Antibodies directed against annexin A2 and obstetric morbidity.

Acquired and inherited thrombophilia have both been reported to be associated with an increased risk of obstetric complications in early or later stages of pregnancy. Annexin A2 (ANXA2) is strongly expressed in vascular and placental tissues and plays a crucial role in fibrinolysis. The aim of the present study was to evaluate the prevalence of antibodies directed against ANXA2 in patients with recurrent miscarriage or obstetric complications. Anti-ANXA2 antibodies (aANXA2) were detected by ELISA in the sera from 46 women with obstetric morbidity, mainly recurrent miscarriage. The cut-off value for positivity was defined as 3 standard deviations above the mean optical density (OD) obtained in the sera from 42 female blood donors. The prevalence of aANXA2 in patients and healthy individuals was 15.2% and 2.3%, respectively. A statistically significant difference was observed between the 2 groups in terms of aANXA2 IgG titers (p=0.01). The highest aANXA2 levels were observed in sera from 2 patients with recurrent miscarriage and one patient with preeclampsia. aANXA2 could play a role in thrombotic mechanisms leading to recurrent pregnancy loss and placental vascular disease. Further studies are needed to determine whether ANXA2 is critical for maintenance of placental integrity.

On the repair of oxidative damage to apoferritin: a model study with the flavonoids quercetin and rutin in aerated and deaerated solutions.

Ferritin (Ft) impairment through (•)O2(-), H2O2, and (•)OH production occurs in the cases of ketoses, diabetes mellitus, acute intermittent porphyria and tyrosinemia. In addition to (•)Trp and TyrO(•) radical production, ferrous iron liberation and Ft synthesis stimulation, site-specific oxidation reactions are induced leading to toxic iron accumulation in organs with high Ft content, for example, liver and brain. To elucidate the potential pathways to Ft recovery, repair of oxidative damage to horse spleen apoferritin (apoFt) and Ft by quercetin (QH) or rutin (RH) was studied in the presence and absence of oxygen. (•)Trp and TyrO(•) radicals were produced in pulse radiolysis through apoFt oxidation by (•)Br2(-) radicals. QH and RH bind to apoFt on eight sites with binding constants of ˜80,000 and ˜32,000 M(-1), respectively. In deaerated solutions, a repair of apoFt radicals is observed involving both bound and free flavonoids. This repair occurs by a fast intra- and a slow inter-molecular electron transfer from bound and free flavonoids, respectively. With QH, the rate constants are 10(4) s(-1) and 3.5 × 10(7) M(-1) s(-1) for the intra- and intermolecular repair reactions, respectively. Oxygen does not interfere with repair of apoFt or Ft by bound QH but inhibits 90% of Ft repair by RH. These results taken together indicate that flavonoid antioxidants may help alleviate Ft impairment in diseases involving an oxidative stress.

Activation of JAK2 by the oxidative stress generated with oxidized low-density lipoprotein.

Atherosclerosis includes a series of cellular and molecular responses characteristic of an inflammatory disease. We provide evidence that cupric-ion-oxidized LDL (CuLDL) or endothelial cell-oxidized LDL (ELDL) induced the activation by Tyr-phosphorylation of JAK2, one of the Janus kinase involved upstream of STATs in the JAK/STAT pathway of cytokine transduction. Oxidized LDL (OxLDL) also initiated STAT1 and STAT3 Tyr-phosphorylation and translocation to the nucleus, with a more marked effect for the extensively modified CuLDL. Genistein, a nonspecific Tyr-kinase inhibitor, and AG490, a specific inhibitor of JAKs, markedly prevented the CuLDL-induced enhancement of STAT1 and STAT3 Tyr-phosphorylation and DNA-binding activity, suggesting that JAKs are the main kinases involved in STATs' activation by oxidized LDL. In addition, the lipid extract of CuLDL increased the intracellular levels of lipid peroxidation products and the Tyr-phosphorylation of JAK2, STAT1, and STAT3, whereas the antioxidant vitamin E prevented all these effects. These results demonstrate that OxLDL induces the activation by Tyr-phosphorylation of JAK2, STAT1, and STAT3 by generation of an intracellular oxidative stress by means of its lipid peroxidation products, and thus include JAK2 within the range of oxidative stress-activated kinases.

Activation of the JAK/STAT pathway by ceramide in cultured human fibroblasts.

Endogenous ceramide (CER) was generated by treatment of cultured fibroblasts with sphingomyelinase (SMase) from Bacillus cereus. A 30 min treatment with 0.1-0.3 U/ml SMase induced a dose-dependent increase in the intracellular level of CER. The activation of the transcription factors signal transducer and activator of transcription (STAT) 1 and STAT3 by SMase was investigated by determination of the phosphorylation state by immunoblot, and of DNA binding activity by electrophoretic mobility shift assay. SMase treatment induced a dose-dependent Tyr-phosphorylation of STAT1/3. SMase also enhanced STAT1/3 DNA binding activity in a dose-dependent manner. Concomitantly, SMase enhanced the Tyr-phosphorylation of Janus kinase (JAK) 2, a Tyr-kinase localized upstream of STATs in the JAK/STAT pathway. The Tyr-kinase inhibitor genistein and the JAK inhibitor AG490 both prevented JAK2 Tyr-phosphorylation, together with STAT1 and STAT3 Tyr-phosphorylation and binding activity. The SMase-induced increase in STAT1/3 binding activity was prevented by methyl-beta-cyclodextrin, a cholesterol binding agent that causes a loss of compartmentalization of the molecules located in caveolae. This increase was also prevented by the MEK inhibitor PD98059, thus demonstrating the role of the MEK/ERK pathway in this system. Besides ERK, SMase activated other signaling kinases such as JNK and p38. Exogenous natural CER also activated STAT1/3 binding activity, which indicates that most probably, endogenous CER is the second messenger involved in the effect of SMase. These results describe a crosstalk between the SMase/CER and the JAK/STAT signaling pathways and include JAK2 within the range of CER-activated intracellular kinases.

UVA radiation stimulates ceramide production: relationship to oxidative stress and potential role in ERK, JNK, and p38 activation.

Exposure of human keratinocytes to UVA radiation induced an increase in ceramide (CER) intracellular content, with a dose-dependent effect within the range of 4-9 J/cm(2). The production of CER reached a maximum 2 h after UVA irradiation. The increase of CER was proportional to the intracellular content of reactive oxygen species, was prevented by the antioxidant vitamin E, and enhanced by the prooxidant buthionine-sulfoximine, suggesting the involvement of an oxidative stress. UVA decreased both neutral and acid sphingomyelinase activities measured in vitro. A direct cleavage of sphingomyelin to CER by UVA, recently described, was not observed under our experimental conditions. We also show that, downstream of CER, UVA activated the Ser/Thr kinases ERK, JNK, and p38. Since ceramide has been shown to play a role in stress kinase activation, our results provide a possible mechanism for UVA-induced activation of stress kinases via ceramide formation. However, the actual mechanisms whereby CER is produced in cultured cells under UVA exposure remain to be specified.

Oxidized LDL induces an oxidative stress and activates the tumor suppressor p53 in MRC5 human fibroblasts.

It is now well established that oxidized LDL (OxLDL) is involved in the progression of the atheromatous plaque via several mechanisms, including its cytotoxicity toward the arterial wall. Our study demonstrates that a 4-h incubation of cultured human fibroblasts with 25-75 microg/ml OxLDL induced a dose-dependent increase in the intracellular levels of reactive oxygen species (ROS) and lipid peroxidation end products (TBARS). This effect was markedly prevented by the antioxidant vitamin E. The lipid extract of OxLDL partially reproduced the action of the LDL particle itself. Concomitantly, OxLDL enhanced the DNA binding activity of p53 measured by electrophoretic mobility shift assay, and the intracellular protein level of p53 determined by immunoblot analysis. Cycloheximide prevented the OxLDL-induced augmentation in both p53 binding activity and intracellular level. Again, the lipid extract of OxLDL reproduced the effect of OxLDL on p53 binding activity, whereas vitamin E prevented it. These results indicate that OxLDL initiates an intracellular oxidative stress by means of its lipid peroxidation products, leading to the activation of the tumour suppressor p53 by enhancement of p53 protein synthesis. This effect might be related to the cytotoxic effect of OxLDL since the activation of p53 is known to lead to cell cycle arrest, necrosis or apoptosis.

Cellular enrichment with polyunsaturated fatty acids induces an oxidative stress and activates the transcription factors AP1 and NFkappaB.

A 48-h incubation of cultured human fibroblasts with 5 x 10(-5) M oleic acid or polyunsaturated fatty acids (PUFA) from the (n-6) (linoleic, gamma-linolenic and arachidonic acids) or (n-3) (alpha-linolenic and eicosapentaenoic acids) series resulted in an enrichment of the cells with the introduced fatty acid. Cell enrichment with PUFA initiated a rise in the intracellular level of reactive oxygen species (ROS) and lipid peroxidation products (thiobarbituric reactive substances TBARS). Simultaneously, cell enrichment with all the studied PUFA induced an increase in AP1 and NFkappaB binding activity measured by electrophoretic mobility shift assay, whereas no significant effect was observed with the monounsaturated oleic acid. Furthermore, the antioxidants vitamin E (alpha-tocopherol) and N-acetyl cysteine prevented both the arachidonic acid-induced increase in intracellular ROS and TBARS, and the activation of AP1 and NFkappaB. These results indicate that the accumulation of PUFA from (n-6) and (n-3) series elicited an intracellular oxidative stress, resulting in the activation of oxidative stress-responsive transcription factors such as AP1 and NFkappaB.

Lipopolysaccharide enhances oxidative modification of low density lipoprotein by copper ions, endothelial and smooth muscle cells.

The effect of lipopolysaccharide (LPS, endotoxin) on low density lipoprotein (LDL) oxidative modification by copper ions, endothelial and smooth muscle cells was studied by determination of the level of lipid peroxidation products (thiobarbituric acid reactive substances or TBARS), the diene level and the electrophoretic mobility of the LDL particle. LPS 25-75 microg/ml induced a dose-dependent increase in LDL oxidation by copper ions, endothelial and smooth muscle cells. At 75 microg LPS/ml, the TBARS content was 1.9, 1.6, and 1.8-fold increased, respectively. The LDL degradation by J774 macrophage-like cells was concomitantly stimulated. Preincubation of the LDL particle with LPS induced a marked increase in the subsequent LDL oxidative modification either by copper ions or by endothelial and smooth muscle cells. In addition, pretreatment of endothelial and smooth muscle cells with LPS also induced an enhancement of LDL oxidative modification performed in the absence of LPS. This effect was accompanied by a parallel increase in superoxide anion release by the cells. These results point at one of the mechanisms involved in the described association between bacterial infection and acute myocardial infarction as well as coronary heart disease.

Oxidized LDL activates STAT1 and STAT3 transcription factors: possible involvement of reactive oxygen species.

The effect of cupric ion-oxidized low density lipoprotein (Cu-LDL) or endothelial cell-oxidized LDL (E-LDL) on STAT1 and STAT3 (signal transducers and activators of transcription) DNA binding activity was investigated by electrophoretic mobility shift assay in human endothelial cells. Both oxidized LDL enhanced STAT1 and STAT3 binding to their respective consensus binding sites. Furthermore, the activation of STATs was proportional to the oxidation degree of LDL in that the highly oxidized Cu-LDL exhibited a more marked effect than E-LDL. Oxidized LDL induced an intracellular oxidative stress, as shown by the increase in the intracellular level of lipid peroxidation products (thiobarbituric acid-reactive substances) and in the level of reactive oxygen species, measured by the fluorescence of dichlorofluorescein diacetate. The binding activity of STAT1 and STAT3 paralleled these two parameters, which suggests that it is dependent upon the redox state of the cell. The activation of STATs by oxidized LDL was almost completely inhibited by the lipophilic antioxidant vitamin E, and partially antagonized by the hydrophilic thiol-containing compound N-acetylcysteine, suggesting that the oxidative stress induced by oxidized LDL is involved in the observed phenomenon. Furthermore, the lipid extract of Cu-LDL also activated STAT1 and STAT3. Since the STAT pathway plays a key role in cytokine and growth factor signal transduction, the activation of STATs by oxidized LDL might be related to their proinflammatory and fibroproliferative effect in the atherosclerotic plaque.

Polyunsaturated fatty acid enrichment enhances endothelial cell-induced low-density-lipoprotein peroxidation.

Oxidative modification of low-density lipoprotein (LDL) is an important feature in the initiation and progression of atherosclerosis. LDL modification by endothelial cells was studied after supplementation of the cells with oleic acid and polyunsaturated fatty acids (PUFA) of the n-6 and n-3 series. In terms of the lipid peroxidation product [thiobarbituric acid reactive substances (TBARS)] content and diene level of the LDL particle, oleic acid had no significant effect, and linoleic acid was poorly effective. Gamma linolenic acid (C18:3,n-6) and arachidonic acid (C20:4,n-6) increased by about 1.6-1.9-fold the cell-mediated LDL modification. PUFA from the n-3 series, alpha linolenic acid (C18:3,n-3), eicosapentaenoic acid (C20:5,n-3) and docosahexaenoic acid (C22:6,n-3), induced a less marked effect (1. 3-1.6-fold increase). The relative electrophoretic mobility of the LDL particle and its degradation by macrophages were enhanced in parallel. Concomitantly, PUFA stimulated superoxide anion secretion by endothelial cells. The intracellular TBARS content was also increased by PUFA. Comparison of PUFA from the two series indicates a good correlation between LDL oxidative modification, superoxide anion secretion and intracellular lipid peroxidation. The lipophilic antioxidant vitamin E decreased the basal as well as the PUFA-stimulated LDL peroxidation. These results indicate that PUFAs with a high degree of unsaturation of the n-6 and n-3 series could accelerate cell-mediated LDL peroxidation and thus aggravate the atherosclerotic process.

Fluid resuscitation in the trauma patient.

Fluid management of the trauma patient can be challenging to the nurse anesthetist. Often the Certified Registered Nurse Anesthetist is unaware of the total blood loss of a trauma victim (ie, how much blood loss occurred at the scene). The anesthetist must quickly assess the patient's vital signs and response to fluid replacement to effectively treat the acute blood loss. In today's healthcare market, when cost of care is consistently scrutinized, the nurse anesthetist must weigh the risks and benefits to a patient when selecting fluid therapies. This article outlines current resuscitative theories and fluid types available for use in resuscitating the trauma patient.

Chronical administration of anti-DNA antibodies by subcutaneous injection of hybridoma clones in BALB/c and NZBxNZW/F1 mice. Experimental model of the pathogenic role of DNA in acute lupus disease.

Several sets of data suggest that specific classes of anti-DNA antibodies could be implicated in the genesis of glomerular lesions in SLE. The goal of this work is to investigate if this pathogenic role could be related to the antibodies' genetic origin--from BALB/c or NZBxNZW/F1 mice--or to their physiological origin--induced either by DNA or by polyclonal B cell activation in normal mice. For this purpose, anti-DNA antibody hybridoma clones produced from different origins were subcutaneously injected in BALB/c or NZBxNZW/F1 female mice, followed by studies of immunological parameters and kidney lesions. Results concur that the induced anti-DNA antibodies can play a role in fatal disease development, related to clonal specificity but not to the way of stimulation which was either polyclonal B cell activation or DNA immunization. Also, they emphasize the possible very lethal role of serum circulating DNA.

Rapidly progressive glomerulonephritis associated with amyloidosis: efficacy of plasma exchange.

This report describes one case of rapidly progressive glomerulonephritis associated with amyloidosis in a 53-year-old woman with rheumatoid arthritis, successfully treated with intensive plasma exchange and immunosuppression. Amyloid deposits were present in all of 20 glomeruli in the kidney biopsy specimen and eight out of nine nonfibrosed glomeruli contained crescents. With intensive plasma exchange and immunosuppressive drugs, renal function improved, and hemodialysis was discontinued. After 2 years, renal function was stable at a moderate level of impairment, but heavy proteinuria persisted.

[Hypothesis: immune complex glomerulonephritis and auto-immunity (author's transl)]. / Glomérulonéphrite à complexes immuns et auto-immunité.

Experimental studies on the NZBxNZW mouse lupus disease and on the development of immune complex (IC) glomerulonephritis in mouse infected with Escherichia coli lead us to state the following hypothesis: two types of factors are implicated in the development of immune complex glomerulonephritis: 1) specific factors leading to the production of IC involving antigens from the triggering agents; 2) non specific factors leading to the production of IC involving auto-antigens.

Immune complex glomerulonephritis in mice infected with Escherichia coli.

C57Bl/6 mice were injected intraperitoneally with 10(8) to 2 x 10(8) living K 38 Escherichia coli (E. coli) and serological changes and kidney involvement were studied. E. coli were found in the blood 45 min to 24 hr after injection. In serum, large amounts of deoxyribonucleic acid (DNA) were present 24 hr after E. coli injection, and thereafter disappeared. Seven days after infection, antibodies directed against E. coli, anti-DNA antibodies and C1q-binding substances were found in serum and the kinetics of the variations of these parameters were studied until day 35. Kidney lesions were evaluated immunochemically and by optical and electron microscopy. In the glomeruli, heavy granular deposits of IgG and IgM were constantly found in mesangium and along capillary walls. In most kidneys slight granular deposits of IgG and IgM were also found in the tubules. Histological studies revealed in the glomeruli mild endocapillary cell proliferation, focal thickening of glomerular basement membrane and dense deposits in mesangial and subendothelial areas and inside the glomerular basement membrane; in the tubules dense deposits were focally observed inside the tubular basement membrane.

Acceleration of glomerulonephritis in NZB x NZW mice by early immunization with DNA and injection of bacterial lipopolysaccharide. Experimental approach to the treatment of lupus nephritis by use of the accelerated model of NZB x NZW mouse disease.

The effects of early immunization with DNA and of injection of bacterial lipopolysaccharide (LPS) on the glomerulonephritis of NZB x NZW mice were studied. Combined injections of DNA complexed to methylated bovine serum albumin (DNA-mBSA) and of LPS appeared to be more efficient in accelerating the disease in NZB x NZW mice than injections of DNA-mBSA or LPS alone. A rapid increase in levels of anti-DNA antibodies, an early appearance of severe renal lesions and a shortened survival were observed in mice injected with both DNA-mBSA and LPS. This new model was found to be suitable for therapeutic studies in mice with accelerated disease treated with cyclophosphamide and heparin. The efficacy of cyclophosphamide for the treatment of NZB x NZW mouse disease was shown by immunological and histological studies in mice younger than 4 months. Heparin appeared to have a beneficial effect by preventing the endocapillary cellular proliferation induced by injections of DNA-mBSA and LPS. The accelerated model of NZB x NZW mouse disease might be a useful tool for experiments on the treatment of lupus nephritis.

[Lupus nephropathy. Treatment with the indomethacin-hydroxychloroquine combination and comparison with corticoids]. / La néphropathie lupique Traitement par l'association indométacine-hydroxychloroquine et comparaison avec les corticoïdes

The authors report their comparative experience of the treatment of proliferative lupus glomerulonephritis using prednisone (16 patients) or the indomethacin-hydroxychloroquine association (12 patients). Prednisone in high dosage was associated in this series with 9 deaths and in 6 patients, with renal failure or an extra-renal complication. By contrast, the indomethacin-hydroxychloroquine association proved to be highly effective, without side-effect. In the endocapillary glomerulonephritis group (8 cases) the authors obtained 7 durable remissions (36.3 months on average) and 1 temporary remission of 24 months, with an average length of treatment of 45.8 months. In the extracapillary glomerulonephritis group (4 cases) the authors obtained 1 remission, 2 improvements and I death, with an average length of treatment of 16.8 months. This combination has a highly significant anti-proteinuric and anti-haematuric action, with a constant efficiency on renal function and on the extra-renal signs of lupus. Its effect is less constant on the immunological disorders. Study of iterative renal biopsies confirms this favourable impression. According to these results, the authors propose a provisional scheme of management of proliferative lupus glomerulonephritis.