Adrenal Medullary Nodules in Beckwith-Wiedemann Syndrome Resemble Extra-Adrenal Paraganglia

Clues to mechanisms regulating development and tumorigenesis may be provided by studies of unusual diseases. Beckwith-Wiedemann syndrome (BWS) is a rare congenital disorder apparently related to abnormal regulation of insulin-like growth factor-2 (IGF-2) production. IGF-2 mRNA has been previously localized to the chief cells of extra-adrenal paraganglia and to adult, but not fetal, adrenal medulla. Expression of IGF-2 by neuroblastomas has been hypothesized to reflect extra-adrenal paraganglionic differentiation. In the adrenals of a fetus with BWS, we have observed both increased numbers of chromaffin cells and organoid nodules resembling extra-adrenal paraganglia. Immunoreactive IGF-2 was observed in both cell types, but was also observed in chromaffin cells in the normal fetal adrenal. The findings suggest autocrine or paracrine influences of IGF-2 in regulating the number and phenotype of cells derived from sympathoadrenal precursors in the developing adrenal medulla as well as in extra-adrenal paraganglia. These results have implications for the interpretation of data from neuroblastoma studies.     

Ly49 gene expression in different inbred mouse strains

Mouse natural killer cells express receptors for class IMHC in the form of the Ly49 family of proteins. The Ly49 family contains at least 13 expressed members (A, B, C, D, E, F, G, H, I, J, L, O, and P) and is further subdivided into activating and inhibitory subfamilies based on intracellular and transmembrane characteristics. The level of sequence identity between different members varies dramatically. However, comparison of the extracellular domain has revealed that several of the Ly49 molecules also form “pairs,” where one member is activating and the other is inhibitory. Until recently, most Ly49 molecules described have come from the C57B1/6 strain of inbred mice. Using molecular cloning and immunochemical analysis we have found that different mouse strains express novel Ly49 molecules. Comparison of the allelic forms of some Ly49 molecules has shown that the dividing line between different genes and different alleles is blurred. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-56000. Animal care was provided in accordance with the procedures outlined in A Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23, 1985). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.     

儿童臀肌挛缩症免疫发病机理的研究

本文对４１例儿童臀肌挛缩症患者外周血及３２例儿童臀肌挛缩症臀肌进行了系统的免疫学研究。结果表明：儿童臀肌挛缩症患者免疫调节功能紊乱，红细胞免疫功能低下，臀肌组织免疫复合物沉积，臀肌小血管数目减少及管壁损伤。由此认为免疫病理因素是儿童臀肌挛缩症的主要发病机理。     

Modulation of natural killing by tumor promoters: the regulatory influence of adherent cells varies with the type of target cell

We have analyzed the regulatory effects of two classes of tumor promoters, phorbol diesters and indole alkaloids, on human natural killer (NK) cell activity in vitro. In accordance with previous reports, we found that 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited natural killing against K 562 targets by unseparated mononuclear cells. Here, suppression of NK required the presence of adherent cells (macrophages). Contrary to the results obtained with K 562, tumor promoter-induced suppression of NK activity tested against U 937, another cell line of known NK susceptibility, was independent of the presence of adherent cells. Thus, NK cytotoxicity of effector cells rigorously depleted of adherent and Ia-positive cells was still inhibited when assayed against U 937, while it was generally enhanced when tested against K 562. Identical results were obtained with teleocidin and dihydroteleocidin B, two members of the recently discovered indole alkaloid class of tumor promoters. Therefore, we demonstrate that the regulatory effect of tumor promoters on human NK activity (suppression or stimulation) is determined not only by macrophages at the effector cell level but also by the type of target cell under study.     

Gene delivery to glioma cells in rat brain by grafting of a retrovirus packaging cell line

Retrovirus vectors only integrate into the genome of dividing cells and can thus be used to selectively infect tumor cells in the adult rat brain. Gene delivery was assessed by using the retrovirus BAG vector, which bears the Escherichia coli lacZ gene under the MoMLV LTR promoter-enhancer element, and by histochemical staining for bacterial beta-galactosidase activity. Direct injection of this vector (90-900 cfu) into the adult rat brain, with or without prior inoculation of C6 glioma cells (2 x 10(5) cells) resulted in labeling of only a few cells as assessed 1 week later. When the psi 2-BAG packaging line was grafted into the brain, labeled psi 2-BAG cells could be found after 1 day, but not after 5 days, following grafting, suggesting that the grafted cells had been rejected and that no endogenous cells had integrated released vector, or that expression of lacZ had been turned off. In contrast, when the psi 2-BAG packaging line was grafted into a brain region, which had been inoculated previously with rat C6 glioma cells (2 x 10(5) cells), beta-galactosidase labeling of these tumor cells, identified by immunocytochemistry for glial fibrillary acidic protein and S100, could be demonstrated 10 days later. Thus, grafting of retrovirus packaging lines into adult brain provides a mean to infect tumor cells in situ. The grafted packaging cells may continue to release retrovirus particles for an extended period, thus infecting more cells at the stage of division appropriate for viral integration, as compared to inoculation of the virus alone. Grafting of retrovirus packaging cell lines could be used to selectively deliver "killer" or "suppressor" genes to tumor cells in the brain to curtail their growth.     

Electrical coupling, without dye coupling, between mammalian astrocytes and oligodendrocytes in cell culture

Evidence of electrical and dye coupling between oligodendrocytes and astrocytes was sought in cultures of mouse spinal cord. Cell identity was verified using cell specific antigenic markers. In most experiments current was injected into oligodendrocytes while recording voltage in nearby astrocytes. Nine of 17 oligodendrocyte-astrocyte cell pairs showed weak electrical coupling; the average estimated coupling ratio was 0.03 +/- 0.06 (cf. 0.11 for oligodendrocyte-oligodendrocyte and 0.44 for astrocyte-astrocyte pairs; Kettenmann and Ransom: Glia, 1: 64-73, 1988). Application of 0.5 mM BaCl2 or 44.6 mM CsCl depolarized astrocytes and oligodendrocytes and was estimated to increase the coupling ratio between these cells 3-5-fold; these effects were rapid in onset and completely reversible. In 5 of 7 cases, oligodendrocyte-astrocyte pairs that appeared uncoupled in normal solution exhibited coupling during Ba++ or Cs+ exposure. The actions of these cations are believed to be mediated by blockade of glial K+ channels. Depolarization, per se, as induced by increasing [K+]o, did not increase coupling ratio. The fluorescent dye lucifer yellow (LY) was injected into 10 oligodendrocytes, 8 of which were electrically coupled to nearby astrocytes, and never passed into astrocytes in detectable quantities. Likewise, astrocytes injected with LY stained other astrocytes, but never oligodendrocytes. These findings document the presence of weak electrical coupling between astrocytes and oligodendrocytes, in the absence of dye coupling. Weak coupling of this sort could subserve metabolic interactions between these cells mediated by the passage of small but important molecules such as cyclic AMP, but would not allow strong electrical interactions. If such coupling among glial cells is widespread, it would constitute a "metabolic syncytium" that could serve to coordinate glial behavior.     

Plasticity of GABA- and glutamate-containing terminals in the mouse thalamic ventrobasal complex deprived of vibrissal afferents: an immunogold-electron microscopic study

GABA and glutamate immunogold staining demonstrated that nerve cells of the thalamic ventrobasal complex (VB) of mice were positive exclusively for glutamate. None of the neuronal perikarya reacted the GABA antibody. By using alternate thin sections of the normal VB, it was also shown that large "specific" somatosensory and small corticothalamic terminals, both of which contained spherical synaptic vesicles, exhibited only glutamate-like immunoreactivity. A third axonal type, containing flat-ovoid synaptic vesicles, stained only for GABA. Seventy-five days after coagulation of the vibrissal follicles in newborn mice, a characteristic multiplication of GABA positive axon terminals was observed. In addition, it was demonstrated that, similarly to modified cortical endings (Hámori et al., J. Comp. Neurol. 254:166-183, '86), many GABA positive terminals appeared as specific afferent endings, replacing the missing "specific" vibrissal afferents. This finding shows a remarkable plasticity of inhibitory GABA axons during developmental synaptogenesis and provides further evidence that the size, location, and the type of attachment of presynaptic terminals are dependent on their postsynaptic target.     

Detection of traumatic myocardial injury by means of simultaneous T1-201/Tc-99m pyrophosphate tomography--report of three cases

Trauma to the chest can result in cardiac damage, which maybe missed by clinical examination because of associated injuries.Routinely performed non-invasive tests may also be non-diagnostic.Tc-99m pyrophos-phate (PPi) tomography, in this study combinedwith T1-201, is a promising addition to non-invasive evaluation.In three patients with cardiac injury, this technique successfullydetected and localized myocardial necrosis.     

Experimental down-regulation of intermediate biomarkers of carcinogenesis in mouse mammary epithelial cells

Summary The polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) is a metabolism-dependent procarcinogen whose tumorigenicity is modified by dietary and endocrine manipulationsin vivo. DMBA initiates molecular and cellular alterations in the mammary tissue, while dietary components and estrogens affect the post-initiational phase of tumorigenic transformation. The mechanism(s) responsible for modulation of tumorigenic transformation remain unclear. This study examines the effects of selected tumor suppressing agents and estradiol (E₂) metabolites onin vitro DMBA carcinogenesis utilizing a newly established mouse mammary epithelial cell line C57/MG. Alteration in DNA repair synthesis, metabolism of E₂ via the C2- and C16-hydroxylation pathways, and acquisition of anchorage-independent growth were utilized as molecular, endocrine, and cellular biomarkers to quantitate the cellular transformation by DMBA and its modulation by tumor suppressing agents and E₂ metabolites. A single 24 hr exposure of 0.78 µM DMBA to C57/MG cells resulted in a 193.9% increase in DNA repair synthesis and a 73.1% decrease in C2/C16 hydroxylation of E₂. The DMBA treated C57/MG cells also exhibited increased anchorage-independencein vitro prior to tumorigenesisin vivo. A simultaneous treatment of cells with DMBA and with the highest non-cytotoxic doses of the tumor suppressing agents 5 µM N-(4-hydroxyphenyl) retinamide (HPR), 50 µM indole-3-carbinol (I3C), or 1 µM tamoxifen (TAM) resulted in a 35.6% to 63.9% decrease in DNA repair synthesis, a 23.8% to 1347.6% increase in C2/C16 hydroxylation of E₂, and a 53.8% to 72.4% decrease in anchorage-independent growth. The E₂ metabolites at the highest non-cytotoxic doses of 0.76 µM estrone (E₁), 0.69 µM 2-hydroxyestrone (2-OHE₁), and 0.66 µM 2-methoxyestrone (2-MeOHE₁) suppressed DMBA-induced DNA repair synthesis by 56.0% to 68.8%. These tumor suppressing agents and E₂ metabolites also effectively suppressed post-initiational, anchorage-independent growth by 24.9% to 72.4%. These results indicate that DMBA induces cellular transformation in part by causing DNA damage, altering C2/C16 hydroxylation in favor of C16-hydroxylation, and inducing anchorage-independent growth prior to tumor development. Effective downregulation of these genotoxic, endocrine and proliferative end points by prototypic tumor suppressing agents and by E₂ metabolites generated via the C2-hydroxylation pathway suggest that these agents may influence mammary tumorigenesis by inhibiting early occurring initiational and/or post initiational events.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - HPR N-(4-hydroxyphenyl) retinamide - I3C indole-3-carbinol - TAM tamoxifen - E₂ 17-estradiol - E₁ estrone - 2-OHE₁ 2-hydroxyestrone - 2-MeOHE₁ 2-methoxyestrone - 16-OHE₁ 16-hydroxyestrone - E₃ estriol - DME/F12 Dulbecco's modified Eagle's medium - F12 Ham's medium - HU hydroxyurea - PBS phosphate buffered saline - NaOH sodium hydroxide - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - [C2-³H] E₂ estradiol labeled at C2 position - [C16-³H] E₂ estradiol labeled at C16 position - ANOVA analysis of variance     

Serotonergic inhibition of the mossy fibre—granule cell glutamate transmission in rat cerebellar slices

Summary The glutamatergic mossy fibre granule cell pathway has been investigated in rat cerebellar slices. Exposure to 35 mM KCI, a concentration of K⁺ known to elicit Ca²⁺-dependent releases of excitatory amino acids from cerebellar slices, raised cGMP levels. The cGMP response was decreased in a concentration-dependent manner by D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5) and by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) indicating the involvement of ionotropic glutamate receptors of both the N-methyl-D-aspartate (NMDA) and the non-NMDA type. The K⁺-evoked production of cGMP was potently inhibited (EC₅₀ = 1.21 nM) by 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a selective 5-HT₂ receptor agonist. The effect of DOI (0.01 M) was antagonized by 0.03 M of the 5-HT₂ receptor antagonists ketanserin and methiothepin. At concentrations higher than 0.1 M, both antagonists increased on their own the cGMP response elicited by high-K⁺. This effect was insensitive to tetrodotoxin.It had been previously shown that rat mossy fibre endings release glutamate upon depolarization and that such release can be inhibited by activation of 5-HT₂ receptors sited on the mossy fibre endings. Altogether the available data suggest the following conclusions: (a) the glutamate/aspartate endogenously released in cerebellar slices during K⁺ depolarization increase cGMP synthesis through the activation of both NMDA and non-NMDA receptors; (b) a portion of the cGMP response can be prevented by 5-HT₂ receptor activation and may reflect the activity of the mossy fibre-granule cell pathway. Thus serotonin is likely to exert a potent inhibitory control of the excitatory mossy fibre input to the cerebellum by acting at receptors of the 5-HT₂ type. Correspondence to M. Raiteri at the above address