Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues.

There is shortage of extensive clinicopathologic studies of cellular senescence because the most reliable senescence biomarker, the detection of Senescence-Associated-beta-galactosidase activity (SA-ß-gal), is inapplicable in archival material and requires snap-frozen tissues. We validated the histochemical Sudan-Black-B (SBB) specific stain of lipofuscin, an aggregate of oxidized proteins, lipids and metals, known to accumulate in aged tissues, as an additional reliable approach to detect senescent cells independently of sample preparation. We analyzed cellular systems in which senescence was triggered by replicative exhaustion or stressful stimuli, conditional knock-in mice producing precancerous lesions exhibiting senescence, and human preneoplastic lesions known to contain senescent cells. In the above settings we demonstrated co-localization of lipofuscin and SA-ß-gal in senescent cells in vitro and in vivo (cryo-preserved tissue), strongly supporting the candidacy of lipofuscin for a biomarker of cellular senescence. Furthermore, cryo-preserved tissues positive for SA-ß-gal were formalin-fixed, paraffin-embedded, and stained with SBB. The corresponding SA-ß-gal positive tissue areas stained specifically for lipofuscin by SBB, whereas tissues negative for SA-ß-gal were lipofuscin negative, validating the sensitivity and specificity of the SBB staining to visualize senescent cells in archival material. The latter unique property of SBB could be exploited in research on widely available retrospective tissue material.

Herpes virus infected spermatozoa following density gradient centrifugation for IVF purposes.

Studies have documented the presence of herpes viruses in semen. The aim of our study was to determine whether they persist in semen samples following two-density gradient centrifugation for IVF purposes. Semen samples were collected from 109 men seeking fertility evaluation, prior to IVF treatment. Routine semen analysis was performed according to WHO guidelines. Each sample was treated in a two-density gradient centrifugation using PureSperm (PS). Both untreated and treated samples were screened for the presence of herpes viruses, using PCR. Kruskal-Wallis, chi-square and binomial statistical tests were used; P ≤ 0.05 was considered statistically significant. No statistically significant associations were observed between semen parameters and viral presence. Viral DNA was detected in 54% of semen samples: HSV1/2 in 32 samples, EBV in 49, CMV in 47, HHV6 in 9, HHV7 in 4 and VZV in none. PS gradient failed to remove CMV in 89.36%, HSV1/2 in 59.38% and EBV in 22.45% of samples, while HHV6 and 7 were completely removed. Especially HSV1/2 and CMV seem to persist even following PS treatment. These observations indicate the possible risk of oocyte becoming infected during insemination, by IVF or intracytoplasmic sperm injection, with unknown sequelae. Further studies are required to determine whether any correlation exists between their presence, implantation rate and the outcome of pregnancy.

Evaluation of claspin as a proliferation marker in human cancer and normal tissues.

Claspin is a nuclear protein involved in DNA replication and the DNA damage response. Its structural and functional properties suggest that it may represent a potentially useful proliferation marker. To this end, a monoclonal antibody was generated and the expression of claspin was investigated in normal fibroblasts and various cancer cell lines, as well as in tumour and normal tissues from patients with primary epithelial carcinomas. Immunoblotting analysis confirmed the specificity of the antibody, while immunohistochemistry demonstrated its applicability in archival material. In normal cells and tissues, claspin expression was weak, whereas increased levels were observed in cancer cell lines and tumour specimens. Claspin staining correlated strongly with Ki67 staining in both normal (p < 0.001) and tumour tissues (p < 0.001). However, the labelling index (LI) of claspin was consistently lower than that of Ki67, suggesting that claspin expression may be limited to a narrower part of the cell cycle. Co-localization assays with cyclin A and cell synchronization experiments indicated that claspin expression coincides with the S phase. Interestingly, the relative increase of the claspin LI in tumour samples compared with normal tissues was significantly higher (14-fold) than that of the Ki67 LI (five-fold), suggesting that claspin may be a more sensitive marker of aberrant proliferation.