Generation of cytotoxic antibodies to the B16 murine melanoma using a formalinized vaccine

The goal of our experiments was to determine the extent to which the humoral response to a melanoma vaccine elicits the production of cytotoxic antibodies in tumor-challenged mice. Mice were immunized with a vaccine produced from formalinized extracellular antigens (FECA) derived from B 16 F 10 melanomas. The production of antibodies that recognized the vaccine preparation was determined by ELISA, as was their cross-reactivity with the B700 melanoma,antigen. The antibodies were shown to be anti-proliferative by inhibition of tritiated thymidine incorporation into the DNA of cultured target cells and cytotoxic by assays for complement-mediated and antibodydependent cellular cytotoxicity. Flow cytometric analyses indicated that-60% of the target cells specifically bound antibody from the immune sera. These results confirm that B700 is a significant antigenic component of the FECA vaccine, and provide encouragement for this approach to developing useful melanoma vaccines.     

Long-term hydroxytamoxifen treatment of an MCF-7-derived breast cancer cell line irreversibly inhibits the expression of estrogenic genes through chromatin remodeling

Antiestrogen resistance is frequently observed in patients after longterm treatment with tamoxifen, a nonsteroidal antiestrogen widely used for endocrine therapy of breast cancer. In vitro studies in resistant cells showed that the expression of natural estrogen-responsive genes is frequently altered. Using MVLN cells, an MCF-7-derived cell model, we previously demonstrated that 4-hydroxytamoxifen (OHT) treatment irreversibly inactivated an estrogen-regulated chimeric luciferase response by a direct effect of the drug and not through a cell selection process (E. Badia et al., Cancer Res., 54: 5860-5866, 1994). In the present study, we present tamoxifen-resistant but still estrogen-dependent clones isolated after long-term treatment of MVLN cells with OHT and show that progesterone receptor (PR) expression was irreversibly decreased in some of these clones, whereas the PRA:PRB ratio of residual PR remained unchanged. The irreversible inactivation of both chimeric luciferase gene and PR gene expression was associated with the disappearance of DNase 1-hypersensitive sites. In the case of the chimeric gene, at least one of these sites was close to the estrogen responsive element. Genomic sequencing analysis of a clone with very low PR content did not reveal any methylation on CpG dinucleotides or any mutation in the PR gene promoter region. In all of the resistant clones tested and independently of their PR content, estrogen receptor expression was only lowered by half and remained functional, whereas pS2 expression was not modified. We also observed that the residual luciferase activity level (1-2%) of the MVLN clones, the luciferase expression of which had been irreversibly inactivated, was raised 4-fold by trichostatin A treatment. We conclude that long-term OHT treatment may modify the chromatin structure and thus could contribute to differentially silencing natural target genes.     

Reporter Cell Lines to Monitor Steroid and Antisteroid Potential of Environmental Samples

In order to monitor the (anti)steroid activity of environmental samples, we established stable cell lines expressing luciferase under the control of estrogens, androgens, progestives and glucocorticoids. The breast cancer MCF-7 cells which express the estrogen receptor (ER), the glucocorticoid receptor (GR) and the progesterone receptor (PR) were transfected by an estrogen (ERE-Glob-Luc) or a glucocorticoid/progestin/androgen (MMTV-Luc) regulated luciferase plasmid in order to enable the detection of compounds which bind both ER, PR and GR (MELN and MMLN cells). Human prostatic cells PC3 were stably transfected by both an androgen receptor gene and the MMTV-luciferase plasmid (PALM cells). These three cellular models were validated as tools to check the estrogenic, progestive, glucocorticoid and androgenic activities using several potential xenohormones and environmental samples. As these environmental samples were fractionated after solid phase extraction to isolate active compounds, we used these cellular models to monitor the different fractions. In the estrogenic model mid-polar fractions of environmental samples were found active while in the androgenic model, the same fractions had antagonist activity.     

Population pharmacokinetics of topotecan: intraindividual variability in total drug

The inter- and intraindividual variabilities in topotecan clearance (CL) were explored using a population pharmacokinetic approach. Total (lactone + hydroxy acid) topotecan plasma concentrations were obtained in 31 women with metastatic epithelial ovarian cancer treated by the 30-min intravenous infusion on 5 subsequent days. The data corresponding to three occasions (days 1 and 5 of cycle 1, and day 1 of cycle 2), were analyzed using the nonlinear mixed effect model program. A large interindividual variability was observed, with CL varying from 9.1 to 42.5 l per hour (mean 21.0). Topotecan CL was related to serum creatinine level, and age. A close relationship was also observed between topotecan CL and creatinine clearance. Intraindividual variability both within cycle 1 and between the two first cycles was limited, with a mean variation of −2 ± 17%, and +5 ± 20%, respectively. A limited sampling strategy using Bayesian estimation based on two samples (5 min before the end of the 30-min infusion, and 4 h after the end of infusion) was developed. The results of this study combine relationships between topotecan pharmacokinetic parameters and patient covariates that may be useful for a priori dose adjustment, and convenient sampling procedure that can be used for further studies and drug monitoring. Received: 23 February 2000 / Accepted: 22 May 2000     

Inpatient Diabetology

Tight glycemic control is now an imperative of outpatient diabetes care. The inpatient arena remains under the influence of an ineffective paradigm characterized by tolerance for hyperglycemia and a reluctance to use insulin intensively. This article is a call to action against the lip service paid to inpatient diabetes care. The compelling in vitro and in vivo evidence for the benefit of intensive insulin-mediated glycemic control is summarized. The linchpin of current inpatient care is a commonly used insulin sliding scale. This autopilot approach as the sole mode of treatment for inpatient hyperglycemia has been strongly condemned. Nevertheless, it continues to survive. The evidence supports the compelling argument that the adverse effect of hyperglycemia on hospital length of stay, morbidity, and mortality is substantial. Clinicians, nurses, administrators, and insurers ought to look critically at the prevailing paradigm and spearhead the much-needed revolution in inpatient diabetology. The issue of glycemic targets, the need for noninvasive blood glucose monitoring, and the role of nursing staff in this revolution are raised. We call for the banning of the insulin sliding scale use as the sole diabetes order. Also, the use of basal insulin via continuous intravenous insulin infusion or subcutaneous insulin analogs should be embraced. Educating nurses, house staff, and other frontline professionals in the adverse consequences of the current paradigm is essential. Inpatient glycemic control matters; clinical and financial outcomes are at stake. It behooves the health care system and the diabetic public to address the contemporary state of inpatient diabetology as soon as possible.     

Skin atrophogenic potential of methylprednisolone aceponate (MPA)

The most prominent and most discussed local side effect of topical corticosteroids is the thinning of the skin. Therefore, the atrophogenic potential is an important indication of the quality of a new corticosteroid. Several studies have been conducted to investigate this parameter. In rats, the effect of breaking strength of the skin, the most appropriate model for evaluating atrophogenicity in animals, showed that MPA and prednicarbate (PC) reduced the breaking strengh only slightly compared to clobetasol propionate (CBP). These results indicate that MPA could be classified as a corticosteroid with low local atrophogenic potential. This was confirmed in humans by a placebo controlled double-blind study comparing intra/interindividuals MPA (cream, ointment and fatty ointment) and bethamethasone-17-valerate (BMV), CBP and PC (cream only) under occlusive dressing over 6 weeks. Three different parameters were assessed (dermal atrophy (clinical picture), surfometric measurement of the dermatoglyphic pattern, visual evaluation of telangiectasia). In all three formulations, MPA is of lower atrophogenic potential than CBP. While there is no statistical difference between BMV and MPA, the atrophogenic potential of MPA is low. In order to reflect more the clinical use of topical corticosteroids, MPA preparations (cream 0.1% and fatty ointment) has been evaluated in comparison to BMV in an 8-week non-occluded application test. MPA was applied once a day (5 days a week) and BMV twice in 20 healthy subjects according to a double-blind randomized design. Assessment of atrophogenic potential was performed weekly using clinical scores (atrophy and telangiectasia) as main criteria and skin thickness measurements (ultrasound imaging) as a second criterion. BMV cream gives higher numbers of telangiectasia than MPA preparations and vehicles. From the skin thickness measurements, MPA treatments once a day has a lower thinning potential than BMV twice a day. These findings were confirmed by clinical trials. In 1145 patients suffering from various types of eczema who used MPA in cream and ointment, mild atrophy of the skin was observed in only one patient. Moreover, clinical signs of atrophy were present in only two out of a group of 673 patients (590 adult and 83 children) treated with MPA fatty ointment. In a group of 66 patients who used this same MPA fatty ointment during 3–4 months, no signs of skin atrophy were observed. Considering animal and human studies, MPA can be classified as a corticoid with low atrophogenic potential.