Identification of proteins undergoing expression level modifications in WI-38 SV40 fibroblasts overexpressing methionine sulfoxide reductase A.

Methionine sulfoxide reductase A overexpressing WI-38 SV40 human fibroblasts have been previously shown to exhibit higher resistance to oxidative stress by decreasing intracellular reactive oxygen species content and oxidative damage to proteins [C.R. Picot, I. Petropoulos, M. Perichon, M. Moreau, C. Nizard, B. Friguet, Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H(2)O(2)-mediated oxidative stress, Free Radic Biol Med 39 (2005) 1332-1341]. In order to get further insight into the molecular mechanisms underlying this resistance to oxidative stress, proteins that are differentially expressed in methionine sulfoxide reductase A overexpressing cells were identified by 2D gel and Western blot quantitative analyses. Five proteins were shown to be differentially expressed and were identified by mass spectrometry, some of them were related to either cellular protection against oxidative stress, apoptosis or premature ageing.

[Quality of life in patients wearing scleral lenses]. / Qualité de vie des patients équipés en verres scléraux.

OBJECTIVE: To evaluate the contribution of scleral lenses in terms of improving the quality of life in the treatment of astigmatism after penetrating keratoplasty or in keratoconus. METHODS: We conducted an observational retrospective study, evaluating quality of life (QOL) of patients who failed to adapt to RPG lenses, fitted with SPOT(®) scleral lenses between October 2007 and March 2011 in the University Hospital of Besançon Department of Ophthalmology. QOL was assessed before and after scleral lens adaptation with the French version of the National Eye Institute Visual Function Questionnaire 25 (NEI-VFQ 25). RESULTS: We included 47 patients (83 eyes) fitted with scleral lenses on one or both eyes: 56 eyes with keratoconus and 27 post-keratoplasty eyes. The average duration of wearing scleral lenses was 18±10months and the average wearing time was 14±3hours per day. The rate of participation in the survey was 86.5% (41 patients). Visual acuity in the better eye progressed from 0.68±0.46 to 0.15±0.17 logMAR at the 6th month after scleral lens adaptation (P<0.0001). The average scores on the NEI-VFQ 25 questionnaire of patients fitted with scleral lenses for at least 6 months were significantly higher than those without scleral lenses, with a global score of 80.2/100 with, versus 48.1/100 without, scleral lenses (P<0.0001). The global score increased by an average of 32.1±4.6 points (-28, 82) (P<0.0001). Statistical analysis found no significant difference in global score between patients in the keratoconus and keratoplasty groups (P>0.05). Scleral lenses showed a significant improvement in quality of life for patients who had failed or are intolerant to conventional rigid gas permeable contact lenses. In our two main optical indications, keratoconus and keratoplasty, they represent an alternative or a step prior to surgery.

Effects of tiludronate on bone loss in paraplegic patients.

Immobilization secondary to spinal cord injury is associated with a marked and rapid atrophy of trabecular bone (disuse osteoporosis). This is due to an early increase of osteoclastic bone resorption associated with a pronounced decreased osteoblastic bone formation. Bisphosphonates are antiosteoclastic compounds and they have been effective in preventing disuse osteoporosis. However, some of them also depress osteoblastic activity and may impair the mineralization process. Tiludronate was shown effective in reducing bone resorption in several metabolic bone diseases without inducing mineralization defects. Twenty paraplegic patients (6 females and 14 males) were randomly assigned to three groups: 6 patients entered the placebo group; 7 patients received tiludronate 200 mg/day; and 7 received 400 mg/day. Histomorphometric analysis was performed on transiliac bone biopsies before and after 3 months treatment. An insignificant decrease of bone volume was observed in the placebo group and the 200 mg group. In patients receiving 400 mg/day, a slight increase was noted. Osteoid parameters changed nonsignificantly in three groups although the 400 mg group exhibited a slight tendency to decrease osteoid volume and thickness. Eroded surfaces increased in all groups. The number of osteoclasts (identified histochemically by TRAP staining) increased in the placebo group but decreased in groups receiving tiludronate. Tiludronate appears effective in reducing bone resorption without impairing bone formation in a manner that preserved bone mass and bone cell coupling.

Efficacy and tolerability of a new formulation of oral tiludronate (tablet) in the treatment of Paget's disease of bone.

We sought to assess efficacy and safety of a new oral formulation (tablet) of tiludronate in Paget's disease of bone. We studied 128 patients with Paget's disease in an open-label uncontrolled trial. Patients received a daily dose of 400 mg oral tiludronate (two tablets). Treatment was for 6 months. Serum alkaline phosphatase activity (SAP) and fasting urinary excretion of hydroxyproline/creatine (OH/Cr) were measured every 3 months, as were biochemical parameters reflecting renal, hepatic, and hematologic functions. Analgesic efficacy was self-evaluated from a visual analog scale (VAS). Statistical analysis revealed a significant reduction from baseline in SAP and OH/Cr levels, as well as VAS scores. In the whole population with evaluation under treatment, there was a reduction in initial SAP activity after 3 months (47.2 +/- 2.2%, mean +/- SEM) and 6 months (58.3 +/- 2.3%). In the population with SAP levels above twice the upper limit at inclusion and with evaluation at month 3 and month 6 (n = 96), the reduction in SAP levels was 49.3 +/- 2.4% after 3 months and of 59.5 +/- 2.6% after 6 months (ANOVA time effect, p = 0.0001). Aside from mild gastrointestinal disturbances, as experienced with other oral bisphosphonates, clinical tolerance was good. Exhaustive biochemical investigation failed to reveal significant toxicity of tiludronate tablets at the dose of 400 mg/day. The dose of 400 mg daily of this new formulation appears to be a satisfactory tiludronate regimen for the treatment of Paget's disease of bone.

Tiludronate therapy for Paget's disease of bone.

A double-blind, randomized, placebo-controlled study was performed to evaluate the effect of oral tiludronate therapy in 139 patients with active Paget's disease of bone. Patients received placebo (N = 48), tiludronate 200 mg (N = 45), or tiludronate 400 mg (N = 46) once daily for 12 weeks. Biochemical and clinical responses were observed during the 12 week treatment phase and during an additional 12 week observation phase of the study. Both the 200 and 400 mg tiludronate groups experienced significant reduction in serum alkaline phosphatase (SAP) and urinary indices of bone resorption. After 12 weeks of therapy, the SAP levels decreased 46% from baseline values in the 200 mg group and 51% from baseline values in the 400 mg group. At the end of the 24 week study, SAP levels were reduced 47% and 58% from baseline in the 200 and 400 mg groups, respectively. The SAP reduction at 24 weeks was greater in the 400 mg group than the 200 mg group (p < 0.05). At the end of 24 weeks, 51% of patients treated with 200 mg and 72% of those who received 400 mg of tiludronate had experienced a reduction in SAP of greater than 50% (p = 0.043), and 7% and 35% of patients in the 200 and 400 mg groups, respectively, had experienced normalization of SAP (p = 0.001). There was no difference in incidence of side effects in patients taking tiludronate or placebo. In conclusion, oral tiludronate is an effective and well-tolerated therapy for patients with Paget's disease of bone. Daily therapy with 400 mg tiludronate for 12 weeks is more effective than a daily dose of 200 mg for 12 weeks.

In situ hybridization and cytofluorometric analysis of cytokine mRNA during in vitro activation of human T cells.

We present an original method for in situ hybridization (ISH) using non isotopic probes and flow cytometry analysis that permits rapid detection of lymphokine transcripts at single cell level in an in vitro activated human Jurkat T cell line and in peripheral blood T cell subsets. After PMA and either ionomycin or ConA stimulation, cells were fixed and hybridized with digoxigenin (DIG)-labelled RNA antisense or sense probes specific for IL-2 and IFN-gamma. The level of cytokine gene expression in individual cells was visualised using FITC-conjugated anti-DIG antibody, and the resultant signal was analysed by flow cytometry. IL-2 mRNA was first detected in activated Jurkat T cells. Addition of cycloheximide 4 hours after the beginning of stimulation increased both the frequency of labelled cells and the amount of mRNA per cell, as determined by the mean fluorescence intensity. The specificity and sensitivity of IL-2 mRNA detection were tested by comparison with Northern blot analysis and in situ hybridization (ISH) with immuno-cytochemical staining. IL-2 and IFN-gamma mRNA were detectable in PBMC as early as 3 hours after in vitro stimulation with PMA and ionomycin. The frequency of positive cells and the amount of mRNA per cell peaked at 6-8 hours, when the percentages of IL-2 and IFN-gamma mRNA-containing cells reached 30-40% and 15-20%, respectively. The two lymphokines were expressed in both CD4+ and CD8+ T cells, but the frequency of IL-2 expressing cells and the amount of IL-2 mRNA per cell were higher in CD4+ (60%) than in CD8+ T cells (25%), whereas IFN-gamma were preferentially transcribed by CD8+ T cells (40%). The results obtained by this method were in accordance with the data obtained by Northern blot analysis, with cellular protein content estimated by immuno-fluorescence staining, and with IL-2 titration by bioassay. We compared the performance of this method with ISH using radioactive probes.

Monoclonal antibody AP-282 recognizes a marker for human polymorphonuclear granules.

Hybridoma AP-282 was produced by fusing mouse plasmacytoma cells with splenocytes of mice immunized against purified human polymorphonuclear cells. The secreted monoclonal antibody (MAb), AP-282, a mouse IgG1, was found to react strongly with all neutrophilic granulocytes, their bone marrow precursors, weakly with blood monocytes and not with eosinophils. The antigen was resistant to formalin fixation but was destroyed by exposure to fixatives containing acetic acid. Using the APAAP technique, antibody AP-282 strongly labelled neutrophils on sections of frozen cut or paraffin embedded tissues. No staining was seen of non hematopoietic tissues. AP-282 recognized an internal antigen associated to cytoplasmic granules. Chemical investigations on dot blots of whole or of purified cellular extracts indicated that the antigen idenfied by MAb AP-282 was different from those recognized by usual antigranulocyte antibodies, i.e. myeloperoxidase, elastase, cathepsin G and lactoferrin. Thus, antibody AP-282 constitutes a new cytoplasmic marker of neutrophils.

Ascites-induced shift along epithelial-mesenchymal spectrum in ovarian cancer cells: enhancement of their invasive behavior partly dependant on αv integrins.

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence. The presence of ascites, which acts as a dynamic reservoir of active molecules and cellular components, correlates with the OC peritoneal metastasis and is associated with poor prognosis. Since epithelial-mesenchymal transition (EMT) is involved in different phases of OC progression, we have investigated the effect of the unique ascitic tumor microenvironment on the EMT status and the behavior of OC cells. The exposure of three OC cell lines to ascites leads to changes in cellular morphologies. Within ascites, OC cells harboring an initial intermediate epithelial phenotype are characterized by marked dislocation of epithelial markers (E-cadherin, ZO-1 staining) while OC cells initially harboring an intermediate mesenchymal phenotype strengthen their mesenchymal markers (N-cadherin, vimentin). Ascites differentially triggers a dissemination phenotype related to the initial cell features by either allowing the proliferation and the formation of spheroids and the extension of colonies for cells that present an initial epithelial intermediate phenotype, or favoring the migration of cells with a mesenchymal intermediate phenotype. In an ascitic microenvironment, a redeployment of αv integrins into cells was observed and the ascites-induced accentuation of the two different invasive phenotypes (i.e. spheroids formation or migration) was shown to involve αv integrins. Thus, ascites induces a shift toward an unstable intermediate state of the epithelial-mesenchymal spectrum and confers a more aggressive cell behavior that takes on a different pathway based on the initial epithelial-mesenchymal cell features.

Evaluation of shellfish consumption in Nha Trang City, Southern coastal Vietnam.

INTRODUCTION: Shellfish consumption may be a significant pathway to food contaminants such as heavy metals, pesticides and phycotoxins. Currently, little information exists about consumption of shellfish in Vietnam. Such data could be of interest in terms of nutritional value or risk assessment. METHODS: Consumption of shellfish was assessed using a food frequency questionnaire and validated by a 7-day recall method. Approximately 1% of the city population of Nha Trang was selected yielding a final sample of 440 participants. The participants were above 18 years of age, in apparently good health and were shellfish consumers. RESULTS: In South coastal Vietnam, the types of shellfish most consumed are green mussel, squid, crab and shrimp. The mean consumption rates of the bivalves, crustaceans, gastropods, cephalopods, echinoderms and all shellfish combined were 39.3, 20.9, 16.4, 11.2, 0.3 and 88.1 g/person/day, respectively. The consumption rate was slightly higher in the age group of 30-54 years, than in the younger (18-29 years) and older (55 years and above) age groups. Shellfish is essentially purchased in the markets and temporary markets, and mostly consumed during the dry season. CONCLUSION: Shellfish consumption in the Southern coastal region of Vietnam is high compared to consumption levels in other countries; it is also high compared to consumption levels of Vietnamese emigrants. Such data may be useful for further investigation on nutrition perspectives and in term of risk assessment of shellfish contaminants.

[Multicentric reticulohistiocytosis (lipoid dermato-arthritis). Apropos of a case with fatal outcome]. / La réticulohistiocytose multicentrique (dermato-arthrite lipoïdique). A propos d'un cas d'évolution mortelle

The authors report the case of a 37-year-old man affected by a seronegative polyarthritis associated at first with erythroderma and then with cutaneous and subcutaneous papulo-nodular structures and a xanthomatous eryption. Histological examination of the cutaneous nodules and of the synovial membrane confirmed the diagnosis of multicentred reticulohistiocytosis by showing the presence of lipid infiltrates in the histiocyte cells and of multinucleate giant cells. A study of the ultrastructure of the histiocytes revealed the presence of numerous intracytoplasmic vacuoles. In spite of antimalaria treatment, with cortisone and then with immuno-depressants, the outcome was fatal with a picture of acute reticulosis and neurological disorders. In discussing this case, the authors also make a general review of the subject.

A double-blind, multicentre, placebo-controlled study of tiludronate in Paget's disease of bone.

A multicentre, randomised, placebo-controlled, dose-ranging study was conducted to investigate the therapeutic activity and sustained efficacy of tiludronate (200 mg, 400 mg and 600 mg once daily) taken orally for 12 weeks in patients with Paget's disease. Serum alkaline phosphatase concentrations were compared with baseline at weeks 12 and 24; treatment success was defined as a 50% reduction compared with baseline. Changes in the hydroxyproline: creatinine ratio were also measured. Pain was assessed using the Huskisson Visual Analogue Scale and by questionnaire. Patients completing at least 11 weeks of treatment were followed-up 18 months later by postal questionnaire. Significantly greater numbers of patients in the tiludronate groups successfully responded to treatment compared with the placebo group. A dose-response was observed; the percentage of patients responding to treatment being 31% (200 mg), 52% (400 mg) and 82% (600 mg) at week 12 and 45% (200 mg), 70% (400 mg) and 82% (600 mg) at week 24. Tiludronate treatment also significantly reduced hydroxyproline: creatinine ratios compared with placebo, again showing a dose response. Dose-related gastrointestinal symptoms were the commonest adverse events, occurring in 2.4%, 11.0%, 5.5% and 18.9% of patients receiving placebo and tiludronate 200, 400 and 600 mg daily, respectively. The response to oral tiludronate was sustained for more than 18 months in some patients and there was evidence of a reduction in the longer term complications of the disease. These results show that oral tiludronate is an effective, well-tolerated treatment for Paget's disease; the 400 mg once daily dose appears to offer the optimum balance of efficacy and tolerance.

[Transient erythroblastopenia in young children]. / Erythroblastopénie transitoire de la petite enfance.

A case of transient erythroblastopenia of childhood is reported. This syndrome occurred in a 13 month-old girl. Initial signs were a 6.1 g/dl hemoglobin level and severe erythroblastopenia. Spontaneous recovery occurred after a few weeks. No etiology could be found.

Expression of the alpha-subunit of CR4 (CD11c) by peripheral blood and crevicular fluid neutrophils.

This work determined the levels of expression of CD11c by neutrophils (PMNs) collected from subjects with various periodontal conditions. The percentages of CD11c-positive crevicular fluid PMNs were significantly lower than those of peripheral blood PMNs, but the levels of CD11c expression were similar in PB-PMNs and CF-PMNs (P<0.001). On the other hand, no significant difference could be found between the groups, either for the percentages of CD11c-positive cells or for the CD11c expression levels.

Distinct pattern of IL-2 and IFN-gamma gene expression in CD4 and CD8 T cells: cytofluorometric analysis at a single cell level using non-radioactive probes.

IL-2 and IFN-gamma gene expression was analyzed using an original method for in situ hybridization (ISH) with non-isotopic probes and flow cytometric analysis (FC). This method permits rapid detection of mRNA at a single cell level among in vitro activated human peripheral blood mononuclear cells (PBMC) and purified CD4 and CD8 T cell subsets. After stimulation with PMA and ionomycin (PMA+Io), cells were fixed at different times and hybridized with digoxigenin (DIG)-labelled RNA antisense or sense probes specific for IL-2 and IFN-gamma. The level of cytokine gene expression in individual cells was visualized using FITC-conjugated anti-DIG antibodies and the fluorescent signal was analyzed by flow cytometry. Specific hybridization with IL-2 and IFN-gamma antisense probes was detected among activated PBMC within lymphoid cells identified by their light scattering properties. Kinetic analysis of the frequency of mRNA producing cells exhibited a biphasic pattern with an early peak at 6-8 hrs. when percentages of IL-2 and IFN-gamma expressing cells reached 35 +/- 7% and 18 +/- 4%, respectively. Similar data were obtained by enzymatic detection on cell smears using AP-conjugated anti-DIG. Combination of ISH with FC was applied to the comparison of the pattern of cytokine gene expression between CD4 and CD8 T cell subsets isolated by negative selection using immunobeads and magnetic separation. IL-2 was expressed by activated CD8 T cells (25-35%), but CD4 T cells were the major producers of IL-2 as assessed by the high frequency of mRNA expressing cells (60%) and the large amount of mRNA per cell relative to the mean fluorescence intensity. In contrast, IFN-gamma mRNA was preferentially expressed by CD8 T cells (27-37%) and a minority of CD4 T cells (17-23%). Despite quantitative differences, kinetic analysis of IL-2 gene expression in CD4 and CD8 T cells showed similar profiles with an early peak at 6-8 hrs. Upregulation of IL-2 gene expression in CD4 T cells by CD28 co-stimulation increases the amount of IL-2 mRNA per cell as visualized by mean fluorescence intensity. In addition the effect of CD28 co-stimulation on IL-2 mRNA stabilisation was demonstrated by the maintenance of a high frequency of IL-2 expressing CD4 T cells and an elevated level of mRNA per cell for prolonged period after PMA+Io stimulation. By contrast CD28 co-stimulation had no obvious effect on IFN-gamma expression.

Adhesion molecule expression and response to chemotactic agents of human monocyte-derived macrophages.

Human monocyte-derived macrophages have been proposed as agents of anti-tumour immunotherapy. The aim of the present study was to investigate in vitro the properties of these cells likely to control their recruitment to the sites of inflammation and tumours. The expression of adhesion molecules involved in the binding of monocytes to endothelial cells was modified during monocyte-macrophage differentiation, with a significant increase in CD11c, CD14 and intercellular adhesion molecule-1 (ICAM-1). Monocyte-derived macrophages were sensitive to chemoattractants, in particular to the monocyte-specific chemokine monocyte chemotactic protein-1 (MCP-1). They responded by an increased expression of adhesion molecules and were attracted by the cytokine in an under-agarose migration assay. The migration response, however, decreased after days 4-5 of monocyte differentiation into macrophage. In conclusion, human monocyte-derived macrophages show alterations of surface structures involved in the recognition of inflammatory endothelium. This may explain why the cells are poorly recruited to the sites of inflammation and tumours when introduced into the circulation.

Enrichment of blood units with young red cells (neocytes) with the IBM 2991 cell washer.

The transfusion of young red blood cells (neocytes) is a complementary approach to the present treatment of thalassaemia major patients. Fresh neocytes were harvested from 34 volunteers with the IBM 2991 cell washer (CW). The upper part of 24 units was separated into 6 aliquots (T1-T6) of 15 ml each and the residue homogenized. Four biological criteria were used to evaluate young red cell quality, the erythrocyte pyruvate kinase activity, the mean corpuscular volume, the reticulocyte count and ektacytometry; each sample (T1-T6) showed an enrichment with neocytes as compared with the standard unit. A real efficiency of this technique with the IBM 2991 CW was obtained with the first third of the unit, corresponding to the T1-T4 fractions. The estimation of the theoretical mean life span of T1-T2 was 270 days. The last 10 units were separated into two halves: the enrichment of the first half was worse than in the first third of the previous technique. We concluded that the transfusional program of thalassaemia patients could profit by the use of the most enriched part of a standard blood unit with the IBM 2991 CW.

Expression of complement components of the alternative pathway by glioma cell lines.

Glioma cell lines express proteins of the complement alternative pathway, namely C3, factor B, factor H, and factor I. Secretion of these proteins was shown by a sensitive and specific ELISA. C3 and factor H were rapidly secreted by glioma cell line CB193 and reached a concentration of 140 ng/ml/10(6) cells after 72 h of culture. Factor B and factor I were secreted at a lower rate and reached concentrations of 25 and 15 ng/ml/10(6) cells, respectively. Western blot and immunoprecipitation experiments showed that secreted proteins were identical to the corresponding plasma proteins. For factor H, besides the well known 150-kDa species, an additional polypeptide of 45 kDa with factor H immunoreactivity was observed. This species corresponded to the N-terminal truncated form found in plasma. In preliminary experiments, we observed control of these syntheses by cytokines. IL-1 beta significantly increased C3 secretion, with no effect on factor H. Secretion of factor H was enhanced by IFN-gamma. These results show that a glioma cell line could be a useful tool to study complement biosynthesis by glial cells in humans.

Ex vivo studies of polymorphonuclear neutrophils from patients with early-onset periodontitis (III). CR3 and LFA-1 expression by peripheral blood and gingival crevicular polymorphonuclear neutrophils.

In this study, we assessed the LFA-1 (CD18/CD11a) and CR3 (CD18/CD11b) expression on peripheral polymorphonuclear leukocytes (PB-PMN) and crevicular fluid polymorphonuclear leukocytes (CF-PMN), by subjects with a healthy periodontium (n = 7), gingivitis (n = 8), early-onset periodontitis (n = 17) and adult periodontitis (n = 8). Using flow cytometry analysis, the %s of CD18, CD11a and CD11b positive cells and the absolute numbers of fluorescent molecules were determined. No significant difference could be found among the 4 groups, for these 2 kinds of parameters, in PB-PMN or CF-PMN. However, a great difference could be noted between the results obtained from PB-PMN and those obtained from CF-PMN. The %s of positive CF-PMN were significantly lower than those of PB-PMN for the 3 sub-units (p < 0.001). The levels of CD18 and CD11b expressed by CF-PMN were higher than those expressed by PB-PMN and the difference was significant for CD11b (p < 0.001). On the contrary, the level of CD11a expressed on CF-PMN was significantly lower than that expressed by PB-PMN (p < 0.001). Hence, our current results show that early-onset periodontitis PMN can be quite normal and this fact is not surprising insofar as, in our study, these cells were perfectly functional and all the subjects were in good health. We concluded that the analysis of the leukocyte adhesion receptors expression on PB-PMN does not appear useful for helping to establish a differential diagnosis between the different forms of periodontitis.(ABSTRACT TRUNCATED AT 250 WORDS)