Various protocols for determining estrogens by the enzymatic method using estradiol dehydrogenase. Respective procedures and advantages

Up to now, more than 40.000 determinations of urinary estrogens (E1 + E2) have been carried out in routine clinical analysis by the enzymatic method using estradiol dehydrogenase. This method makes use of the transhydrogenating activity of the placental enzyme: this enzyme transfers hydrogen from NADP to NAD with recycling of the specific substrate (E1 + E2). For several years the necessary reagents have been commercially available in the form of a kit. Nonetheless, various improvements have been made to the measurement of reduced NAD, which accumulates in the reaction medium and is directly proportional to the concentration of the two estrogens. Three protocols are available at present: Spectrophotometric measurement at 340 nm (initial technique); Colorimetric measurement at 492 nm. The pink colour measured arises from the reduction of a tetrazolium salt (INT) by reduced NAD in a coupled system using diaphorase; Measurement by bioluminescence of the light energy liberated on the reduction of flavin derivatives by NADH. The reaction is mediated by various enzymes isolated from marine bacteria (FMN oxidoreductase and luciferase) in the presence of an aliphatic aldehyde (decanal). The procedure for each of these protocols is described as well as the means for controlling the linearity of the reaction. The choice of protocol is determined by the biological fluid available, the speed of response desired and the cost of the analysis.     

Expression of HLA-DR and common acute lymphoblastic leukemia antigens on glioma cells

The expression on several established human glioma cell lines of two well-defined differentiation antigens, HLA-DR and the common acute lymphoblastic leukemia antigen (CALLA) has been demonstrated. Rabbit anti-CALLA antiserum and monoclonal anti-la antibodies specifically lysed glioma cells in the presence of complement. Absorption of anti-CALLA antiserum and anti-Ia antibodies by glioma cells abolished their cytotoxicity against blasts isolated from a common acute lymphoblastic leukemia. Immunoprecipitation of solubilized glioma cells by monoclonal anti-Ia antibodies revealed two polypeptide chains of 28 and 33 kDa, whereas the anti-CALLA antiserum precipitated a single polypeptide chain of 100 kDa.     

Simplified enzyme-linked immunosorbent assay for specific antibodies to respiratory syncytial virus.

A simplified and reliable enzyme-linked immunosorbent assay (ELISA) was applied to the detection of serum antibodies against respiratory syncytial virus (RSV). RSV-infected cells were fixed and dried on 96-well microtiter plates and kept at 4 degrees C. The titers of reference sera were determined by endpoint dilution. A linear relation was found between the titers and the logarithm of absorbance values of sera diluted to 1:1,000 (r = 0.93, P less than 0.001). Measurement of RSV antibodies was done by using a single serum dilution (1:1,000) in conjunction with a standard curve. A strong correlation was found between complement fixation and ELISA results (r = 0.89, P less than 0.001). In addition, the ELISA method exhibited higher titers and a greater sensitivity than did complement fixation, although the applicability of the assay is limited with positive serum samples of low titer.     

Family and Illness Predictors of Outcome in Pediatric Brain Tumors

Investigated the prediction of cognitive and behavioral outcomesin 63 children with heterogenous brain tumors. Hierarchicalmultiple regression analyses were used to determine how family-relatedvariables added to the prediction of children's outcome overand above illness measures. The best predictors of children'sbehavior problems and adaptive behavior were family and demographicvariables, whereas the best predictors of achievement were illnessand demographic variables. A combination of family and illnessvariables, however, was the best predictor of intellectual functioning.In addition to identifying specific predictors of cognitiveand behavioral outcome in children with brain tumors, theseresults lend initial support for the inclusion of contextualfactors such as family stress, maternal coping, number of parentsin the home, and family SES measures in studies of how diseasefactors affect outcomes in pediatric brain tumor patients.     

Epithelial cell heterogeneity in the guinea pig thymus: immunohistochemical characterization of four thymic epithelial subsets defined by monoclonal anti-keratin antibodies

Keratins are a family of related polypeptides constitutive of the cytoskeleton of epithelial cells and are never found in nonepithelial tissues. Thymic epithelial cells (TEC), known to induce T cell differentiation, are the keratin-containing cells within the thymus. Using four monoclonal anti-keratin antibodies (KL1, KL4, AE2, AE3) directed against keratins of different molecular weight, we have investigated the guinea pig thymic epithelium. The immunohistochemical analysis of thymic cryostatic sections revealed that the keratin expression of TEC varied according to their location in the thymic lobula; the thymic cortex was specifically stained by AE3 whereas the thymic medulla and the subcapsular cortex were recognized by KL4. In addition, KL1 and AE2 exclusively labeled Hassall's corpuscles. The biochemical analysis of keratins extracted from the thymus showed that each TEC subset was characterized by an unique pattern of keratin polypeptides. This study extends the concept of thymic epithelium heterogeneity and suggests that anti-keratin antibodies which allow the typing of TEC subsets may be valuable tools for studying the differentiation of thymic epithelium and its in vitro function on T lymphocytes.     

Long-term reduction of vasopressin excretion induced by the central injection of an immunoconjugate (antibody to vasopressin linked to ricin a chain)

We have previously demonstrated that vasopressin-producing neurons are the target of monoclonal antibodies to vasopressin microinjected into the brain tissue. At the same time, this central microinjection of vasopressin-monoclonal antibody into the supraoptic nuclei produced hydro-osmotic disorders mimicking the effects of a central diabetes insipidus. In order to investigate the increase in both duration and amplitude of the biological effects seen after the injection of vasopressin-monoclonal antibody, an immunoconjugate was constructed with the vasopressin-monoclonal antibody IgG1k isotype and the cytotoxic part of the ricin molecule, the ricin A chain. The biological parameters, such as diuresis and urine osmolality which are directly regulated by vasopressin, and vasopressin excretion, were measured after the central injection of this immunotoxin/immunoconjugate. The consequences of immunotoxin injection were also studied when immunotoxin was co-injected with monensin (50 nM) which has been shown to decrease the intracellular degradation of immunotoxin, and plasma complement, which has been shown to increase the neuronal uptake of immunotoxin. Single injection of immunotoxin near the hypothalamic supraoptic nuclei significantly increased diuresis and decreased vasopressin excretion. However, these effects were only transient and disappeared 24 h later. Four successive injections of immunotoxin (one per day) with monensin induced a decrease of vasopressin excretion which was still observed after a resting period of four days after the fourth injection. The long-term reduction of vasopressin excretion was induced in rats receiving four successive injections of a mixture consisting of immunotoxin with monensin and plasma complement. In such experiments, the vasopressin content of urine remained low (55% under the baseline value), two weeks after the fourth injection of immunotoxin. At the same time, the diuresis was increased (80% above the baseline value) and urine osmolality lowered (45% under the baseline value). When non-specific IgG replaced specific antibody, vasopressin excretion, diuresis as well as urine osmolality were unchanged.

The results of this study demonstrated that the use of a specific immunotoxin results in a local interference with the vasopressinergic neurons and induces a long-term reduction of vasopressin secretion.