Proteomic analysis of rabbit fresh and cryopreserved semen provides an important insight into molecular mechanisms of cryoinjuries to spermatozoa.

Cryoinjury and protein changes are a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, viability and fertilizing ability. However, potential proteomic changes in rabbit semen throughout the cryopreservation process have never been previously investigated. The aim of the present study was to compare the whole proteome of fresh and cryopreserved rabbit semen (spermatozoa and extracellular fluid), to examine the effects of freeze-thawing on proteins changes in semen. Comparative analysis and identification of proteins was carried out using 2-dimensional difference in-gel electrophoresis coupled with a matrix-assisted laser desorption/ionization mass spectrometry. Proteomic raw data are available via ProteomeXchange with identifier PXD034832 for spermatozoa and PXD034853 for extracellular fluid. Respectively, 107 and 28 proteins differed in abundance in spermatozoa and extracellular fluid between fresh and frozen groups. Most of these proteins were involved in pathways related to energy metabolism and protein quality control under stress conditions, reproductive processes and mechanisms of cell death/survival regulation, resulting in a significant decrease of motility and viability of post-thawing rabbit sperm and its potential fertilizing ability. These results broaden the understanding of the effects of cryopreservation on rabbit semen and represent a new starting point for the development of improved freezing procedures.

Effect of acute lung injury on VLA-4 and CXCR4 expression in resident and circulating hematopoietic stem/progenitor cells.

BACKGROUND: The effect of acute lung injury on adhesion molecule expression in hematopoietic stem/progenitor cells (HSPCs) is poorly understood. OBJECTIVES: The aim of this study was to determine whether there is a relationship -between pulmonary inflammation, expression of VLA-4 (CD49d), LFA-1 (CD11a), L-selectin (CD62L), CXCR4, and chemotaxis in resident HSPCs, as well as the level of circulating HSPCs. METHODS: Following intratracheal administration of a single LPS bolus in C57Bl/6 mice, the number of inflammatory cells, differential counts, and amounts of cytokines/ chemokines were studied in cytospins and bronchoalveolar lavage fluid (BALF) specimens. Expressions of adhesion -molecules and CXCR4 were analyzed in HSPCs by flow cytometry, as well as SDF-1-directed chemotaxis. Levels of HSPCs in the blood were studied in ungated and circulating subpopulations. RESULTS: In coincidence with a peak of airway neutrophils, cytokine (IL-1ß, TNF-α, and IL-6), chemokine (KC, MIP-2, and SDF-1) levels in BALF and the number of marrow HSPCs expressing CD49d and CXCR4 significantly increased at 48 h. The number of CD49d- and CXCR4-positive HSPCs dropped at 72 h. The HSPC subset comprising bigger cells behaved the same for CD49d. Chemotaxis of the marrow HSPC subset of bigger cells was higher in LPS-treated animals than in controls at 72 h. Finally, we could detect a significant decrease in circulating Sca-1(+) cells in the mononuclear population at 72 h in LPS-treated mice. CONCLUSIONS: Our data provide evidence for a temporal relationship between pulmonary inflammation, CD49d and CXCR4 expression fluctuation in resident HSPCs, and the level of circulating HSPCs.

Isolation and characterization of microparticles in sputum from cystic fibrosis patients.

BACKGROUND: Microparticles (MPs) are membrane vesicles released during cell activation and apoptosis. MPs have different biological effects depending on the cell from they originate. Cystic fibrosis (CF) lung disease is characterized by massive neutrophil granulocyte influx in the airways, their activation and eventually apoptosis. We investigated on the presence and phenotype of MPs in the sputum, a rich non-invasive source of inflammation biomarkers, of acute and stable CF adult patients. METHODS: Spontaneous sputum, obtained from 21 CF patients (10 acute and 11 stable) and 7 patients with primary ciliary dyskinesia (PCD), was liquefied with Sputasol. MPs were counted, visualized by electron microscopy, and identified in the supernatants of treated sputum by cytofluorimetry and immunolabelling for leukocyte (CD11a), granulocyte (CD66b), and monocyte-macrophage (CD11b) antigens. RESULTS: Electron microscopy revealed that sputum MPs were in the 100-500 nm range and did not contain bacteria, confirming microbiological tests. CF sputa contained higher number of MPs in comparison with PCD sputa. Levels of CD11a+-and CD66b+-, but not CD11b+-MPs were significantly higher in CF than in PCD, without differences between acute and stable patients. CONCLUSIONS: In summary, MPs are detectable in sputa obtained from CF patients and are predominantly of granulocyte origin. This novel isolation method for MPs from sputum opens a new opportunity for the study of lung pathology in CF.

Fcgamma receptors mediate internalization of anti-Ro and anti-La autoantibodies from Sjögren's syndrome and apoptosis in human salivary gland cell line A-253.

BACKGROUND: The presence of serum anti-Ro and anti-La autoantibodies directed against the ribonucleoproteins Ro and La has been associated with Sjögren's syndrome (SS), an autoimmune rheumatic disease that targets salivary and lachrymal glands. There is increasing evidence of the direct involvement of autoantibodies in the pathogenesis of tissue injury and correlation of their presence with clinical manifestations in SS. The focus of this work was to explore the cellular apoptotic pathway triggered by binding and penetration of anti-Ro and anti-La autoantibodies in human salivary gland cell line A-253 and to identify the membrane receptors through which anti-Ro and anti-La could exert their effect. METHODS: Anti-Ro and anti-La autoantibodies were purified from IgG fractions, obtained from eleven healthy volunteers and patients with primary Sjögren's syndrome, using Sepharose 4B-Ro and Sepharose 4B-La affinity columns. Flow cytometry, RT-PCR, western blot and confocal microscopy analysis were used to visualize the FCgammaRI, FCgammaRII and FCgammaRIII receptors on the A-253 cell membrane. DNA laddering and western blot analysis of caspases activation were studied to evaluate in A-253 cells treated with anti-Ro and anti-La autoantibodies. RESULTS: The results yeilded the evidence of the presence of members of the Fcgamma receptors (FcgammaRs) family on the cell membrane of the human salivary gland cell line A-253. Furthermore, we demonstrated that, in the A-253 cell line, anti-Ro and anti-La autoantibodies can access the cells probably through Fcgamma receptors, and trigger apoptotis. CONCLUSIONS: We conclude that anti-Ro and anti-La autoantibodies have pathogenic effects that could depend on binding to Fcgamma receptors.