More powerful procedures for multiple significance testing

The problem of multiple comparisons is discussed in the context of medical research. The need for more powerful procedures than classical multiple comparison procedures is indicated. To this end some new, general and simple procedures are discussed and demonstrated by two examples from the medical literature: the neuropsychologic effects of unidentified childhood exposure to lead, and the sleep patterns of sober chronic alcoholics.     

Measurement of estradiol-17-fatty acid esters in human tissues.

We have developed a gas chromatographic/mass spectral method for the sensitive and reproducible measurement of estradiol-17-fatty acid esters in human tissues and blood. To provide an internal standard for quantification, a trideuterated analog of a representative estradiol ester is added to the tissues. Estradiol (E2) released from the nonpolar ester fraction by alkaline hydrolysis is derivatized to form the ditrimethylsilyl ether and then analyzed by gas chromatographic/mass spectral, monitoring the molecular ions mass per U charge of the ditrimethylsilyl derivative of E2 and [2H3]E2. There are low but detectable levels of E2 ester in the blood of cycling females; there are none in urine. While the E2 ester is present in breast cyst fluid, its concentration, 77-140 pmol/L, is considerably less than E2, 110-2,863 pmol/L. But there is a large amount of E2 ester in fat. In premenopausal women the average E2 ester in fat (sc and omental) is 957 +/- 283 38 fmol/g (SEM); in women who are menopausal less than 12 yr, the E2 ester in fat is 669 +/- 158 fmol/g; in women who are menopausal at least 15 yr, the fat level is 399 +/- 146 fmol/g. Muscle from the same women have lower concentrations of the ester; in 8 out of 12 muscle specimens it was not detectable. The E2 esters are extremely potent estrogens. Although they are hormonally active they require enzymatic hydrolysis to exert their hormonal action. These studies show that these long chain esters of E2 are sequestered in fatty tissues, wherein they represent a protected store of preformed hormone. Under the proper stimulation, adipose tissue can activate the estrogenic signal through the action of hormonally sensitive esterases. Thus, through signaling between estrogen sensitive tissues and neighboring fat cells, a local paracrine loop may exist.     

Interactions between growth hormone and dexamethasone in skeletal growth and bone structure of the young mouse

Summary The present study investigated the interactions of growth hormone (GH) and glucocorticoid on skeletal growth and bone structure in young mice. The purpose of this study was to examine the possible prevention by GH of the damage inflicted by dexamethasone (Dex) at sites of skeletal growth and ossification. Dex (1 mg/kg) with or without rat GH (rGH) or bovine GH (bGH), 1 mg/kg, was given for 4 weeks, from age 3–7 weeks, to female ICR mice. Tibiae, humerus, and vertebrae were analyzed morphometrically and biochemically. Growth, as determined by the mouse weight, tibial length, and humerus protein content was found to be compromised by dexamethasone. This was prevented by rGH or bGH. The epiphyseal growth plate width, trabecular bone volume, cortical bone width, mineral bone content, and alkaline and acid phosphatase activity were decreased by dexamethasone. These were prevented by rGH or by bGH. The findings of the present study suggest that in the mouse, GH can decrease or even avoid some of the pathological features in growing bones inflicted by high-dose glucocorticoid treatment.     

Studies on the Level of Natural Antibodies Reactive with Various Tumor Cells during Urethane Carcinogenesis in BALB/c Mice

Serum from young normal BALB/c mice was found to contain IgM antibodies able tomediate complement-dependent lysis of certain syngeneic or allogeneic tumor target cells. The titer of such naturally occurring antitumor antibodies (NATA) was found to increase with aging.A longitudinal serological study comparing the cytotoxicity potential of NATA fromnormal and from urethan-treated BALB/c mice was performed. It was found that urethan-treated mice that did not develop primary lung-adenomas within the duration of the experi-ment had significantly lower NATA titers, against one out of 4 target cells assayed, than urethan-treated animals that developed lung adenomas. This difference was evident in two independent experiments. The results suggested that the lower NATA activity of the urethan-treated mice that did not develop tumors existed even before exposure to the carcinogenic insult. This raises the possibility that certain populations could be segregated according to their natural antibody profile into those individuals which will develop primary tumors within a certain period if exposed to a subthreshold amount of carcinogen, and those which will not.     

The product of the imprinted H19 gene is an oncofetal RNA.

AIMS/BACKGROUND: The H19 gene is an imprinted, maternally expressed gene in humans. It is tightly linked and coregulated with the imprinted, paternally expressed gene of insulin-like growth factor 2. The H19 gene product is not translated into protein and functions as an RNA molecule. Although its role has been investigated for more than a decade, its biological function is still not understood fully. H19 is abundantly expressed in many tissues from early stages of embryogenesis through fetal life, and is down regulated postnatally. It is also expressed in certain childhood and adult tumours. This study was designed to screen the expression of H19 in human cancer and its relation to the expression of H19 in the fetus. METHODS: Using in situ hybridisation with a [35S] labelled probe, H19 mRNA was detected in paraffin wax sections of fetal tissues from the first and second trimesters of pregnancy and of a large array of human adult and childhood tumours arising from these tissues. RESULTS: The H19 gene is expressed in tumours arising from tissues which express this gene in fetal life. Its expression in the fetus and in cancer is closely linked with tissue differentiation. CONCLUSIONS: Based on these and previous data, H19 is neither a tumour suppressor gene nor an oncogene. Its product is an oncofetal RNA. The potential use of this RNA as a tumour marker should be evaluated.     

Purification and characterization of placental heparanase and its expression by cultured cytotrophoblasts

The role of different extracellular matrix (ECM)-degrading enzymesin the normal functioning of the placenta is well documented.Heparan sulphate proteoglycan (HSPG) is an integral constituentof the placental and decidual ECM. Because this proteoglycanspecifically interacts with various macromolecules in the ECM,its degradation may disassemble the matrix. Hence, in the caseof the placenta, this may facilitate normal placentation andtrophoblast invasion. Crude placental specimens were collectedfrom first and third trimester placentas. Heparanase (endo-P-glucuronidase)was isolated and purified by ammonium sulphate precipitationfollowed by sequential chromatographies on carboxymethyl-, heparin-and ConA-Sepharose columns. The placental enzyme was furthercharacterized for its molecular weight and specific inhibitionby heparin, and was shown to resemble heparanase expressed byhighly metastatic tumour cells and activated cells of the immunesystem. In order to locate the source of heparanase activityin the placenta, primary cytotrophoblast cultures were established.Intact cells, as well as conditioned medium and cell lysates,were analysed for heparanase activity using metabolically sulphate-labelledECM as a natural substrate. Heparanase was highly active inlysates of cytotrophoblasts. This activity was also expressedby intact cytotrophoblasts seeded on ECM, but no activity couldbe detected in the culture medium. Incubation of the cytotrophoblastsin contact with ECM resulted in release of ECM-bound basic fibroblastgrowth factor (bFGF). We propose that the cytotrophoblasticheparanase facilitates placentation, through cytotrophoblastextravasation and localized neovascularization. cytotrophoblast/extracellular matrix/heparanase/heparan sulphate proteoglycan/placenta     

Endothelial nitric oxide synthase immunoreactivity in early gestation and in trophoblastic disease.

AIMS: To study the localisation of the endothelial nitric oxide synthase (eNOS) in the normal placenta, with special emphasis on the implantation site in the first trimester of pregnancy, and in the different subtypes of trophoblastic cells in gestational trophoblastic disease. METHODS: The immunoperoxidase technique with an antibody directed against eNOS was applied to paraffin sections from first and second trimester placentas, placenta accreta, partial and complete hydatidiform moles, and choriocarcinoma. Immunoperoxidase staining for human placental lactogen (hPL) was performed on parallel sections. RESULTS: Prominent immunoreactivity for eNOS was found to be present in the intermediate trophoblastic cells of the cell columns of the anchoring villi and in trophoblastic cells at the implantation site. Staining was also present in the syncytiotrophoblast, most conspicuous at the apical cell border. In trophoblastic disease, proliferating large mononuclear cells, which were strongly positive for hPL, were found to be immunoreactive for eNOS. CONCLUSIONS: eNOS immunoreactivity is strongly positive in the extravillous trophoblastic cells and to a lesser extent in the syncytiotrophoblast. In the former it may play a role in implantation and vascular invasion. Cells with differentiation to intermediate trophoblast in complete hydatidiform mole and choriocarcinoma also show high levels of eNOS, which may be associated with the haematogenous mode of spread of trophoblastic disease.     

Intravenous pulsed steroids in rheumatoid arthritis: a comparative dose study

This prospective, double blind study was undertaken to test the efficacy of intravenous "minipulse" (100 mg) methylprednisolone (MP) therapy versus standard pulse (1000 mg) MP therapy in rheumatoid arthritis (RA). Thirty-six patients with RA synovitis flares were randomized to receive either 100 or 1000 mg MP IV QD x 3 doses. These 2 universally comparable groups exhibited no statistically significant differences in their striking prompt and sustained clinical improvement. These data suggest that minipulse MP is as efficacious as conventional pulse MP in the treatment of RA flares.