Mechanical clearance of red blood cells by the human spleen: Potential therapeutic applications of a biomimetic RBC filtration method.

During their lifespan, circulating RBC are frequently checked for their deformability. This mechanical quality control operates essentially in the human spleen. RBC unable to squeeze though narrow splenic slits are retained and cleared from the blood circulation. Under physiological conditions this prevents microvessels from being clogged by senescent, rigid RBC. Retention of poorly deformable RBC is an important determinant of pathogenesis in malaria and may also impact the clinical benefit of transfusion. Modulating the splenic retention of RBC has already been proposed to support therapeutic approaches in these research fields. To this aim, the development of microplates for high throughput filtration of RBC through microsphere layers (microplate-based microsphiltration) has been undertaken. This review focuses on potential therapeutic applications provided by this technology in malaria chemotherapy and transfusion.

Waldenström's macroglobulinemia harbors a unique proteome where Ku70 is severely underexpressed as compared with other B-lymphoproliferative disorders.

Waldenström's macroglobulinemia (WM) is a clonal B-cell lymphoproliferative disorder (LPD) of post-germinal center nature. Despite the fact that the precise molecular pathway(s) leading to WM remain(s) to be elucidated, a hallmark of the disease is the absence of the immunoglobulin heavy chain class switch recombination. Using two-dimensional gel electrophoresis, we compared proteomic profiles of WM cells with that of other LPDs. We were able to demonstrate that WM constitutes a unique proteomic entity as compared with chronic lymphocytic leukemia and marginal zone lymphoma. Statistical comparisons of protein expression levels revealed that a few proteins are distinctly expressed in WM in comparison with other LPDs. In particular we observed a major downregulation of the double strand repair protein Ku70 (XRCC6); confirmed at both the protein and RNA levels in an independent cohort of patients. Hence, we define a distinctive proteomic profile for WM where the downregulation of Ku70-a component of the non homologous end-joining pathway-might be relevant in disease pathophysiology.

Secretome of human bronchial epithelial cells in response to the fungal pathogen Aspergillus fumigatus analyzed by differential in-gel electrophoresis.

BACKGROUND: For years, the analysis of innate responses to the major mold pathogen Aspergillus fumigatus has been restricted to specialized cells, such as professional phagocytes. More recently, the contribution of the airway epithelial barrier has been assessed and studies have shown that it was able to sense and react to the Aspergillus infection, for example, by producing cytokines. METHODS: To further explore the reaction of the respiratory epithelium to the fungus, we analyzed the proteome response of a human bronchial epithelial cell line to Aspergillus infection using difference gel electrophoresis. We studied the protein pattern of BEAS-2B cell culture supernatant after interaction of the cells with Aspergillus during a 15-hour coculture. RESULTS: We found formerly unknown aspects of bronchial cell behavior during Aspergillus infection: bronchial cells are able to develop both cellular defense mechanisms (ie, thioredoxin system activation) and immune reactions (ie, lysosomal degranulation and cathepsin activation) in response to the fungal aggression. CONCLUSIONS: Bronchial epithelial cells appear to be a more important effector of antifungal defense than expected. Degranulation of lysosomal enzymes that might be responsible for both fungal growth inhibition and host cell damage suggests that inductors/inhibitors of these pathways may be potential targets of therapeutic intervention.

DIGE enables the detection of a putative serum biomarker of fungal origin in a mouse model of invasive aspergillosis.

Invasive aspergillosis (IA) is a major threat for immunocompromised patients. Diagnostic difficulties often delay specific treatment initiation, which increases mortality. Finding new biomarkers to improve and speed accurate diagnosis is thus vital. To investigate the ability of proteomic methods for discovering new biomarkers of IA, we used a DIGE approach to perform a proteomic analysis on both bronchoalveolar lavages (BAL) and sera at different time-points of infection in a mouse model of invasive pulmonary aspergillosis. Progression of the infection was monitored using a bioluminescent strain of Aspergillus fumigatus. Sera proteins were enriched using the ProteoMiner kit (Biorad). This method allowed us to identify a fungal protein, the A. fumigatus major allergen Asp f 2, in sera of mice one day after the infection. However, this fungal protein was not detected three days after the infection. Importantly, in BAL, this work provides evidence of an in vivo complement evasion mechanism through the cleavage of C3b into three fragments during aspergillosis. Finally, our results underlining the inflammatory host response to IA in both lung and blood compartments at different times of infection may provide new insights into the pathophysiology of this disease.

[Unexpected in vitro fertilization failure in patients with normal sperm: a proteomic analysis]. / Infertilité masculine chez les patients normospermiques : analyse protéomique des spermes normaux non fécondants en fécondation in vitro classique.

OBJECTIVE: Despite normal sperm parameters, 5% of in vitro fertilization (IVF) attempts result in an unpredictable failure of fertilization. In 56% of the cases, there is no obvious oocyte anomaly, but lack of sperm binding to the zona pellucida. This study aims to contribute to clarify the male molecular causes of failures in IVF, which are undetected by classical sperm analysis. PATIENTS AND METHODS: The spermatic proteomic profiles of patients, with a complete failure of fertilization and no spermatozoa bound to the zona pellucida, is compared to controls (patients with normal fertilization and cleavage rates after a classical IVF for tubal indication). All samples are analysed by 2 Dimensional Electrophoresis-Differential In Gel Electrophoresis (2DE-DIGE) after being divided into three fractions according to their isoelectric point (acid, intermediate and basic). RESULTS: Fourteen proteins differentially expressed between all the cases and all the controls were highlighted. Twelve of these proteins were identified by mass spectrometry (six from the acid fraction and six from the basic fraction). Two of these proteins may have an interest in gametic interaction: the laminin receptor LR67 and the L-xylulose reductase. DISCUSSION AND CONCLUSION: More investigation is needed to understand the involvement of the identified proteins in the IVF fertilization failure of the infertile patients in this study.

Proteomic Analysis of Membrane-associated Proteins from the Breast Cancer Cell Line MCF7.

BACKGROUND: Proteins associated with cancer cell membranes represent targets of choice for humoral immune response as well as potential tumour marker proteins in human malignancies. However, proteomic analysis of these proteins, and more generally of low-soluble proteins, remains difficult. MATERIALS AND METHODS: The breast cancer cell line MCF7 was selected to evaluate a sequential extraction method that enables simple fractionation of human cell proteins according to their subcellular localization, yielding subproteomes enriched in cytosolic and membrane-associated proteins, respectively. A crude plasma membrane preparation was followed by the solubilisation of proteins using trifluoroethanol (TFE) as co-solvent. RESULTS: Cross-matching and statistical analysis performed for each set of two-dimensional electrophoresis (whole-cell, membrane and soluble extracts) and between the different sets highlighted the reproducibility of the extraction process and its usefulness for proteomic analysis. Eighty-three % of the spots of the gels corresponding to the membrane fraction were not found in the gels of the soluble fraction. CONCLUSION: Due to its simplicity, the approach described here appears well suited for membrane proteomic investigation of human cancer cells and detection of potential biomarkers undetected by current techniques.

Compression wood-responsive proteins in developing xylem of maritime pine (Pinus pinaster ait.).

When a conifer shoot is displaced from its vertical position, compression wood (CW) is formed on the under side and can eventually return the shoot to its original position. Changes in cell wall structure and chemistry associated with CW are likely to result from differential gene/protein expression. Two-dimensional polyacrylamide gel electrophoresis of differentiating xylem proteins was combined with the physical characterization of wooden samples to identify and characterize CW-responsive proteins. Differentiating xylem was harvested from a 22-year-old crooked maritime pine (Pinus pinaster Ait.) tree. Protein extracted from different samples were revealed by high-resolution silver stained two-dimensional polyacrylamide gel electrophoresis and analyzed with a computer-assisted system for single spot quantification. Growth strain (GS) measurements allowed xylem samples to be classified quantitatively from normal wood to CW. Regression of lignin and cellulose content on GS showed that an increase in the percentage of lignin and a decrease of the percentage of cellulose corresponded to increasing GS values, i.e. CW. Of the 137 studied spots, 19% were significantly associated with GS effect. Up-regulated proteins included 1-aminocyclopropane-1-carboxylate oxidase (an ethylene forming enzyme), a putative transcription factor, two lignification genes (caffeic O-methyltransferase and caffeoyl CoA-O-methyltransferase), members of the S-adenosyl-L-methionine-synthase gene family, and enzymes involved in nitrogen and carbon assimilation (glutamine synthetase and fructokinase). A clustered correlation analysis was performed to study simultaneously protein expression along a gradient of gravistimulated stressed xylem tissue. Proteins were found to form "expression clusters" that could identify: (a) Gene product under similar control mechanisms, (b) partner proteins, or (c) functional groups corresponding to specialized pathways. The possibility of obtaining regulatory correlations and anticorrelations between proteins provide us with a new category of homology (regulatory homology) in tracing functional relationships.

Separation and characterization of needle and xylem maritime pine proteins.

Two-dimensional gel electrophoresis (2-DE) and image analysis are currently used for proteome analysis in maritime pine (Pinus pinaster Ait.). This study presents a database of expressed proteins extracted from needles and xylem, two important tissues for growth and wood formation. Electrophoresis was carried out by isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second. Silver staining made it possible to detect an average of 900 and 600 spots on 2-DE gels from needles and xylem, respectively. A total of 28 xylem and 35 needle proteins were characterized by internal peptide microsequencing. Out of these 63 proteins, 57 (90%) could be identified based on amino acid similarity with known proteins, of which 24 (42%) have already been described in conifers. Overall comparison of both tissues indicated that 29% and 36% of the spots were specific to xylem and needles, respectively, while the other spots were of identical molecular weight and isoelectric point. The homology of spot location in 2-DE patterns was further validated by sequence analysis of proteins present in both tissues. A proteomic database of maritime pine is accessible on the internet (http://www.pierroton.inra.fr/genetics/2D/).