Treatment of human sperm with serine protease during density gradient centrifugation

PURPOSE Seminal pathogens can bind specifically or nonspecifically to spermatozoa, rendering semen decontamination procedures ineffective, whereby vertical or horizontal transmission of the infection could occur. Serine proteases have been demonstrated to effectively inactivate viruses and to break pathogen-sperm bonds. However, the addition of a protease to density gradient layers during semen processing could negatively impact on sperm parameters. This study investigated the effect of the addition of a recombinant, human-sequence protease (rhProtease) on sperm parameters during density gradient centrifugation. METHODS (i) Pooled semen samples (n09) were split and processed by density gradient centrifugation, with the top density layers supplemented, or non-supplemented with rhProtease at three different concentrations (diluted 2, 10 and 20 times). Sperm parameters were then analysed by flow cytometry and computer-assisted semen analyses. (ii) Semen samples (n05) were split and similarly processed using PureSperm® Pro, with rhProtease in the 40 % density gradient layer, or standard PureSperm® not supplemented with rhProtease (Nidacon, International) respectively. The Hemizona assay was then utilized to compare sperm-zona binding post processing. RESULTS Evaluation of sperm parameters indicated that rhProtease did not, at any of the tested concentrations, have an impact on (i) mitochondrial membrane potential, vitality, motility, or (ii) zona binding potential. CONCLUSION We report that the addition of rhProtease to density gradients is a non-detrimental approach that could improve the effectiveness of semen processing for the elimination of seminal pathogens, and benefit assisted reproduction outcome.The MRChttp://www.springerlink.com/content/104689

Elimination of bacteria from human semen during sperm preparation using density gradient centrifugation with a novel tube insert

The occurrence of bacteria in sperm samples intended for in vitro fertilization, can compromise the outcome of assisted reproductive techniques. Effective semen processing procedures should therefore be implemented to remove bacteria from semen. Unfortunately, technique failure does occur whereby bacteria can be found in processed sperm preparations. To improve the effectiveness of semen processing, a novel centrifuge tube insert was developed to facilitate the layering of density gradients and semen, and to prohibit the reinfection of purified sperm pellets. The purpose of this study was to: 1) determine the prevalence and type of bacteria present in semen of patients participating in the Unit’s assisted reproduction program, and 2) evaluate the effectiveness of density gradient centrifugation with the novel tube insert, for the elimination of bacteria and yeast from spiked human semen samples. A survey in 2007-2010 indicated that 50% of semen samples were found to have positive bacterial cultures. Semen processing by means of density gradient centrifugation with the novel tube insert, eliminated significantly more in vitro derived (spiked) bacteria and yeast from semen compared to processing without the insert (P<0.004). Therefore, it is highly recommended that the centrifuge tube insert, ProInsertTM, be incorporated into assisted reproductive programs.Medical Research Council (MRC)http://www.blackwell-synergy.com/loi/an

A review of hyperfibrinolysis in cats and dogs

The fibrinolytic system is activated concurrently with coagulation; it regulates haemostasis and prevents thrombosis by restricting clot formation to the area of vascular injury and dismantling the clot as healing occurs. Dysregulation of the fibrinolytic system, which results in hyperfibrinolysis, may manifest as clinically important haemorrhage. Hyperfibrinolysis occurs in cats and dogs secondary to a variety of congenital and acquired disorders. Acquired disorders associated with hyperfibrinolysis, such as trauma, cavitary effusions, liver disease and Angiostrongylus vasorum infection, are commonly encountered in primary care practice. In addition, delayed haemorrhage reported in greyhounds following trauma and routine surgical procedures has been attributed to a hyperfibrinolytic disorder, although this has yet to be characterised. The diagnosis of hyperfibrinolysis is challenging and, until recently, has relied on techniques that are not readily available outside referral hospitals. With the recent development of point‐of‐care viscoelastic techniques, assessment of fibrinolysis is now possible in referral practice. This will provide the opportunity to target haemorrhage due to hyperfibrinolysis with antifibrinolytic drugs and thereby reduce associated morbidity and mortality. The fibrinolytic system and the conditions associated with increased fibrinolytic activity in cats and dogs are the focus of this review article. In addition, laboratory and point‐of‐care techniques for assessing hyperfibrinolysis and antifibrinolytic treatment for patients with haemorrhage are reviewed

The multiplex bead array approach to identifying serum biomarkers associated with breast cancer

Introduction Breast cancer is the most common type of cancer seen in women in western countries. Thus, diagnostic modalities sensitive to early-stage breast cancer are needed. Antibody-based array platforms of a data-driven type, which are expected to facilitate more rapid and sensitive detection of novel biomarkers, have emerged as a direct, rapid means for profiling cancer-specific signatures using small samples. In line with this concept, our group constructed an antibody bead array panel for 35 analytes that were selected during the discovery step. This study was aimed at testing the performance of this 35-plex array panel in profiling signatures specific for primary non-metastatic breast cancer and validating its diagnostic utility in this independent population. Methods Thirty-five analytes were selected from more than 50 markers through screening steps using a serum bank consisting of 4,500 samples from various types of cancer. An antibody-bead array of 35 markers was constructed using the Luminex (TM) bead array platform. A study population consisting of 98 breast cancer patients and 96 normal subjects was analysed using this panel. Multivariate classification algorithms were used to find discriminating biomarkers and validated with another independent population of 90 breast cancer and 79 healthy controls. Results Serum concentrations of epidermal growth factor, soluble CD40-ligand and proapolipoprotein A1 were increased in breast cancer patients. High-molecular-weight-kininogen, apolipoprotein A1, soluble vascular cell adhesion molecule-1, plasminogen activator inhibitor-1, vitamin-D binding protein and vitronectin were decreased in the cancer group. Multivariate classification algorithms distinguished breast cancer patients from the normal population with high accuracy (91.8% with random forest, 91.5% with support vector machine, 87.6% with linear discriminant analysis). Combinatorial markers also detected breast cancer at an early stage with greater sensitivity. Conclusions The current study demonstrated the usefulness of the antibody-bead array approach in finding signatures specific for primary non-metastatic breast cancer and illustrated the potential for early, high sensitivity detection of breast cancer. Further validation is required before array-based technology is used routinely for early detection of breast cancer.Kenny HA, 2008, J CLIN INVEST, V118, P1367, DOI 10.1172/JCI33775Shah FD, 2008, INTEGR CANCER THER, V7, P33, DOI 10.1177/1534735407313883Carlsson A, 2008, EUR J CANCER, V44, P472, DOI 10.1016/j.ejca.2007.11.025Nolen BM, 2008, BREAST CANCER RES, V10, DOI 10.1186/bcr2096Brogren H, 2008, THROMB RES, V122, P271, DOI 10.1016/j.thromres.2008.04.008Varki A, 2007, BLOOD, V110, P1723, DOI 10.1182/blood-2006-10-053736Madsen CD, 2007, J CELL BIOL, V177, P927, DOI 10.1083/jcb.200612058Levenson VV, 2007, BBA-GEN SUBJECTS, V1770, P847, DOI 10.1016/j.bbagen.2007.01.017VAZQUEZMARTIN A, 2007, EUR J CANCER, V43, P1117GARCIA M, 2007, GLOBAL CANC FACTS FIMoore LE, 2006, CANCER EPIDEM BIOMAR, V15, P1641, DOI 10.1158/1055-9965.EPI-05-0980Borrebaeck CAK, 2006, EXPERT OPIN BIOL TH, V6, P833, DOI 10.1517/14712598.6.8.833Zannis VI, 2006, J MOL MED-JMM, V84, P276, DOI 10.1007/s00109-005-0030-4Jemal A, 2006, CA-CANCER J CLIN, V56, P106Silva HC, 2006, NEOPLASMA, V53, P538Chahed K, 2005, INT J ONCOL, V27, P1425Jain KK, 2005, EXPERT OPIN PHARMACO, V6, P1463, DOI 10.1517/14656566.6.9.1463Abe O, 2005, LANCET, V365, P1687Paradis V, 2005, HEPATOLOGY, V41, P40, DOI 10.1002/hep.20505Molina R, 2005, TUMOR BIOL, V26, P281, DOI 10.1159/000089260Furberg AS, 2005, CANCER EPIDEM BIOMAR, V14, P33Benoy IH, 2004, CLIN CANCER RES, V10, P7157Song JS, 2004, BLOOD, V104, P2065, DOI 10.1182/blood-2004-02-0449Schairer C, 2004, J NATL CANCER I, V96, P1311, DOI 10.1093/jnci/djh253Hellman K, 2004, BRIT J CANCER, V91, P319, DOI 10.1038/sj.bjc.6601944Roselli M, 2004, CLIN CANCER RES, V10, P610Zhou AW, 2003, NAT STRUCT BIOL, V10, P541, DOI 10.1038/nsb943Hapke S, 2003, BIOL CHEM, V384, P1073Miller JC, 2003, PROTEOMICS, V3, P56Amirkhosravi A, 2002, BLOOD COAGUL FIBRIN, V13, P505Bonello N, 2002, HUM REPROD, V17, P2272Li JN, 2002, CLIN CHEM, V48, P1296Louhimo J, 2002, ANTICANCER RES, V22, P1759Knezevic V, 2001, PROTEOMICS, V1, P1271Di Micco P, 2001, DIGEST LIVER DIS, V33, P546Ferrigno D, 2001, EUR RESPIR J, V17, P667Webb DJ, 2001, J CELL BIOL, V152, P741Gion M, 2001, EUR J CANCER, V37, P355Schonbeck U, 2001, CELL MOL LIFE SCI, V58, P4Blackwell K, 2000, J CLIN ONCOL, V18, P600Carriero MV, 1999, CANCER RES, V59, P5307Antman K, 1999, JAMA-J AM MED ASSOC, V281, P1470Loskutoff DJ, 1999, APMIS, V107, P54Molina R, 1998, BREAST CANCER RES TR, V51, P109Bajou K, 1998, NAT MED, V4, P923Chan DW, 1997, J CLIN ONCOL, V15, P2322Chu KC, 1996, J NATL CANCER I, V88, P1571vanDalen A, 1996, ANTICANCER RES, V16, P2345Yamamoto N, 1996, CANCER RES, V56, P2827KOCH AE, 1995, NATURE, V376, P517HADDAD JG, 1995, J STEROID BIOCHEM, V53, P579FOEKENS JA, 1994, J CLIN ONCOL, V12, P1648GEARING AJH, 1993, IMMUNOL TODAY, V14, P506HUTCHENS TW, 1993, RAPID COMMUN MASS SP, V7, P576DECLERCK PJ, 1992, J BIOL CHEM, V267, P11693GABRIJELCIC D, 1992, AGENTS ACTIONS S, V38, P350BIEGLMAYER C, 1991, TUMOR BIOL, V12, P138DNISTRIAN AM, 1991, TUMOR BIOL, V12, P82VANDALEN A, 1990, TUMOR BIOL, V11, P189KARAS M, 1988, ANAL CHEM, V60, P2299, DOI 10.1021/ac00171a028LERNER WA, 1983, INT J CANCER, V31, P463WESTGARD JO, 1981, CLIN CHEM, V27, P493TROUSSEAU A, 1865, CLIN MED HOTEL DIEU, V3, P654*R PROJ, R PROJ STAT COMP1