Isoflurane applied during ischemia enhances intracellular calcium accumulation in ventricular myocytes in part by reactive oxygen species.

BACKGROUND: Isoflurane applied before myocardial ischemia has a beneficial preconditioning effect which involves generation of reactive oxygen species (ROS); ROS, however, have been implicated in critical cytosolic calcium ([Ca2+]i) overload during ischemia. We therefore investigated isoflurane's effects on intracellular Ca2+ handling in ischemic ventricular myocytes and the association with ROS. METHODS: Simulated ischemia was induced in electrically stimulated rat ventricular myocytes for 30 min (ischemia). Isoflurane-treated cells were additionally exposed to 1MAC of isoflurane (ischemia + iso). To determine the contribution of ROS to Ca2+ homeostasis during ischemia in both groups, the intracellular ROS scavenger, N-mercaptopropionylglycine (MPG), was added to the superfusion buffer. The fluorescent ratiometric Ca2+ dye fura-2 was employed to determine [Ca2+]i. RESULTS: Resting and peak [Ca2+]i increased in the ischemia and the ischemia + iso group. However, Ca2+ accumulation was most prominent in isoflurane-treated cardiomyocytes (P < 0.05) and could be mitigated by MPG in both groups (P < 0.001). Isoflurane also decreased the rate constant of the Ca2+ transient decline but did not further diminish the amplitude of the transient during ischemia. CONCLUSION: Isoflurane when applied during ischemia appears to worsen [Ca2+]i overload, which is caused by impeding Ca2+ clearance. As MPG mitigated the increase in [Ca2+]i, isoflurane seems to enhance ROS-mediated effects on intracellular Ca2+ handling in cellular ischemia.

Clinical relevance of immunohistochemical expression of p53-targeted gene products mdm-2, p21 and bcl-2 in breast carcinoma.

The present study was designed to investigate the clinical/prognostic relevance of immunohistochemical expression of p53-targeted genes mdm-2, p21WAF1 and bcl-2 alone and in combination with p53 for the indirect assessment of p53 gene status in breast cancer. 141 archival breast carcinomas were immunostained, and the putative mutational status of the p53 gene was defined in 21 of them, as a control for immunohistochemistry, using the polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP) analysis. Genetic changes of p53 correlated significantly with p53 protein overexpression (p = 0.01) but did not do so with any of the related molecules. Immunohistochemical p53 status was directly correlated with mdm-2 (p = 0.0001), p21 (p = 0.0004) and inversely with bcl-2 (p = 0.005) expression. bcl-2 proved to be an independent marker of prognosis, p53 only in the group of node-positive carcinomas, whereas bcl-2-/p53+ tumours revealed the worst prognosis. Mdm-2 and p21 expression was of prognostic significance neither alone nor in combination. We conclude that the detection of down-stream regulators of p53 does not increase the efficacy of immunohistochemistry in assessing the functional status of p53 in breast cancer; however, their combined analysis may help to select subgroups of patients at the extremes of risk for recurrence, or those with greater chances for survival.

Standardized in situ AgNOR analysis in breast pathology: diagnostic and cell kinetic implications.

The aim of the present study was to assess the diagnostic value of the recently standardized morphometric analysis of silver-stained nucleolar organizer region-associated proteins (AgNORs) [30] in a variety of 155 routinely processed benign and malignant breast lesions. 5 normal breast samples, 21 adenoses, 20 ductal hyperplasias, 10 atypical ductal hyperplasias, 20 in situ and 43 invasive ductal carcinomas, 10 in situ and 26 invasive lobular carcinomas were investigated. A statistically highly significant difference was found between normal/ordinary hyperplastic and neoplastic breast lesions with all 4 consensus AgNOR parameters (mean area, mean number, CV of area, CV of number) evaluated. AgNOR quantity was significantly related to histological grade of both in situ and invasive carcinomas. However, variable overlap was found between AgNOR values in different diagnostic groups. We conclude that standardized AgNOR analysis is a prerequisite for objective and reproductible AgNOR assessment in archival tissues. Despite its limited diagnostic utility for individual breast lesions, standardized AgNOR analysis bears a significant potential for characterizing cell kinetic and metabolical activity of breast lesions. This may give insight into the biological background of breast carcinogenesis, differentiation and tumor progression and may also underlie the independent prognostic value of AgNORs in breast cancer.

Immunophenotypic and prognostic analysis of E-cadherin and beta-catenin expression during breast carcinogenesis and tumour progression: a comparative study with CD44.

AIMS: This study was performed to investigate whether immunohistochemical expression of E-cadherin (E-cad) and beta-catenin (beta-cat) in conjunction with CD44 may correlate with the clinical evolution and prognosis of breast cancer. METHODS AND RESULTS: One-hundred and forty-two routinely processed breast tissue samples including normal breast, benign lesions, in situ and invasive carcinomas were investigated. E-cad and beta-cat were strongly expressed by luminal and basal cells in normal glands, benign proliferative and early neoplastic intraductal lesions. Contrarily, CD44 was expressed exclusively by myoepithelial cells in normal breast, whereas different isoform expression patterns were observed in premalignant and malignant lesions. Simultaneous lack of E-cad/beta-cat expression was detected in in situ and invasive lobular carcinomas in contrast to ductal lesions, in which the differential loss of the molecules was associated with poorer differentiation, irrespective of CD44 immunophenotype. Reduced E-cad (P = 0.003), beta-cat (P = 0.03) and increased CD44v4 (P = 0.005) and v7 (P = 0.007) expression were significantly associated with positive lymph node status. Decreased E-cad and lack of CD44v6 expression correlated with poor survival. There was no difference between the expression of either molecule in in situ and invasive components within the same tumour. CONCLUSIONS: Our results indicate that changes in E-cad, beta-cat and CD44 expression occur early in breast carcinogenesis; they are involved in tumour differentiation, but events additional to their deranged expression are needed to acquire an invasive phenotype.

Gains and losses of CD44 expression during breast carcinogenesis and tumour progression.

AIMS: This study was performed to investigate whether the CD44 immunophenotype of breast lesions correlates with the clinical evolution and prognosis of breast cancer. METHODS AND RESULTS: One-hundred and fifty-two routinely processed normal, benign and malignant breast tissue samples were investigated by the following monoclonal antibodies: CD44s (F10-44-2), CD44v3 (3G5), CD44v4 (11.10), CD44v5 (VFF-8), CD44v6 (VFF-18), CD44v7 (VFF-9), CD44v9 (11-24) after wet autoclave pretreatment for antigen retrieval. We found that: (1) in normal breast tissues luminal epithelial cells lacked detectable CD44 in contrast to basal cells, which constitutionally expressed CD44s, v3, v5 v6 and v9 isoforms; (2) in the intraductal compartment of benign hyperplastic lesions, there was scattered or focal staining for CD44s, v5, v6, v7 and v9 isoforms; (3) in neoplastic lesions restricted neo-expression of CD44v3 and v4 was detected; and (4) the CD44 immunophenotype of invasive breast carcinomas was influenced largely by differentiation grade, steroid receptor status of the tumours and significantly correlated with metastatic involvement of the axillary lymph nodes. CONCLUSIONS: Qualitative and quantitative changes of CD44 expression are implicated in early stages of breast carcinogenesis. The restricted neo-expression of certain CD44 isoforms in breast neoplasias suggests that CD44 might be a potential target for future antibody-based tumour therapy.

[Effect of pre-existing lipids on calcinosis of swine heart valve bioprostheses. A study of a dynamic in vitro model]. / Einfluss präexistenter Lipide auf die Verkalkung von Schweinebioklappenprothesen. Eine Untersuchung am dynamischen In-vitro-Modell.

Calcification is one of the main causes of failure of porcine bioprosthetic heart valves, a problem which has not been satisfactorily solved to date. Previous studies have shown that calcification is decreased in porcine heart valves that have undergone treatment for lipid extraction. The relation between lipid distribution and calcification of seven new commercially available porcine bioprosthetic heart valves (St. Jude Medical, Bioimplant) is assessed by Sudan red III staining to show preexisting lipids. All porcine bioprosthetic valves contained lipids to different degrees. Five valves were subjected to the pulsatile accelerated calcification process for 4 weeks. Distribution and level of calcification were evaluated by radiography and compared to the Sudan red-stained fatty areas. Comparison of the calcifications detected by radiography and the macroscopically detected lipids showed a coincidence of 57-66%. Two porcine bioprosthetic valves were histologically prepared and stained with Hematoxylin and von Kossa's stain, respectively. By light microscopy, the von Kossa staining revealed microcalcifications and polarizing microscopic investigation showed birefractive substances in areas with lipid deposits. These preexisting substances in porcine valve tissue may play a determinative role in the calcification of the tissue. Substantial studies are necessary to further determine the role of preexisting lipids in calcification.