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Stumpf, Walter E |
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Degrees
Dr.med. 1952, Humboldt-University, Berlin
Ph.D. Pharmacology 1967, University of Chicago, Chicago
Dr.rer.biol.hum.h.c. 1987, University of Ulm, Ulm
Specialty in Neurology and Psychiatry 1957, Humboldt-University, Berlin
Awards
- Examination for Medical Doctor all grades A (summa cum laude), 1952
- Berlin Laboratories Fellowship, 1968-1969
- Robert-Feulgen-Lecture, Histochemische Gesellschaft, Gargellen, 1982
- Bissendorf-Lecture, European Anatomical Society, Antwerpen, 1987
- Pfizer Traveling Fellow, Clin. Res. Inst., Montreal, 1989
- Humboldt Senior Scientist Award, Ulm, 1989-1990
- Monbushou Visiting Professor, Shinshu University, Matsumoto, 1990
Biography
Studied medicine at the University of Leipzig (1946-1950) and Berlin (1950-1952). Medical residency at the Charité-Hospital, Department of Neurology and Psychiatry. in Berlin (1952-1957) - including four semesters at the Berlin Institut fuer Psychotherapie, and at the Department of Neurology and Psychiatry of the University of Marburg (1958-1961). Research in radiobiology and nuclear medicine at the Strahlenklinik of the University of Marburg (1961-1962).
In 1963 moved to the United States on a one-year research fellowship of the Deutsche Forschungsgemeinschaft in the Department of Pharmacology (with L.J. Roth) at the University of Chicago to develop a method for the localization of drugs in the brain. Extended research with completion of a Ph.D. in Pharmacology (1967). Continued at the University of Chicago as Assistant Professor in Pharmacology (1967-1970).
From 1970 to 1995 at the University of North Carolina Chapel Hill as Professor of Cell Biology and Pharmacology, with appointments in the Department of Anatomy (Cell Biology and Anatomy) and in the Department of Pharmacology. Member of the Laboratories for Reproductive Biology (with H.S. Bennett) and Neurobiology. Teaching in Histology and Neuroendocrinology. Research on sites and mechanisms of action of estrogens, androgens, adrenal steroids, thyroid hormone, others, with focus on brain, spinal cord, and female and male reproductive organs. Providing brain maps of target neurons and circuits for estradiol, progestin, androgen, corticosterone, aldosterone, ecdysteroid, vitamin D, 2-deoxyglucose. From the applications of receptor microautoradiography and related follow-up many discoveries and new concepts resulted.
Between 1992 to 1995 part-time leave from the university, working as in-house research advisor at Chugai Pharmaceutical Company in Tokyo, Japan, for the study of vitamin D analogues.
Since 1995 Professor Emeritus. From 2001 to 2003 invited Research Professor at the University of Sao Paulo, Brazil (with T. Zorn) for the study of estrogen receptors in the uterus during implantation and early pregnancy. In 2003 conducting a three-day LEICA-workshop on “Drug Localization in Tissues and Cells” at the University of Heidelberg.
Development of methods and applications
Localization of drugs and other diffusible compounds with dry-mount and thaw-mount autoradiography
For the in vivo cellular and subcellular localization of non-covalently bound drugs (diffusible compounds) no method existed. Development of a suitable histochemical method was considered very difficult (C.P. Leblond), even impossible (H. Levi). Adequate preservation of tissue structure and simulataneous retention of diffusible constituents and drugs posed seemingly insurmountable problems.
As a clinical neurologist and psychiatrist, being confronted with severe side effects of newly introduced psychotropic drugs, I recognized the need for better understanding sites and related mechanisms of action. I abandoned work at the bedside in order to focus on research of drug receptor binding and deposition.
In association with Lloyd Roth at the University of Chicago, during a three-year period, I developed two approaches: dry-mount and thaw-mount autoradiography that excluded any fluid phase during tissue preparation, preserving tissue structure for cellular resolution, and retaining tissue constituents at original in vivo sites. Several technical innovations and breakthroughs provided the foundation, such as, simultaneous freeze-mounting on tissue holders, avoiding disruptive ice crystal formation and translocation, cutting thin frozen sections at low temperatures previously considered impossible. Common multi-step dehydration-infiltration with liquid fixation and embedding, considered necessary by histologists, were eliminated. A brief single-step staining replaced the common multistep H&E staining. Tissue treatment was kept minimal with the goal to study ‘unmolested tissue' in order to gain authentic in vivo information.
It was emphasized that recommendation of a method for the localization (imaging) of drugs should be preceded by tests with two diffusible compounds known localized. Pictorial evidence for sensitivity, resolution, and authenticity of data - avoidance of artifacts - must be provided as validation before publication of any application for a new compound. Such postulate, a conditio sine qua non and valid to date, was based on the abundance of misleading data with unrecognized false positives and false negatives published in the literature.
Discoveries related to development of methods
Freezing of tissue
Development of procedure to prevent or minimize ice crystal disruption of tissue structure, considering tissue size, temperature and speed of freezing in experiments with liquified propane, isopentane, liquid nitrogen, or dry ice. For details see monograph (Stumpf, Drug Localization in Tissues and Cells).
Frozen sectioning at low and ultra-low temperatures
First demonstration of feasibility of frozen sectioning at temperatures below-45° C, as low as -105° C. Recognition of relationship between temperature and section thickness (Nature 1965), providing a basis for the development of ultra-cryotomy (acknowledged by U. Fernandez-Moran). Frozen sectioning below -45° C has been previously considered impossible (A.G.E. Pearse, 1963, 1968).
Freeze drying of frozen sections
First demonstration of freeze-dried frozen sections suitable for microscopy. Development of conditions for freezing and freeze-drying (Stumpf and Roth 1966). Design of a portable Cryosorption-Pump. Freeze-dried sections have been used for biochemical studies (O. Lowry) and whole body autoradiography (S.Ullberg), however, without preserving microscopic structure.
Dry-mounting on microscopic slides of freeze-dried frozen section – without exposure to fluids or moisture – was considered essential in order to exclude loss or translocation of any tissue constituents. Dry-mounted freeze-dried frozen sections served as a basis for authentic (representative in vivo) microscopic localization of drugs and as a control for less pristine.approaches. Diffusible compounds known localized served as test substances, including estradiol in the uterus and mesobilirubinogen in the liver (Stumpf and Roth, 1964-1967). In addition, the “extracellular space indicator” inulin was tested and found not exclusively restricted to extracellular space.
Thaw-mounting of frozen sections
Results obtained with freeze-dried sections were compared with those from thaw-mounted sections and found similar if sections 4 micrometer thin or less used, thus possible artifacts due to interaction of tissue components with nuclear emulsion avoided or minimized. Large scale use of the thaw-mount procedure during several decades established its utility as a standard approach designated as Receptor Microscopic Autoradiography. Cutting and thaw-mounting of thin frozen sections are delicate steps that require skill and experience. Attention to detail is required in the execution of all steps of any method of high sensitivity and resolution.
Receptor microautoradiography can be combined with other histochemical techniques. For instance: combination of high resolution autoradiography with fluorescence microscopy (e.g., 3H-estradiol and catecholamines) or immunocytochemistry (e.g., 3H-vitamin D and antibodies to receptor protein; thyrotropin, insulin) for characterization of receptor binding sites.
Receptor Microscopic Autoradiography presently is the method of choice for the localization (high-resolution imaging) of drugs at the cellular-subcellular and tissue-organ level. Discoveries and changes of paradigm resulted from information gained that is difficult or impossible to obtain otherwise. Important leads for new drug development have been provided.
Because of time and effort required, some consider such methods non-expedient. However, evidence indicates, results with high-resolution histochemical methods may turn out highly informative, essential, time and money saving, compared to quick “expedient” procedures alone that often turn out as costly failures.
Discoveries and new concepts
With receptor microscopic autoradiography – as the method of thaw-mount autoradiography with 4 micrometer sections has been named – many discoveries were made with radiolabeled steroid hormones, drugs, and metabolic indicators. New targets were identified, characterized by their topographic location and through combined autoradiography-immunocytochemistry with radiolabeled compounds and antibodies to cellular products. Detailed target maps of brain, spinal cord and other organs provided. Discoveries gave rise to new concepts, e.g.:
- Estrogen targets throughout the female and male body in specific locations (e.g., heart atrium, testis Leydig cells, ovarian granulosa and theca cells, hair dermal papillae). Some experts initial comments: ‘it has no meaning, it's everywhere, it is only localization.'
- First demonstration of estrogen, androgen, progestagen, corticosterone and aldosterone receptor neurons in regions of brain and spinal cord.
- Identification of estrogen-neuron circuits, for instance, stria terminalis (Science, 1968).
- Concepts of steroid hormone target ABC-Circuits (Allocortex-Brain-Core) and MAHS (Multiple Activation of Heterogeneous Systems) for endocrine-autonomic regulation of aminergic-peptidergic neurons, extending the ‘Limbic System' concept. Critique of the anatomical definition of the Limbic System and the concept of the Hypothalamic Hypophyseotropic Area.
- Quantitative assessment of differential receptor binding (hierarchies) in pituitary and other target cell populations.
- Discovery of vitamin D targets in brain, pituitary, skin, heart, adrenal medulla, etc, together in over 50 tissues (Science, 1979; Histochem.Cell Biol., 1995), not only in the ‘classical target organs' intestine, kidney, bone and parathyroid.
- Identification and mapping of brain and spinal cord: vitamin D neuronal circuits in contrast to a blood-brain barrier with biochemical assays and whole body autoradiography.
- New paradigm of the main biological role of vitamin D: Multifunctional seasonal adaptation and regulation of growth, cell proliferation and differentiation, reproduction, endocrine and exocrine secretions, immune and stress responses, cardio-vascular and gastro-intestinal functions. Regulation of calcium-homeostasis is part of 1,25 (OH)2 vitamin D3-soltriol) functions, especial related to bone growth and development.
- Thyroid hormone localization in brain regions and pituitary.
- Identification of retinoic acid target cell populations in brain and other tissues.
- Co-localization of progestin receptor and hormone, demonstrating hormone-independent nuclear presence of receptor (with J.-M. Gasc).
- Localization of steroid hormone binding sites in early chick embryos (with J.-M. Gasc).
- First demonstration and mapping of ecdysteroid binding sites in insect tissues and brain (with H-J. Bidmon).
- Identification of 2-deoxyglucose high activity neurons in medulla oblongata (with G. Duncan).
- Drug Homunculus for profiling (finger-printing, “body-printing”) of drug sites of receptor binding (target identification), deposition, and related function.
- Use of high resolution autoradiography and related imaging can be facilitated if detection of cellular-subcellular radiation can be achieved through digital recording. However, to-date sensitivity and resolution in a topographic context (!), uniquely demonstrated through nuclear emulsion recording, have not been matched. The image is the evidence!
Applications that require high resolution and high sensitivity
Receptor Microautoradiography currently is the general method of choice, providing high sensitivity and high resolution with cellular-subcellular information simultaneously with tissue and organ overviews.
- Measurement of Target Bioavailability and Target Pharmacokinetics,time and dose comparisons
- Low dose versus high dose quantitative target distribution (receptor binding)
- Simultaneous imaging of cellular and organ distribution (facilitating comprehension of molecular and systems pharmacology)
- Prediction of Action, Side Effects, and Toxicity,
- Validation of In Vitro and High-Throughput Procedures
- Comparison with and validation of ‘expedient’ low resolution imaging methods
- Knock-out animals, assessment of knock-out effects
- Construction of Drug-Target Homunculus from qualitative-quantitative microautoradiographic data enables functional finger-printing of a specific drug, facilitates comparisons among drugs and analogs, and provides convenient surveys of sites of action for pharmaceutical companies, regulatory agencies, clinicians, and even patients.
- Assessment of low-dose versus high-dose sites and related differential (enantiodromic) mechanisms of action, and for the determination of enantiodromic thresholds (see article: The Dose Makes the Medicine, 2006).
Regulatory agencies and pharmaceutical companies are challenged to seek information on target identification and target pharmacokinetics for the approval of new drugs. Such information is essential for the understanding of mechanisms of action (see article: Memo to the FDA and ICH, 2007).
Reviews by others
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Roth LJ and Stumpf WE, eds. AUTORADIOGRAPHY OF DIFFUSIBLE SUBSTANCES. 371 pp, Academic Press, New York, 1969.
Stumpf WE and Grant LD, eds. ANATOMICAL NEUROENDOCRINOLOG,
472 pp, S. Karger, Basel, 1975.
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Stumpf WE and Solomon H, eds. AUTORADIOGRAPHY AND CORRELATIVE IMAGING. Academic Press, San Diego, 1995.
Stumpf WE. DRUG
LOCALIZATION IN TISSUES AND CELLS. IDDC Press, Chapel Hill, NC,
2003.
300 + (Medline recorded), 550 + (total)
Selected Articles:
StumpfWE. “Vitamin D: Beyond Bone.” Address to Conference. New York Academy of Science, September 21, 2012.
StumpfWE. Vitamin D and the scientific calcium dogma: understanding the ‘Panacea’ of the sun. Editorial. Europ. J. Clinic. Nutr., 2012; 66: 1080-1081.
StumpfWE. Vitamin D in Relations to the Dao – Life, Death, and Return. Metaphysics of the one- and two- phasic actions toward maintenance of life (Yang Sheng) and Cyclicity (Huan[Yuan]-Tao[Dao] ‘Go-around’ des Lueshih chunqiu (Spring and Autumn Analects).
StumpfWE. Vitamin D Beziehungen zur Philosophie des Dao – Leben, Tod und Wiederkehr. Zur Metaphysik der ein- und zweiphasischen Aktionen zur Erhaltung des Lebens (Yang Sheng) und Zyklizitaet (Huan[Yuan] –Tao[Dao] ‘Go-around’ [circle] des Lueshi chunqiu (Fruehling und Herbst Analekte).
StumpfWE. Drugs in the brain – cellular imaging with receptor microscopic autoradiography. Progress in Histochemistry and Cytochemistry 47 (2012) 1–26.
Stumpf WE. In vivo target recognition with high-resolution imaging: significance for drug development. Eur J Drug Metab Pharmacokinet. 2010 Sep;35(1-2):15-22.
Stumpf WE. Vitamin D and the digestive system. European Journal of Drug Metabolism and Pharmacokinetics 2008; Vol. 33, No. 2, 85–100.
Vitamin D Digestive System Schematic
Stumpf WE., Hayakawa, N., Bidmon, H-J. Skin research and drug localization with receptor
microscopic autoradiography. Experimental Dermatology 2008; 17: 133–138.
Stumpf WE., Hayakawa, N. Salivary glands epithelial and myoepithelial cells are major vitamin D targets. European Journal of Drug Metabolism and Pharmacokinetics 2007, Vol. 32, No. 3, pp. 123-129.
Stumpf WE. Memo to the FDA and ICH: appeal for in vivo drug target identification and target pharmacokinetics Recommendations for improved procedures and requirements. Drug Discov Today. 2007 Aug;12(15-16):594-8.
Stumpf WE. Editorial. The main role of vitamin D: seasonal regulation of vital functions. High-resolution target recognition leads to a new paradigm and advanced drug development. Eur J Drug Metab Pharmacokinet. 2007 Jan-Mar;32(1):1-6.
Stumpf WE. The dose makes the medicine. Drug Discovery Today. 2006 June; 11(11-12):550-5.
Stumpf WE. Drug
localization and targeting with receptor microscopic autoradiography.
J Pharmacol Toxicol Methods. 2005 Jan-Feb;51(1):25-40.
Hayakawa N, Kubota N, Imai N, Stumpf WE. Receptor microscopic autoradiography for the study of percutaneous absorption, in vivo skin penetration, and cellular-intercellular deposition. J Pharmacol Toxicol Methods. 2004 Sep-Oct;50(2):131-7.
Stumpf WE. Corpora
non agunt nisi in loco. Interactions between things do not "take place"
unless at "proper locus".
Drug Metab Dispos. 1996 May;24(5):507-8.
Stumpf WE. Vitamin
D sites and mechanisms of action: a histochemical perspective.
Reflections on the utility of autoradiography and cytopharmacology for
drug targeting. Histochem Cell Biol. 1995 Dec;104(6):417-27.
Birmingham MK, Sar M, Stumpf WE. Dexamethasone
target sites in the central nervous system and their potential
relevance to mental illness.
Cell Mol Neurobiol. 1993 Aug;13(4):373-86.
Stumpf WE, Bidmon HJ, Li L, Pilgrim C, Bartke A, Mayerhofer A, Heiss C. Nuclear receptor sites for vitamin D-soltriol in midbrain and hindbrain of Siberian hamster (Phodopus sungorus) assessed by autoradiography. Histochemistry 1992 98:155-164.
Stumpf WE, Bidmon HJ, Murakami R. Retinoic acid binding sites in adult brain, pituitary, and retinaNaturwissenschaften. 1991 Dec;78(12):561-2.
Stumpf WE, Privette TH. The steroid hormone of sunlight soltriol (vitamin D) as a seasonal regulator of biological activities and photoperiodic rhythms. J Steroid Biochem Molec Biol 1991 39(2):283-289.
Bidmon HJ, Gutkowska J, Murakami R, Stumpf WE. Vitamin D receptors in heart: effects on atrial natriuretic factor. Experientia. 1991 Sep 15;47(9):958-62.
Duncan GE, Kaldas RG, Mitra KE, Breese GR, Stumpf WE. High activity neurons in the reticular formation of the medulla oblongata: a high-resolution autoradiographic 2-deoxyglucose study. Neuroscience. 1990;35(3):593-600.
Stumpf WE, Denny ME.
Vitamin
D (soltriol), light, and reproduction.
Am J Obstet Gynecol. 1989 Nov;161(5):1375-84.
Stumpf WE, Privette TH. Light, vitamin D and psychiatry. Role of 1,25 dihydroxyvitamin D3 (soltriol) in etiology and therapy of seasonal affective disorder and other mental processes. Psychopharmacology (Berl). 1989;97(3):285-94.
Stumpf WE. Vitamin D-Soltriol. The heliogenic steroid hormone: somatotrophic activator and modulator. Discoveries from histochemical studies lead to new concepts. 89:209-219, 1988
Shrughrue PJ, Stumpf WE and Sar M. The distribution of progesterone receptor in the 20-day old fetal mouse: An autoradiographic study with [125I] progestin. Endocrinol. 123:2382-2389, 1988.
Stumpf WE and O'Brien LP. 1,25 (OH)2 Vitamin D3 sites of action in the brain: an autoradiographic study. Histochem.87:393-406, 1987.
Stumpf WE, Clark, SA, O'Brien, LP and Reid, FA. 1,25 (OH)2 vitamin D3
sites of action in spinal cord and sensory ganglion. Anat. Embriol.
177:307-310, 1988.
Stumpf WE, Jennes, L. The A-B-C (Allocortex-Brainstem-Core) Circuitry of Endocrine-Autonomic Integration and Regulation. Peptides. Vol. 5, Suppl 1, pp 221-226, 1984.
Stumpf WE, Jennes, L. The A-B-C (Allocortex-Brainstem-Core) Circuitry - schematic
Stumpf WE, Sar M, Narbaitz R, Huang S, DeLuca HF. Autoradiographic localization of 1,25-dihydroxyvitamin D3 in rat placenta and yolk sac. Horm Res. 1983;18(4):215-20.
Heritage AS, Stumpf WE, Sar M, Grant LD. Brainstem catecholamine neurons are target sites for sex steroid hormones. Science. 1980 Mar 21;207(4437):1377-9.
Stumpf WE. "Peer"
review.
Science. 1980 Feb 22;207(4433):822-3.
Stumpf WE, Sar M, Reid FA, Tanaka Y, DeLuca HF. Target cells for 1,25-dihydroxyvitamin D3 in intestinal tract, stomach, kidney, skin, pituitary, and parathyroid. Science. 1979 Dec 7;206(4423):1188-90.
Sar, M and Stumpf, WE. Neurons of the hypothalamus concentrate3H
progesterone or metabolites of it. Science. 182:1266 1268, 1973.
Examples of autoradiograms prepared by Receptor Microscopic Autoradiography after injection of tritium-labeled vitamin D. The same preparation yields low-resolution surveys and high-resolution cellular-subcellular detail with quantitative differences of uptake and retention of radiolabeled compound. Among the over fifty target tissues identified and characterized by autoradiography a hierarchy of uptake and retention is recognizable. (For details see Stumpf: Drug Localization in Tissues and Cells, 2003; Drug localization and targeting with receptor microscopic autoradiography, 2005.)
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Brain amygdala central nucleus (upper picture) and spinal cord
lamina IX (lower picture) with labeled target neurons. Note the
differential nuclear concentration of radio-labeled compound. Most but
not all motor neurons are labeled.
Pituitary target cell population survey (upper picture). Colocalization with TSH-antibodies (brown cytoplasm in lower picture) characterizes many of the heavily labeled target cells as thyrotropes.
Duodenum with strong nuclear concentration of radiolabeled compound in absorptive epithelial cells – but not in Goblet cells (upper picture). In the low-magnification survey (lower picture) labeling exists in the nuclear regions of epithelium of intestinal villi and crypts but not in the muscularis. High level of radioactivity in the intestinal lumen - probably mostly liver-bile derived metabolites - with barrier between luminal content and villi.
Adrenal with radioactive labeling in medullary cells and blood
vessels, recognizable at low magnification (upper picture). At high
magnification (lower picture) nuclear concentration in medullary cells
can be clearly identified. Also relatively high radioactivity in
capillary sinusoids. In cortex zona reticularis (dark cells at left) no
nuclear concentration of labeled compound exists under the conditions of the
experiment.
Target identification derived from the application of receptor microautoradiography allows composition of a “drug homunculus” for drug-specific finger-printing, overviews, links for functional detail, facilitating functional and clinical follow-up, prediction of actions, side-effects, and toxicity.
The results thus obtained and insights gained suggest a change of concept. Although systemic calcium homeostasis is an important function of vitamin D, calcium binding proteins cannot be viewed as general guide to vitamin D sites and mechanisms of actions – as indicated in our comparative studies. Our data suggest:
The Main Biological Role of Vitamin D is Seasonal Adjustment of Vital Functions.
These include regulation of growth, reproduction, survival stress response; endocrine and exocrine secretion, cell proliferation, cognition and mood; neuro-motor, neuro-endocrine, and neuro-sensory functions, immune response, cardio-vascular and gastro-intestinal functions, regulation of calcium and other mineral levels, cell proliferation and protein synthesis-differentiation, others.
Comparative data with different steroid hormones and target-overlap further suggest interactive (cooperative and/or antagonistic) regulation of DNA by steroid messengers (Stumpf, 1995):
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Gonadal Steroids |
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Adrenal Steroids |
From the microautoradiographic target recognition and related actions it follows that vitamin D has healing potential for prevention and treatment of various deficiencies and ailments, including old age: a PANACEA? If there is any compound that deserves being designated a panacea, the multifunctional heliogenic vitamin D appears a suitable candidate.
Philosophical consideration: “Vitamin D”, the term does not reflect its significance. I have used instead SOLTRIOL in several publications as a more appropriate designation. – Is there not a link to HERACLITUS' emanation of “ ever-living fire ”? The cosmic solar fire as the sustaining life force, providing wave length energies for Temperature , Visible Light , and Ultraviolet B (Soltriol).
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Contact: stumpfwe@email.unc.edu
Fec. April 2005
Updated November 2012