Human bone cell cultures in biocompatibility testing. Part I: osteoblastic differentiation of serially passaged human bone marrow cells cultured in alpha-MEM and in DMEM

Well-characterised human osteoblastic bone marrow cell cultures are a useful in vitro tool to analyse bone tissue/biomaterials interactions. In this work, human bone marrow was cultured in experimental conditions described to favour osteoblastic differentiation and, serially passaged cells were cultured in two widely used culture media, minimum essential medium Eagle, alpha modification (alpha-MEM) and Dulbecco's modified Eagle's medium (DMEM). Cultures were grown for 35 d and compared concerning morphologic appearance on scanning electron microscopy (SEM), cell viability/proliferation, total protein content, activity of alkaline phosphatase (ALP) and ability to form calcium phosphate deposits. Results showed that cell proliferation was similar in cultures grown in the two media but ALP activity and ability to form mineralised deposits were lower in DMEM cultures. In both experimental situations, osteoblastic parameters were strongly reduced on cell passage, particularly from the first to the second subculture. In the experimental conditions used (presence of ascorbic acid, sodium beta-glycerophosphate and dexamethasone in the primary and secondary cultures), osteoblastic differentiation was observed in the first and second subcultures grown in alpha-MEM and in the first subculture grown in DMEM. These results underline the importance of the definition of the experimental conditions in studies involving bone cell cultures.     

DNA damage in cytologically normal urothelial cells of patients with a history of urothelial cell carcinoma

In order to determine if patients with a history of previous urothelial cell carcinoma (UCC) but with current normal urinary cytology have DNA damage in urothelial cells, the single-cell gel electrophoresis (comet) assay was conducted with cells obtained by urinary bladder washings from 44 patients (28 with a history of previous UCC). Increased DNA damage was observed in cytologically "normal" urothelial cells of patients with a history of UCC when compared with referents with no similar history and after correcting the data for smoking status and age (P < 0.018). Increased DNA damage also correlated with the highest tumor grade, irrespective of time or course of the disease after clinical intervention (Kendall tau correlation, 0.37, P = 0.016). Moreover, aneuploidy, as assessed by DNA content ratio (DCR; 75th/25th percentile of total DNA fluorescence of 50 comets/patient) was unaltered by smoking status, but increased with UCC grade: 1.39 +/- 0.12 (median +/- 95% confidence interval; referents); 1.43 +/- 0.11 (Grade I UCC; P = 0.264, against referents); 1.49 +/- 0.16 (Grade II UCC; P = 0.057); 1.57 +/- 0.16 (Grade III UCC; P = 0.003). Micronucleated urothelial cells (MNC) were also scored on Giemsa-stained routine cytological smears and were found not to correlate with DNA damage or DCR. MNC frequencies were higher for patients with a history of UCC and/or smoking than referents with neither history, but there was no statistical difference between groups. Taken together, these results suggest that the normal-appearing urothelium of patients resected for UCC still harbor genetically unstable cells.     

A 3 year retrospective review of intrauterine insemination, using cryopreserved donor spermatozoa and cycle monitoring by urinary or serum luteinizing hormone measurements

Insemination with donor spermatozoa is an integral part of infertility treatment. For the last 3 years in our unit, intrauterine insemination with donor spermatozoa (IUID) has been used in preference to vaginal insemination. In this retrospective study, patients were offered an initial course of five single intrauterine inseminations with cryopreserved donor spermatozoa and treatment was then reviewed. A total of 389 patients received 1465 inseminations. In all, 1119 cycles were monitored using luteinizing hormone serum analyses and 346 cycles using the urine home test kits. The clinical pregnancy rate per insemination for the cycles monitored by the serum assay was 18.0% (202/1119) compared with the urine cycles (13.7%, 46/346) (P <05). The pregnancy loss rate was not significantly different (14.4%, 29/202 and 21.7%, 10/46) (serum and urine cycles respectively). The viable clinical pregnancy rate was significantly higher (P <03) for the serum cycles than for the cycles using the urinary monitoring (15.5%, 173/1119 and 10.4%, 36/346 respectively). The cycles monitored by serum assay had a significantly higher cumulative viable clinical pregnancy rate (P <0001) of 70.2% after nine inseminations compared with the urine monitored cycles of 54.8%. The majority of patients opted for the serum cycles, with a minority self-selecting the urine cycles mainly for travelling convenience. The explanation for the significant differences between the viable clinical pregnancy rates per insemination and the cumulative viable clinical pregnancy rates may be due to the sensitivity of the urine home test kit or the patients' interpretation of the result.      

B lymphocytes secreting IgG linked to latent transforming growth factor- beta prevent primary cytolytic T lymphocyte responses

B lymphocytes secreting IgG linked to latent transforming growth factor (TGF)-beta (IgG-TGF-beta) prevent cytolytic T lymphocyte (CTL) responses to unrelated antigens in mixed lymphocyte cultures (MLC) so long as resting resident macrophages and functional Fc receptors are present. This was shown using IgG-secreting plaque-forming cells (PFC) to sheep erythrocytes (SRBC) obtained from popliteal lymph nodes of mice injected repeatedly in foot pads with SRBC. Remarkably, as few as approximately 300 PFC prevented CTL responses of 5 x 10(5) normal syngeneic spleen cells in MLC. Supranatants of short-term cultures of PFC also prevented CTL responses, and suppression was prevented by eliminating or dissociating IgG and TGF-beta present in supranatants or by antibody against active TGF-beta. Furthermore, the latency- associated peptide of latent TGF-beta was detected in approximately 10% of foci of IgG captured from single PFC, indicating that at least some B lymphocytes secrete IgG-TGF-beta as a complex. Resting resident macrophages (which do not produce latent TGF-beta) and functional Fc receptors were required for suppression, consistent with idea that IgG- TGF-beta is taken up through Fc receptors for IgG and that active TGF- beta, cleaved from latent TGF-beta of the complex, is delivered directly to potentially responding CTL. If CTL responses in man are similarly regulated by B lymphocytes, then an ongoing B cell response in patients with chronic viral infections or bearing immunogenic cancers may prevent effective therapeutic vaccination.