cytosolic HSP72 and HSP73, and the reticulum-associated GRP94. Experiments were carried out on "Wistar'' female rats daily injected with 4 mg NiCl2 per kg body weight for 1, 3, 5, and 10 days. Another set of experiments were carried out using cell lines, derived from the monkey kidney (COS-7), and from human tumors of the lung (A549) and liver (HepG2). Cells were cultured for 4 days in the permanent presence of 100, 200, or 400 microM NiCl2. In control rats, stress proteins pattern was found to be tissue specific: two protein bands of 96 and 94 kDa were immunodetected with the anti-GRP94 antibody in kidney and liver extracts, whereas only the 96 kDa band was present in ovary extracts. HSP73 was present in kidney, liver, and ovary whereas HSP72 was only found in kidney. In kidney of nickel-treated animals, HSP73 and the 96 kDa proteins were overexpressed whereas HSP72 was strongly down regulated. No such effect was observed in liver or ovary. Similarly, in nickel-treated cell lines, HSP72 was downregulated and GRP94 (96 kDa protein) was overexpressed. HSP73 expression appeared moderately increased in A549 cells but decreased in COS-7 cells. Because long-term caloric restriction was reported to reduce free radical generation in cells, the effect of 1 month food restriction (50%) was tested in rats as a possible way to lower oxidative damages induced by Ni. No significant effect on HSP expression was observed.

Volatile organic compounds cytotoxicity and expression of HSP72, HSP90 and GRP78 stress proteins in cultured human cells.

The aim of this study was to determine whether overexpression of stress proteins (SPs) could be a sensitive biomarker for cell injury due to exposure to low doses of volatile organic compounds (VOCs) such as benzene, ethylbenzene, toluene, xylene, and chlorinated derivatives (ClB). Sublethal and cytotoxic threshold concentrations of the VOCs were determined by studying the growth rate of normal (fibroblasts) or tumor-derived human cell lines (A549, HepG2) exposed for 4 days to VOCs. Changes in SP expression as a function of concentrations were investigated by Western blotting.VOC toxicity was found to be correlated with their degree of chlorination and their hydrophobicity. Cytotoxic threshold concentrations (no-observed effect concentration, NOEC) were found to be similar for the three cell lines. It was observed that using a mixture of VOCs, each of them at concentration below the NOEC, resulted in an actual toxicity to the cells. This finding reveals a synergistic effect and should be taken into account when assessing threshold risk and exposure limit values in the worker's environment when several pollutants may be present. HSP72 and HSP90 expression levels were not affected whereas GRP78 expression was increased by all the VOCs. Taking into account the specific molecular function of GRP78, it suggests that VOC exposure results in misfolded or underglycosylated protein accumulation in the endoplasmic reticulum. GRP78 overexpression was closely related to the magnitude of growth inhibition due to increasing concentrations of each VOC. The overexpression was found to be significant for concentrations 5 to 30 times higher than NOEC, indicating that, under our experimental conditions, GRP78 expression cannot be considered as a sensitive biomarker of exposure to environmental VOCs.

Effect of chronic lead exposure on kidney function in male and female rats: determination of a lead exposure biomarker.

Several cytotoxic chemical pollutants inducing peroxidative damages are liable to induce kidney failure. Among these pollutants we find heavy metals such as: lead, nickel, cadmium, vanadium and mercury. Lead is one of the most dangerous metals because it is widely spread in the environment, and because it may be a source of several nervous diseases. The aim of this study is to provide evidence concerning the effect of this metal on the renal function and to try to determine a storage corner in the organism which serves as an indicator of a lead intoxication. Lead acetate was administered by oral route in the drinking water to adult rats aged three months at the rate of 0.3% (P1) and 0.6% (P2). Reference rats received distilled water to drink under the same conditions. The treatment continued for 15, 30, 45, 60 and 90 days. The creatinemia, uremia, glycemia and creatinuria are determined by colorimetric techniques. Lead concentration in blood as well as the lead content of the tail are determined by atomic absorption after nitroperchloric mineralization at the liquid stage. The results showed an increase of creatinemia on the 30th day of the experiment for both sexes in (P1 and P2). The same happened for ureamia. The increase of these two parameters would indicate a renal deficiency which is confirmed by a decrease of creatinuria and urinary pH observed mainly on and after the 45th day of the experiment. An increase of the renal relative weight was noticed in P1 and P2 on the 30th day of the treatment. The determination of the concentration of lead in the blood shows that this factor increases among treated subjects in a constant way, independently of the dose and the duration of the treatment. Nevertheless, the rate increase of lead in the tail seems to be dose-dependent. In conclusion, lead administered by oral route causes a renal deficiency to the rat without distinction between males and females. In addition, the tail seems to be a reliable exposure biomarker that demonstrates lead intoxication. The tail seems to be a dosimeter of lead bio-accumulation. It constitutes an endogenous source of lead impregnation. The concentration of lead in the blood is only an indicator of recent exposure.

Implication of free radicals and glutathione in the mechanism of cadmium-induced expression of stress proteins in the A549 human lung cell-line.

Cellular mechanisms underlying the expression of stress proteins (HSP) were studied in the human cell-line A549 submitted to a pollutant, cadmium, in the presence of several agents which modulate the glutathione level and, supposedly, the effects of this metal in the cell. It was observed that HSP 90, HSP 72 and HSP 27 are significantly over-expressed after exposure to cadmium chloride for 24 h. Low cadmium concentrations (i.e. from 1 to 10 microM) also triggered a slight accumulation of glutathione, whereas this compound was depleted after exposure to higher cadmium concentrations (25-100 microM). When 50 microM diethyl-maleate, which traps glutathione, was added together with cadmium, the over-expression of HSP 72 and HSP 90 was much stronger. Treatment of cells with 20 or 40 mM N-acetyl-L-cysteine, which traps free radicals, was found to increase by 30% the glutathione level and to suppress the HSP over-expression. From our results, it is suggested that HSP induction by cadmium in A549 cells is due, at least in part, to the oxidative stress consisting in formation of reactive oxygen species and inhibition of peroxides detoxification. Due to this oxidative status within the cell, more proteins would be damaged inducing the HSP over-expression.

Pattern of stress protein expression in human lung cell-line A549 after short- or long-term exposure to cadmium.

Exposure to cadmium is associated with the development of pulmonary damage such as emphysema and lung cancer. This metal is also a powerful inducer of stress proteins in many biologic models. The present study was undertaken to evaluate whether an overexpression of the heat shock protein (hsp)72 stress protein, which indicates repair of damaged proteins, could be a sensitive and early biomarker of environmental pollution by Cd. In comparative studies, we examined the effects of exposure to Cd (as CdCl(2)) on the growth rate of the A549 pulmonary cell line, and (by Western blot analyses) on the induction of the hsp72 stress protein and metallothioneins (MTs). CdCl(2) exposure was studied for periods of 2 hr to 1 month. For short-term exposure (2-6 hr) to Cd concentrations higher than 50 microM, an overexpression of hsp72 appeared 6 hr later, suggesting that hsp72 might be considered an early biomarker of acute exposure to Cd. For exposures lasting more than 4 days, lower doses of Cd (0.1-10 microM) similar to levels encountered in occupational exposure induced a significant increase of the hsp72 level. Because the increase of hsp72 occurs for doses that did not affect cell proliferation, our work supports the idea that its overexpression might be used as a sensitive indicator of occupational exposure to Cd. However, increased resistance to Cd appeared in A549 cells exposed for 1 month and overexpression of hsp72 disappeared simultaneously. It is possible that, in vivo, cell adaptation also occurs throughout chronic exposure to Cd, with a decrease of hsp induction as a consequence. A dose-related increase of MTs was found after 4 days of exposure to Cd concentrations ranging from 0.1 to 10 microM without change of overexpression during chronic exposure, suggesting that MT expression could be a more constant indicator of Cd pollution. Because 0.1 microM Cd (11 microg/L) induces hsp72 expression, showing the presence of damaged proteins, our work suggests that the maximum allowable biologic exposure limit should be lowered.

Supernatant cytotoxicity and proteolytic activity of selected oral bacteria against human gingival fibroblasts in vitro.

The purpose of this study was to determine if endodontic bacterial act in vitro on human gingival fibroblast functions via extracellular products. The bacteria used were Prevotella nigrescens, Capnocytophaga ochracea, Peptostreptoccocus micros and Actinobacillus actinomycetemcomitans. Supernatants were collected from bacterial cultures at the beginning of the stationary phase when their density was similar. Toxins that inhibited fibroblast proliferation were found in all culture supernatants of Gram-positive or Gram-negative bacterial strains, except for Prev. nigrescens. The cytotoxicity of A. actinomycetemcomitans supernatant was about 1000 fold higher than the others. This supernatant diluted to 1/1000 led to total fibroblast growth inhibition whereas only 25% growth inhibition was obtained with Capn. ochracea and Pept. micros diluted to 1/10. Bacterial supernatant proteolytic activity was investigated in confluent fibroblast cultures that were incubated for 48 hr with each of the supernatants diluted to 1/2 except for A. actinomycetemcomitans supernatant diluted to 1/20. Indirect immunofluorescence studies of extracellular-matrix molecules, followed by immunoelectrophoretic analysis of extracts of whole-cell layers, demonstrated that only conditioned medium of Prev. nigrescens had a proteolytic activity capable of degrading the greater part of type I collagen and fibronectin fibres in the extracellular matrix.

Hsp72 mRNA production in cultured human cells submitted to nonlethal aggression by heat, ethanol, or propanol. Application to the detection of low concentrations of chromium(VI) (potassium dichromate).

The HT29 and HepG2 human cell lines have been shown to express stress proteins (heat shock proteins, HSP) when submitted to a variety of sublethal environmental aggressions. In the present study, these cells were submitted to standardized mild aggression by heat, ethanol, or propan-1-ol in vitro. Subsequent formation of the hsp72 mRNA was measured by a very specific RNase protection method using a radiolabeled antisense RNA probe. The accumulation of the mRNA coding for the HSP72 stress proteins was found to be maximum within 3 h after the aggression. Results were obtained faster and were much more interpretable than those from the classical method involving the autoradiography of electrophoretically separated 35S-labeled proteins, especially in the case of very weak, threshold-level, aggressions. When this model was used as a biological system for the detection of low concentrations of chromium(VI) (Cr2O7(2-)), it was possible to detect concentrations as low as 0.5 mumol/L. This indicates that measuring indices of stress induction in human cultured cells can be several orders of magnitude more sensitive than the commercial Microtox assay used for detecting low levels of pollution.

Normal human epidermal keratinocytes express in vitro specific molecular forms of (pro)filaggrin recognized by rheumatoid arthritis-associated antifilaggrin autoantibodies.

BACKGROUND: The so-called antikeratin antibodies and the antiperinuclear factor are the most specific serological markers of rheumatoid arthritis (RA). They were recently shown to be largely the same autoantibodies and to recognize human epidermal filaggrins and profilaggrin-related proteins of buccal epithelial cells (collectively referred to as (pro)filaggrin). MATERIALS AND METHODS: To further characterize the target antigens, we investigated their expression by normal human epidermal keratinocytes cultured in differentiating conditions, using immunofluorescence and immunoblotting with RA sera and three different monoclonal antibodies to (pro)filaggrin. RESULTS: On the cornified, stratified epithelial sheets obtained in vitro, RA sera with anti(pro)filaggrin autoantibodies (AFA) produced granular staining of the stratum granulosum and diffuse staining of the stratum corneum. The antigens recognized by RA sera strictly colocalized with (pro)filaggrin in keratohyalin granules. Following sequential extraction of the proteins from the epithelial sheets, the RA sera and the three monoclonal antibodies to (pro)filaggrin, recognized a series of low-salt-soluble molecules, including a neutral/acidic isoform of filaggrin and several proteins with sizes and pI intermediates between this isoform and profilaggrin. They also recognized urea-soluble high-molecular-weight profilaggrin-related molecules. CONCLUSIONS: These results show that in vitro epidermal keratinocytes express various molecular forms of (pro) filaggrin that bear epitopes targeted by AFA of RA sera, and that some of these are absent from epidermis. Moreover, these epitopes, which are present on the keratohyalin granules of buccal epithelial cells but not on those of epidermal cells, are present on the granules of the cultured keratinocytes. This work completes the molecular characterization of the proteins targeted by AFA.

Modulation by hypergravity of extracellular matrix macromolecules in in vitro human dermal fibroblasts.

In vitro human dermal fibroblasts were submitted to normal gravity (1 g) or to chronic hypergravity (20 g) over a period of 8 days. Changes in organization of extracellular matrix molecules were seen by indirect immunofluorescence. In the fibronectin layer, bundles of fibrils were gathered together leading to a disorganisation of the normal parallel pattern of fibers seen in control cultures. Type I collagen fibrils appeared with wooly outlines in controls whereas thick fibers were closely packed in 20-g cultures. A moderate increase of type III collagen fibril density was observed. No elastic fibers were seen in control or in 20-g cultures. In the culture medium, the release of soluble elastin (ELISA) and type I and III collagens (RIA) was undisturbed. Assays of enzymes involved in the remodeling of extracellular matrix showed an increase of cellular elastase activity (10%) and a decrease of the spontaneously active collagenase. Nevertheless, the total collagenase activity, (activated by trypsin), was increased by up to 30%. These data show a significant rise of the latent collagenase activity and suggest that release of the tissue inhibitor of metalloproteinase (TIMP1) was enhanced by hypergravity.

Effects of hypergravity on the cell shape and on the organization of cytoskeleton and extracelluar matrix molecules of in vitro human dermal fibroblasts.

In vitro human dermal fibroblasts were submitted to normal gravity (1 g) or to chronic hypergravity ranging from 2 to 20 g for 8 days. Changes only appeared above 15 g. The majority of 20 g-subjected cells showed fine filipods in the shape of a star whereas most control cells had rounded shapes and spread by forming lamellipodia. Indirect immunofluorescence staining of vinculin, alpha-actinin and actin stress fibers showed changes of the arrangement anchoring points of stress fibers under hypergravity. Tubulin staining showed that the centrosomal material generally located above the nucleus in control cells had migrated to the nucleus side in 20 g-exposed cells. After 8 d of culture under 20 g hypergravity the thickness of fibronectin network seemed to be increased and bundles of fibrils appeared linking ordered arrays of fibers. The fibrils of collagen I formed better delimited and thicker bundles of fibers. We may assume that 20 g hypergravity can induce changes in fibroblast cell shape, migration way, and anchorage leading to a reorganization of extracellular matrix without concomitant change of cell proliferation.

The expression of desmosomal and corneodesmosomal antigens shows specific variations during the terminal differentiation of epidermis and hair follicle epithelia.

Using five monoclonal antibodies (MAb), we studied by indirect immunofluorescence the desmosomes and a junctional structure specific to cornified layers, the corneodesmosome, in normal and plantar epidermis and in the various sheaths of the anagen hair follicle. The monoclonal antibodies DP1&2.2-15, PG5.1, and DG3.10, specific for desmoplakins I/II, plakoglobin, and desmoglein I, respectively, were used to study the desmosome antigens, and G36-19 and G20-21 to study the corneodesmosome antigens. The distribution and sequence of expression of the five antigens allowed the nine epithelial differentiation pathways studied to be merged into four distinct families: non-plantar epidermis, characterized by the absence of desmosome and corneodesmosome antigens in the stratum corneum; the outer root sheath of the hair follicle, which behaves like the viable layers of the epidermis with regard to the desmosome antigens but does not express the corneodesmosome antigens; plantar epidermis and the three components of the inner root sheath in which the corneodesmosome antigens are present up to the desquamating layer; and the three components of the hair shaft, which are characterized by the absence of expression of both the desmosome and the corneodesmosome antigens in its mature portion.