Fusion of bone marrow-derived stem cells with cardiomyocytes in a heterologous in vitro model.

OBJECTIVE: Recent studies have demonstrated that transplanted bone marrow-derived stem cells (BMCs) possess a broad differentiation potential and are able to form new cardiomyocytes. However, the identity of BMCs as true cardiomyocytes is still ambiguous. Therefore, we investigated the fate of transplanted fluorescence labeled BMCs and cardiomyocytes in co-culture. METHODS: For cell tracking we used two different fluorescent probes, Vybrant/DiO and Vybrant/DiI. BMCs were taken from human sternal marrow, purified using a Ficoll-gradient-centrifugation, treated with 5-azacytidine and stained with Vybrant/DiO. Furthermore, isolated spontaneous beating cardiomyocytes of neonatal rats (CM) were labeled with Vybrant/DiI. Thereafter, the BMCs were transplanted into CM-cultures and investigated on day 1, 4, 7, 14 and 28 using two-color fluorescence phenotyping by laser-scanning-cytometry (LSC). Two-color positive cells were harvested by patch-clamp technique and beta-MHC mRNA expression was analyzed by single-cell PCR. RESULTS: Two different morphological phenotypes were observed by LSC. First, isolated DiO labeled BMCs without contact or with direct cell contact to DiI labeled CMs. Second, some BMCs and CMs were double positive for DiO/DiI spontaneously forming hybrids. This population increased by 18% from day 1 to 4 and decreased only slightly until day 28. Additionally, few two-color positive cell formations expressed both human and rat specific beta-MHC mRNA as well as only human beta-MHC mRNA indicating that cell-fusion and transdifferentiation has occurred. CONCLUSION: These observations provide in vitro evidence for spontaneous cell fusion and transdifferentiation of BMCs in co-culture, raising the possibility that the observed phenomenons may contribute to development or maintenance of these cell types.     

DNA in human glioblastomas. A flow-fluorescence cytometrical examination of 96 tumors

The Flow-Fluorescence Cytometric Method (FCM) was applied to investigate the DNA content and the ploidy outlines of each of 96 glioblastomas. No specific DNA pattern was detected, possibly because of the tangle morphology of these variable tumors. Due to their capricious growth the DNA distribution proved to fluctuate greatly. Thus, the series, arranged according to increased PI (proliferation index) values, exhibited a wide spread within a total range from 7.1–97.15% (mean 39.3%) PI. A threefold subdivision of main types (I–III) appears to be of practical use for clinical prognostic assessment: diploid tumors with a PI range up to 10% (N=7) are followed by abnormal chiefly tretra- and hyper-tetraploid tumors up to PI values about 30% (N=21). The third category includes cases showing excessive aneuploidy combined more and more with polyploidy and valid stemlines, up to the PI maximum of about 97 rel.% (N=68). Thus, in 89 tumors clear pathological changes of DNA content can be decoded; of these 68 (76.4%) express a considerable aneuploidy and polyploidy respectively.Dedicated to Prof. Dr. HJ Bauer for his 75th birthday, March 31, 1989     

Extended stereotactic brain biopsy in suspected primary central nervous system angiitis: good diagnostic accuracy and high safety

Journal of Neurology - To evaluate the diagnostic accuracy and safety of extended stereotactic brain biopsy (ESBB) in a single center cohort with suspected primary angiitis of the central nervous...     

Immunohistochemical and clinical evaluation of cathepsin expression in soft tissue sarcomas

Lysosomal proteases are known to enhance the spread of epithelial tumour cells, but little is known of the possible role of proteases in the growth of soft tissue sarcomas (STS). We investigated the expression of cathepsins D, B, S, H, L and procathepsin L in frozen sections of 34 STS from 34 patients by immunohistochemistry (IHC). Cathepsins D, B and H were relatively highly expressed in STS (77–91%). The expression rate of cathepsins S and L and of procathepsin L was lower (40–66%). Cathepsin S and L expression showed a moderate (P = 0.078 andP = 0.019) and procathepsin L a strong (P = 0.00001) correlation with the survival rate of STS patients. Cathepsin S expression is also correlated with the local recurrence rate (P < 0.01). Lysosomal proteases may play a role in STS progression, and cathepsin expression may also have, significance as a prognostic factor in STS.     

Expression of telomerase genes in thyroid carcinoma

Telomerase (T) is a ribonucleoprotein complex that includes the telomerase RNA component (hTR), telomerase associated protein (TP1) and the telomerase catalytic subunit (hTERT). Telomerase has been shown in stem cells and found to be activated in tumor tissues and immortalized cells. We wanted to test whether the expression of the telomerase complex subunits correlate with the enzyme activity in human thyroid tissue. Hence, we determined the expression of hTERT, hTR and TP1 mRNA by RT-PCR and compared the results to telomerase activity as detected by the telomeric repeat amplification protocol (TRAP) assay. Fifteen benign goiters (G), 11 follicular carcinomas (FTC) including 2 oncocytic follicular carcinomas (also called Hurthle cell carcinoma, oFTC), 12 papillary carcinomas (PTC) including 3 microcarcinomas (mPTC), and 12 undifferentiated anaplastic thyroid carcinomas (UTC) were investigated. Experienced pathologists performed histological and pTNM classification in each specimen. RT-PCR analysis revealed that TP1 was ubiquitously expressed in all G and carcinomas. hTR was expressed in 4 out of 15 G, in 2 out of 3 mPTC, in 5 out of 9 PTC, in 5 out of 9 FTC, in all oFTC and in 9 out of 12 UTC samples. Regarding all carcinomas, no statistically significant correlation was observed between hTR-expression and tumor stage, lymph node or distant metastasis. hTERT-expression was associated with malignancy and tumor stage. All mPTC and 13 out of 15 G did not express hTERT, whereas all samples of pT3-4 tumor stage of FTC, PTC, UTC and all oFTC were positive for hTERT. No telomerase activity could be detected in G. Telomerase activity in carcinoma was only measurable in tissues that expressed the catalytic subunit hTERT. Our data indicate that telomerase activity is up-regulated in neoplastic cells. In contrast to TP1 and hTR, hTERT and telomerase activity may be of help in identifying invasive tumors and may be additional markers for classification of benign goiter and malignant thyroid carcinoma.     

Detrimental Immunologic Effects of Preoperative Chemoradiotherapy in Advanced Rectal Cancer

PURPOSE: Preoperative chemoradiotherapy for advanced rectal cancer has been an important therapeutic tool to improve the long-term results of curative resection. It is not known whether preoperative chemoradiotherapy for advanced rectal cancer influences the perioperative course of immune parameters. METHODS: Thirty patients with rectal cancer underwent surgery with (study group, n = 15) or without (control group, n = 15) preoperative chemoradiotherapy (2 cycles of 5-fluorouracil, 45 Gy). Blood samples were taken before neoadjuvant therapy, preoperatively, and on Days 1, 2, and 5 after surgery. Cell numbers of lymphocyte subpopulations, granulocytes, monocytes, and natural killer cells were determined by flow cytometry; tumor necrosis factor- and interleukin-6 serum levels were measured with enzyme-linked immunosorbent assay. RESULTS: Significant differences between study and control patients (P < 0.05) were detected regarding circulating interleukin-6 and tumor necrosis factor- levels, with depression of the proinflammatory response to surgery in study patients. Similarly, granulocytosis and monocytosis after surgery were significantly lower in patients after neoadjuvant therapy. Furthermore, cell counts of total T lymphocytes, T helper cells, B lymphocytes, and natural killer cells were significantly reduced after preoperative chemoradiotherapy. This depression of cell-mediated immunity in study patients was even more pronounced after surgery. CONCLUSIONS: Preoperative chemoradiotherapy for advanced rectal cancer results in a significant preoperative and postoperative immune dysfunction as indicated by depression of lymphocyte subpopulations, monocytes, granulocytes, and proinflammatory cytokine release. These findings are of importance because increased perioperative morbidity and mortality rates have been observed after preoperative chemoradiotherapy.