A novel method for analysis of nuclear receptor function at natural promoters: peroxisome proliferator-activated receptor gamma agonist actions on aP2 gene expression detected using branched DNA messenger RNA quantitation.

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, plays an essential role in the mediation of the actions of antidiabetic drugs known as thiazolidinediones (TZDs). PPARgamma activates many target genes involved in lipid anabolism including the adipocyte fatty acid binding protein (aP2). In this study, induction of aP2 gene expression by PPARgamma agonists was examined in both cultured cells and diabetic mice using branched DNA (bDNA)-mediated mRNA quantitation. bDNA technology allows for the direct measurement of a particular mRNA directly within cellular lysate using a 96-well plate format in a time frame comparable to a reporter gene assay. In cultured human subcutaneous preadipocytes, the TZDs, troglitazone and BRL-49653, both rapidly induced aP2 mRNA as detected with the bDNA method. In these cells, the effect of BRL-49653 on aP2 mRNA levels was detectable as early as 30 min after treatment (47% increase) and was maximal after 24 h of treatment (12-fold increase). The effects of troglitazone on aP2 mRNA induction were similar to those of BRL-49653 except that the maximal level of induction was consistently lower (e.g. 24 h treatment = 4-fold increase). Dose-response relationships for both of the TZDs were also determined using the 24-h treatment time point. EC50s for both BRL-49653 and troglitazone were estimated to be 80 nM and 690 nM, respectively. A natural PPARgamma ligand, 15-deoxy-delta12,14-PGJ2, was also active in this assay with a maximal induction of aP2 mRNA of approximately 5-fold when tested at 1 microM. Since the PPARgamma:retinoid X receptor (RXR) heterodimer has been characterized as a permissive heterodimer with respect to RXR ligands, the ability of 9-cis-retinoic acid (9-cis-RA) to induce aP2 mRNA was examined. Although 9-cis-RA had very low efficacy (2-fold induction), the maximal effect was reached at 100 nM. No synergism or additivity in aP2 mRNA induction was detected when 9-cis-RA was included with either of the TZDs used in this study. Significant induction of aP2 mRNA in bone marrow of db/db mice treated with either troglitazone or BRL-49653 was also detected, indicating that the bDNA assay may be a simple method to monitor nuclear receptor target gene induction in vivo.

Nonsteroidal progesterone receptor ligands. 2. High-affinity ligands with selectivity for bone cell progesterone receptors.

A novel series of nonsteroidal heterocycles was discovered which display cell-type selective, high-affinity (nanomolar) binding to the progesterone receptors from TE85 osteosarcoma cells but > 1 microM binding affinity to the progesterone receptors from T47D and ZR75 human breast carcinoma cells. Structure-activity relationships were developed for a set of these compounds, and a representative analog 1-(3,4-dichlorobenzoyl)-3-phenyl-1,4,5,6-tetrahydropyridazine++ + (1i, RWJ 25333) was chosen for further evaluation. RWJ 25333 stimulated the in vitro proliferation of human osteoblast-like cells but not human breast cells.

Characterization of the progestin receptors in the human TE85 and murine MC3T3-E1 osteoblast-like cell lines.

Progestins are believed to exert positive effects on bone density through receptors located in osteoblasts. In the present studies, the binding characteristics and regulation of the progestin receptors in two osteoblast-like cell lines were compared with those in human breast lines. Human TE85 and murine MC3T3-E1 osteoblast-like cells contain a single, high-affinity progestin binding site whose affinity and concentration are lower than in human breast cells. The osteoblastic progestin binding sites showed the expected steroid specificity and associated with the cell nuclei when occupied by ligand. The progestin receptors in osteoblastic cells also had sedimentation coefficients similar to those receptors in breast cells. The regulation of the progestin receptor in the osteoblast-like cells was explored by treating them with estradiol. In contrast to the large, rapid change seen in the breast cells, the progestin receptor levels in the MC3T3-E1 cells showed only a small, delayed up-regulation with estradiol treatment. The progestin receptor number in the TE85 cells was unaffected by estradiol. Down-regulation of the progestin receptors was explored by treating the cells with the progestin, norethindrone (NET). NET administration produced a rapid drop in progestin binding sites in the breast cells and a smaller, more gradual decline in MC3T3-E1 progestin binding. While the maximal decrease in receptor number occurred within 24 h in the breast cells, the receptor number was still continuing to fall after 72 h in the MC3T3-E1 cells. The data presented here demonstrate that both human and murine osteoblast-like cells contain a functional progestin receptor whose binding characteristics and regulation are similar, but not identical, to those receptors in other progestin target tissues such as the breast.

Intestinal lactase, sucrase, and alkaline phosphatase in 373 patients with coeliac disease.

Lactase, sucrase, and alkaline phosphatase activities were measured in 833 peroral small intestinal biopsies from 373 patients with coeliac disease. Enzyme activities decreased with increasing degrees of mucosal damage. Enzyme activities in mucosae of patients with coeliac disease in remission were lower than in control groups matched for age, sex, and site of biopsy. Enzyme activities were measured in 81 patients when the mucosa was severely damaged and later when considerable improvement had occurred. Lactase activity remained low in 13% of patients under the age of 18 and in 33% of those over 18 years. Sucrase activity usually improved with histological recovery, but alkaline phosphatase activity tended to remain depressed in patients in whom lactase activity failed to improve.

Pseudomonas aeruginosa cross-infection following endoscopic retrograde cholangiopancreatography.

In a 6 week period, three of 50 patients developed Pseudomonas aeruginosa septicaemia following Endoscopic Retrograde Cholangiopancreatography (ERCP). Pseudomonas aeruginosa serotype 10 was isolated from each of the patients and from the endoscope. The outbreak was related to inadequate disinfection of the air and water channel of the endoscope. Following the introduction of a modified decontamination technique, which involved rinsing the air and water channel with glutaraldehyde, no further cases of pseudomonas infection occurred, and the organism could not be isolated from the instrument. Obstruction of the biliary tract was a predisposing factor in the development of infection; and administration of antibiotics immediately following the procedure failed to prevent it. This may have been due to inadequate dosage. We suggest that patients presenting for ERCP, in whom obstruction of the biliary tract is suspected, should come prepared for immediate drainage of the obstructed system at the time of the procedure.

A case-control study of obstetric complications and later autistic disorder.

The precise etiology of autism remains unclear. Obstetric adversity has been described as one factor that may increase the risk for the disorder. We examined the contemporaneous birth records of 49 children satisfying DSM-III-R criteria for autistic disorder, at four Dublin maternity hospitals, using the previous same-sex live birth in that hospital as a control. Data were evaluated blind to subject status using two obstetric complication (OC) rating scales. No significant differences in obstetric adversity were found between index and control groups. Autistic individuals did not differ from controls in terms of previously described risk factors for this disorder (maternal age, maternal parity, birth order, and low birth weight) in autism. These data do not support the view that OCs increase the risk for later autism.

Pharmacological profile of a novel, non-TZD PPARgamma agonist.

AIM: The purpose of this study was to characterize a novel, non-thiazolidinedione (TZD) peroxisome proliferator-activated receptor (PPAR)gamma agonist, RWJ-348260, via both in vitro and in vivo approaches. METHODS: The in vitro PPARgamma activities of RWJ-348260 were assessed in PPARgamma-GAL4 co-transfection assay, PPARgamma receptor binding assay, aP2 gene induction assay and preadipocyte differentiation assay. The in vivo efficacy of the compound was determined in rodent genetic diabetes models [ob/ob mouse, db/db mouse and Zucker diabetic fatty (ZDF) rat] following multiple days of oral administration. RESULTS: RWJ-348260 selectively activated PPARgammain vitro. In vivo, RWJ-348260 produced significant decreases in plasma glucose, HbA1c, insulin and triglyceride levels. RWJ-348260 also dose-dependently improved oral glucose tolerance. In db/db mice, the compound up-regulated PPARgamma target genes in white adipose tissues. RWJ-348260 produced a lower extent of hepatocyte lipid deposition and a smaller increase in liver weight compared to rosiglitazone in db/db mice. While RWJ-348260 effectively normalized hyperglycaemia and dyslipidaemia, it did not change haematocrit, transaminase, alkaline phosphatase, total bilirubin levels or liver weights in ZDF rats. CONCLUSIONS: RWJ-348260 is a potent PPARgamma agonist with efficacious antidiabetic activity in diabetic animal models. The compound has an improved side-effect profile compared to rosiglitazone.

Obsessive-compulsive disorder and paraphilia in a monozygotic twin pair.

We report OCD and paraphilia in two male members of triplets (the two males being monozygotic twins), and discuss the possible aetiological factors for this previously unreported occurrence. We suggest that patients presenting with paraphilia should be examined for OCD and that a detailed sexual history should be obtained in all patients with OCD.

Secretion of a growth inhibitory factor by ZR-75-1 human breast cancer cells.

Cultures of the human mammary carcinoma line ZR-75-1 secrete a growth inhibitory factor (GIF) that, when diluted, slows the growth of MDA-MB-231 and MCF-7 cells. Undiluted "conditioned" media prevents cell division from occurring in both human breast cancer lines. ZR-75-1 cells are unaffected by this factor. The amount of GIF in the culture media is related to the confluency of the ZR-75-1 cells. The activity of this GIF is not altered by DNAse or RNAse but is destroyed by heating or trypsin. Growth inhibition is 85-90% reversible if conditioned media is replaced with fresh media.

Decreasing incidence of coeliac disease.

Between 1960 and 1974 the incidence of coeliac disease in children under 12 years in County Galway remained fairly constant, but since 1975 it has fallen by 62% and the lowered incidence seems well established. The number of those who were breast fed and the age at which first gluten feeding took place during the 22 years both increased significantly and from the mid-70s total protein content and osmolarity of proprietary cow's milk formulas were reduced. All three factors may be relevant. A negative correlation with the incidence of gastroenteritis was found.

Congenital neutropenia and low serum immunoglobulin A: description and investigation of a large kindred.

A kindred is described in which neutropenia and low serum immunoglobulin A levels has occurred. The affected members have variable clinical courses which do not appear to relate to the degree of neutropenia. The bone marrows show a maturation delay in granulocyte production, with increased numbers of lymphocytes. In vitro cultures of the bone marrows failed to produce granulocyte colonies. Studies of peripheral blood and bone marrow lymphocyte subpopulations give inconsistent results, although a general increase in T suppressor lymphocytes compared with T helper lymphocytes emerges. A therapeutic trial of corticosteroid and then of lithium carbonate was without effect on one of the patients. The pathogenetic basis for this probably X-linked disorder remains unclear.

A branched DNA signal amplification assay to quantitate messenger RNA of human uncoupling proteins 1, 2, and 3.

Uncoupling proteins (UCP) are inner mitochondrial membrane transporters which dissipate the proton gradient, releasing stored energy as heat. Three subtypes of UCP have been identified so far. The regulation of UCP expression is mainly controlled at the transcriptional level, thus making the measurement of UCP mRNA beneficial for both diagnosis and research of weight disorders and diabetes. We have developed an assay using the branched DNA signal amplification assay (bDNA assay) to quantitatively measure the mRNA levels for human UCP1, 2, and 3. UCP-subtype-specific primers were designed for the assay. RNA transcripts of each UCP generated by in vitro transcription were used to validate the specificity and sensitivity of the assay. The quantitative measurement of UCP mRNA was further demonstrated with cultured cells and human tissue. A comprehensive survey of UCP expression from 17 human tissues measured by the newly developed assay is provided. The method described here offers a rapid, sensitive, specific, and quantitative assay for measurement of human UCP mRNA.

Mouse ovarian tumor cells: an experimental model for progestin-mediated radiotherapy.

Mouse ovarian tumor (MOT) cells have been grown in C3HeB/FeJ mice as an ascites and as a subcutaneous tumor and in cell culture as a suspension. These cells contain saturable, high-affinity, specific progesterone receptors. Estrogen receptors were not detectable in these cells. MOT cells can be used as both an in vivo and an in vitro model for progestin-mediated radiotherapy.

Celiac sprue and immunodeficiency states: a 25-year review.

Immunoglobulin deficiency, especially deficiency of IgA, has been described in patients with celiac sprue (CS). Our study was performed in an area of high prevalence of CS to determine the prevalence of immunodeficiency states in patients with CS, to examine their clinical characteristics, response to treatment, and HLA phenotypes compared with a group of age- and sex-matched persons with CS but without immunoglobulin deficiency. Fourteen of 604 patients with CS were identified as being selectively deficient in IgA, whereas one had common variable immunodeficiency. At diagnosis, anemia was present in 8 of 14 IgA-deficient patients compared with 10 of 42 controls (p = 0.047), whereas abdominal pain was more common in controls with CS. Autoimmunity and recurrent infection were more prevalent in the IgA-deficient group. Response to gluten-free diet was similar in both groups in terms of histologic structure and recovery of intestinal brush-border enzyme activity. IgA-deficient participants with CS had no increased risk of associated malignancy or lymphoma. HLA phenotypes were similar in both groups. The prevalences of selective IgA deficiency and common variable immunodeficiency in this series of patients with CS are 2.31 in 100 and 0.16 in 100, respectively. Although this group is unique in character, close follow-up coupled with conscientious compliance with a gluten-free diet, remains the mainstay of treatment for these patients.

Immunoglobulins in healthy controls: HLA-B8 and sex differences.

Serum immunoglobulins were measured in one hundred and eighty-four healthy controls. One hundred and fifty-nine of these were also HLA typed. IgG levels did not differ with sex or HLA-B8 status. IgM levels did not differ with HLA-B8 status but were significantly higher in females than males. IgA levels were lower in females than males; they were also lower in HLA-B8 positive compared with HLA-B8 negative individuals; the lowest IgA levels were found in HLA-B8 positive females. The IgA variation may be relevant to the higher incidence of certain disorders among HLA-B8 positive individuals and among females.