P P Liberski

P Brown

Kuru: its ramifications after fifty years

Keywords:

Kuru was the first human neurodegenerative disease in the group of transmissible spongiform encephalopathies, prion diseases or, in the past, slow unconventional virus diseases. It was reported to Western medicine in 1957 by Gajdusek and Zigas. Kuru was spread by endocannibalism and because of this the ratio of affected women and children to men was excessive. The hallmark of kuru neuropathology is the amyloid plaque. We may speculate what would happen if kuru had not been discovered or did not exist. The infectious nature of Creutzfeldt-Jakob disease (CJD) would probably not have been suspected until the beginning of the variant CJD (vCJD) outbreak in the UK. Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker disease would have remained for decades as obscure neurodegenerations of merely academic interest. The familial forms of CJD would not have benefited from PRNP gene (a gene encoding for prion protein) analysis, but only later would have been studied by linkage analysis and reverse genetics probably. The study of kuru would have probably been of minimal interest to veterinarians and anthropologists until the BSE epidemic began to exert its devastating effect. The discovery of vCJD would have been delayed, as no surveillance would have been initiated for CJD. And perhaps most importantly, the realization of 'protein-misfolding diseases', including not only the neurodegenerative but also an increasing number of non-neurological disorders, would have been delayed by decades.

Kuru [START_REF] Gajdusek | Experimental transmission of a kuru-like syndrome to chimpanzees[END_REF], the first human neurodegenerative disease classified as a transmissible spongiform encephalopathy (TSE), prion disease or, in the past, slow unconventional virus disease (Gajdusek, 1977a(Gajdusek, , 1977b[START_REF] Gajdusek | Observations on the early history of kuru investigation[END_REF] was first reported to Western medicine in 1957 by Gajdusek (Fig. 1) and Zigas [START_REF] Gajdusek | Degenerative disease of the central nervous system in New Guinea. The endemic occurrence of "kuru" in the native population[END_REF][START_REF] Hadlow | Scrapie and kuru[END_REF][START_REF] Gajdusek | Studies of kuru. I. The ethnologic setting of kuru[END_REF]. A complete bibliography of kuru through 1975 has been published by [START_REF] Alpers | Bibliography of kuru[END_REF].

The solution of the kuru riddle opened a novel field of biomedical sciences and initiated more than a quarter of century of research that has already resulted in two Nobel prizes (to Gajdusek in 1976 andto Prusiner in 1997) and was linked to a third (Wüthrich who determined the structure of the prion protein in 2002). Kuru research has impacted the concepts of nucleation-polymerization and "conformational disorders" [START_REF] Gajdusek | Transmissible and nontransmissible dementias: distinction between primary cause and pathogenetic mechanisms in Alzheimer's disease and aging[END_REF].

"Kuru" in the Fore language on Papua New Guinea (Fig. 2 -3) means to shiver from fever or cold [START_REF] Alpers | Kuru: a clinical study[END_REF][START_REF] Gajdusek | Observations on the early history of kuru investigation[END_REF][START_REF] Gajdusek | Kuru. Early letters and field-notes from the collection of D. Carleton Gajdusek[END_REF]. The Fore used the nominative form of the verb to describe the fatal disease which decimated their children and adult women, but rarely men [START_REF] Alpers | Epidemiology and ecology of kuru[END_REF][START_REF] Alpers | Changing pattern of kuru: epidemiological changes in the period of increasing contact of the Fore people with western civilization[END_REF]Gajdusek et al., 1977aGajdusek et al., , 1977b;;[START_REF] Klitzman | The natural incubation period of kuru and the episodes of transmission in three clusters of patients[END_REF][START_REF] Zigas | Clinical, pathological and epidemiological study of a recently discovered acute progressive degenerative disease of the central nervous system reaching "epidemic" proportions among natives of the Eastern Highlands of New Guinea[END_REF]. It was and still is restricted to natives of the Foré linguistic group at Papua New Guinea's Eastern Highlands and neighboring linguistic groups that exchange women with Fore people (Auiana, Awa, Usurufa, Kanite, Keiagana, Iate, Kamano, Kimi; Fig. 2). Neighboring groups into which kuru-affected peoples did not settle through marriage or adoption, such as the Anga (Kukukuku), and remote Iagaria, Kamano and Auiana people, were not affected. It seems that Kuru first appeared at or shortly after the turn of 20 th century [START_REF] Glasse | Fieldwork in the South Fore: the process of ethnographic inquiry[END_REF] in Uwami village of Keiagana people and spread from there to the Awande in the North Fore where the Uwami had social contacts. Within 20 years it had spread further into the Kasokana (Lindebaum 1979) and Miarasa villages of North Fore, and a decade later had reached the South Fore at the Wanikanto and Kamira villages. Kuru became endemic in all villages which it entered and became hyperendemic in the South Fore region. All native informants stressed the relatively recent origin of kuru [START_REF] Glasse | Fieldwork in the South Fore: the process of ethnographic inquiry[END_REF]. Interestingly, when kuru first appeared, it was considered poetically by Foré as similar to "the swaying of casuarinas tree" and kuru was labeled cassowary disease to stress the similarity between cassowary quills and "waving casuarinas fronds" [START_REF] Glasse | Fieldwork in the South Fore: the process of ethnographic inquiry[END_REF]).

Gajdusek's introduction to kuru was via Scragg from a report sent by Zigas to Gunter in 1956 [START_REF] Gajdusek | Kuru. Early letters and field-notes from the collection of D. Carleton Gajdusek[END_REF]. Gajdusek then joined Zigas in 1957 in Papua New Guinea.

Cannibalism

Ritualistic endocannibalism (eating of relatives as part of a mourning ritual) was practiced not only in the kuru area but in many surrounding Eastern Highland groups in which kuru never developed [START_REF] Alpers | Kuru: implications of its transmissibility for the interpretation of its changing epidemiologic pattern[END_REF][START_REF] Lindenbaum | Kuru sorcery. Disease and danger in the New Guinea Highlands[END_REF][START_REF] Glasse | Fieldwork in the South Fore: the process of ethnographic inquiry[END_REF]. The Fore learnt the practice from Keiagana and Kamano [START_REF] Gajdusek | Kuru and scrapie[END_REF]. This type of cannibalism is designated in the Western world as "ritualistic" but, according to Lindebaum (1979), such a label is misleading "When a body was considered for human consumption, none of it was discarded except the bitter gall bladder. In the deceased's old sugarcane garden, maternal kin dismembered the corpse with a bamboo knife and stone axe. They first removed hands and feet, then cut open the arms and legs to strip the muscles. Opening the chest and belly, they avoided rupturing the gall bladder, whose bitter content would ruin the meat. After severing the head, they fractured the skull to remove the brain. Meat, viscera, and brain were all eaten. Marrow was sucked from cracked bones, and sometimes the pulverized bones themselves were cooked and eaten with green vegetables. In North Fore but not in the South, the corpse was buried for several days, then exhumed and eaten when the flesh had "ripened" and the maggots could be cooked as a separate delicacy".

The first European who observed kuru was Ubank in 1936 [START_REF] Lindenbaum | Kuru sorcery. Disease and danger in the New Guinea Highlands[END_REF]. In the late 1930's and 1940's, many miners, missionaries, and government officials made contacts with the northern periphery of the kuru region, and became thoroughly familiar with the ritual endocannibalism of Eastern Highland tribes. In the early 1950's, kuru was observed by Berndt and Berndt [START_REF] Gajdusek | Observations on the early history of kuru investigation[END_REF], and the first mention of kuru (skinguria in Pidgin) was included in reports of patrol officers in 1953. Gajdusek in response to when he first linked cannibalism to kuru replied "anyone would come to the conclusion that a disease endemic among cannibals must be spread through eating corpses". The hypothesis of cannibalism was thus not widely discussed because it was taken for granted, not because of any lack of insight [START_REF] Gajdusek | Observations on the early history of kuru investigation[END_REF], and it was proved in 1965 by the transmission of kuru to chimpanzees (Gajdusek andGibbs, 1964, 1966;[START_REF] Beck | Kuru and Creutzfeldt-Jakob disease: neuropathological lesions and their significance[END_REF][START_REF] Hainfellner | Pathology and immunohistochemistry of a kuru brain[END_REF] (Fig. 5).

In subsequent years, the number of kuru cases has steadily declined, with the youngest patients becoming progressively older, and the disease is now virtually extinct.

Among the Foré, kuru was believed to result from sorcery [START_REF] Lindenbaum | Kuru sorcery. Disease and danger in the New Guinea Highlands[END_REF]. To cause kuru, a would-be -sorcerer would need to obtain a part of the victim's body (nail clippings, hair) or excreta, particularly feces-or urine-soaked vegetation, saliva, blood, or partially consumed food such as sweet potato peelings, or clothing. These were packed with leaves and made into a "kuru bundle" and placed partially submerged into one of numerous in the Fore regions swamps. Subsequently, the sorcerer shook the package daily until the sympathetic kuru tremor was induced in his victim. As a result, kinsmen of a kuru victim attempted to identify and subsequently kill a suspected sorcerer if they could not bribe or intimidate him to release a victim from the kuru spell.

Divination rituals helped to identify a sorcerer. One method was to collect water for the kuru victim from different sources; if one "induced" vomiting, it was considered to be near the sorcerer's residence. Another method was to place hair clippings from a kuru victim into a bamboo cylinder, and a freshly killed possum in another cylinder. Calling the name of a suspected sorcerer while shaking the cylinders, a member of the victim's family placed the possum-containing cylinder into a fire. The sorcerer was identified if the liver of the possum, believed to be the residence of his soul, remained uncooked. Still another rite was the roasting of small rats, each in a separate bamboo cylinder, each one having been given the name of a hamlet or village in which the suspected sorcerer lived. Careful inspection of the rat's viscera helped to identify the sorcerer.

Killing of a sorcerer -tukabu -was a ritualistic form of vendetta; it included crushing with stones the bones of the neck, arm, and thigh, as well as the loins, biting the trachea, and grinding the genitalia with stones and clubs. As sorcerers were mostly adult men, whereas kuru victims were mostly women and children of both sexes, killing of male sorcerers contributed somewhat to maintaining the sex ratio in a population devastated by the kuru deaths of their women.

Kuru etiology

Although on epidemiological grounds the etiology of kuru was thought to be infectious, patients had no obvious signs of infection: no meningoencephalitic signs or symptoms (fever, confusion, convulsions, or coma), no cerebrospinal fluid pleocytosis or elevated protein level and, on autopsy, no perivascular cuffings or other signs of inflammatory brain pathology. Moreover, all attempts to transmit kuru to small laboratory animals or to isolate a bacterium, leptospirum, fungus, rickettsia, or virus using tissue cultures or embryonated hen's eggs were unsuccessful. In other wide ranging investigations, neither exhaustive genetic analyses nor the search for nutritional deficiencies or environmental toxins resulted in a tenable hypothesis.

In 1959 Gajdusek was informed by Hadlow [START_REF] Hadlow | Scrapie and kuru[END_REF] of theanalogies between kuru and scrapie, a slow neurodegenerative disease of sheep and goats known to be endemic in the United Kingdom since the 18 th century and experimentally transmitted in 1936.

Hadlow, having observed kuru plaques reported his findings (Haldow, 1959). In response to his insight, Gajdusek replied that "we are pursuing the matter of possible infectious etiology extensively", and took up Hadlow's recommendation to hold small laboratory rodents and (especially) monkeys for longer periods of observation than had been done previously. He also renewed attempts to obtain optimal tissue for inoculation from rapidly autopsied kuru [START_REF] Gajdusek | Kuru and scrapie[END_REF]. Gajdusek also concluded that "We too believe that toxic and/or infectious factors will still prove to be important in the disease but as yet we are operating largely on suspicion".

In 1961, Gajdusek presented "Kuru: an appraisal of five years of investigation. With a discussion of the still undiscardable possibility of infectious agent", [START_REF] Gajdusek | Kuru: an appraisal of five years of investigation[END_REF] in which he said: "In spite of all the genetic evidence, both the pathological picture and the epidemiological peculiarities of the disease persistently suggest that some yet-overlooked, chronic, slowly progressive, microbial infection may be involved in kuru pathogenesis.

Similar suspicion prevails in our current etiological thinking about a number of less exotic and less rare chronic, progressive degenerative diseases of the central nervous system in man. Thus, […], amyotrophic lateral sclerosis, Schilder disease,leukoencephalitis, Koshevnikoff's epilepsy syndrome in the Soviet Union, the Jakob-Creutzfeldt syndromes, acute and chronic cerebellitis, and even many forms of Parkinsonism, especially the Parkinsonism dementia encountered among the Chamorro population in Guam, continue to suggest the possibility that in man there may be infections analogous to the slow infections of the nervous system of animals which were intensively studied by Bjorn Sigurdsson, the Icelandic investigator who formulated the concept of "slow virus infections" ". This comments preceded the discovery of kuru transmissibility by more than 4 years [START_REF] Gajdusek | Experimental transmission of a kuru-like syndrome to chimpanzees[END_REF][START_REF] Gibbs | Spongiform encephalopathies -slow, latent, and temperate virus infections -in retrospect[END_REF]. Furthermore, many of the diseases mentioned by Gajdusek are now grouped together under the umbrella of "protein conformational disorders".

Clinical manifestations

Kuru is an invariably fatal cerebellar ataxia accompanied by tremor, choreiform and athetoid movements (Fig. 6) [START_REF] Alpers | Kuru: a clinical study[END_REF][START_REF] Hornabrook | Kuru and clinical neurology[END_REF]. In contrast to the neuropathological picture (Klatzo and Gajdusek, 1959;[START_REF] Scrimgeour | A clinico-pathological study of case of kuru[END_REF][START_REF] Hainfellner | Pathology and immunohistochemistry of a kuru brain[END_REF], it is remarkably uniform in clinical signs, symptoms and evolution. The progressive dementia that is so characteristic of sporadic CJD, is barely noticeable in patients with kuru, and then only late in the course of the illness. This absence of cognitive signs and symptoms is very similar to the clinical picture of CJD following peripheral inoculation with contaminated human growth hormone. However, kuru patients often displayed emotional changes, including inappropriate euphoria and compulsive laughter (the journalistic "laughing death" or "laughing disease"), or apprehension and depression. Kuru is divided into clinical three stages: ambulant, sedentary and terminal (the Pidgin expressions, wokabaut yet; i.e. "is still walking", sindaun pinis; i.e. "is able only to sit" and slip pinis; i.e. "is unable to sit up") [START_REF] Alpers | Kuru: a clinical study[END_REF]. There is an ill defined prodromal period characterized by the presence of headache and limb pains, often in the joints, abdominal pains and loss of weight. Fever and other signs of infectious disease are never seen. The prodromal period is followed by the "ambulant stage", the end of which is defined when the patient is unable to walk without a stick. This period is characterized by the onset of subtle signs of gait unsteadiness that are usually only recognized by the patient, but which over a period of a month or so progress to severe astasia and ataxia. A fine 'shivering' truncal tremor, amplified by cold, is often followed by titubation and other types of abnormal movements. Attempts to maintain balance result in clawing of the toes and curling of the feet. A horizontal convergent strabismus may be present. Plantar reflex is always flexor while clonus, in particular ankle clonus but also patellar clonus, are hallmarks of the clinical picture. The second "sedentary" stage begins when the patient is unable to walk without constant support and ends when he or she is unable o sit without it. Postural instability, severe ataxia, tremor and dysarthria progress endlessly through this stage. Deep reflexes may be increased but the plantar reflex is still flexor. In the terminal stage, the patient is bedridden and incontinent, with dysphasia and primative reflexes, and eventually succumbs in a state of advanced starvation.

Neuropathology

The first systematic examination of kuru neuropathology (12 cases) was published by Klatzo and Gajdusek in 1959. Pathological changes were confined to the brain and spinal cord, with the cerebellum, pontine nuclei, thalamus, and spinal cord bearing most of the burden. Macroscopically, some brains were oedematous and leptomeninges were congested.

Microscopically, neuronal changes observed in anterior motor neurons of the spinal cord, in different brain stem nuclei, in the cerebellum, and in the cerebral cortex were non-specific in nature but nonetheless sufficient for Klatzo et al. to draw a parallel between kuru and Creutzfeldt-Jakob disease.

Numerous neurons were either shrunken and hyperchromatic or, to the contrary, pale with dispersion of Nissl substance or intracytoplasmic vacuoles not unlike those seen in scrapie, bovine spongiform encephalopathy (BSE), and chronic wasting disease, but which are rather infrequent in human TSE. In the striatum, some neurons were vacuolated to such a degree that they looked "moth-eaten". Neuronophagia was observed. A few binucleated neurons were visible and torpedo formation was noticed in the Purkinje cell layer, along with empty baskets that marked the presence of degenerated Purkinje cells [START_REF] Beck | Kuru and Creutzfeldt-Jakob disease: neuropathological lesions and their significance[END_REF]). In the medulla, neurons of the vestibular nuclei and the lateral cuneatus were frequently affected; the spinal nucleus of the trigeminal nerve and nuclei of 6 th , 7 th , and motor nucleus of the 6 th cranial nerves were affected less frequently while nuclei of the 12 th cranial nerve, the dorsal nucleus of 10 th cranial nerve and nucleus ambiguous were relatively spared.

In the cerebral cortex, the deeper layers were affected more than the superficial layers, neurons in the hippocampal formation were normal. In the cerebellum, the paleocerebellar structure (vermis and flocculo-nodular lobe) was most severely affected, and spinal cord pathology was most severe in the corticospinal and spinocerebellar tracts. Astro-and microglial proliferation was widespread; the latter formed rosettes and appeared as rod-or amoeboid types or as macrophages (gitter cells). Myelin degradation was observed in 10 of 12 cases. Interestingly, the significance of vacuolar changes was not appreciated by Klatzo and Gajdusek (1959), but "small spongy spaces", were noted in 7 of 13 cases studied by [START_REF] Beck | Kuru and Creutzfeldt-Jakob disease: neuropathological lesions and their significance[END_REF].

The most striking neuropathologic feature of kuru was the presence of numerous amyloid plaques, described as "spherical bodies with a rim of radiating filaments" and found in 6 of 12 cases studied by Klatzo and Gajdusek (1959), and in "about three quarters" of the 13 cases of [START_REF] Beck | Kuru and Creutzfeldt-Jakob disease: neuropathological lesions and their significance[END_REF]; they became known as "kuru plaques" (Fig. 91011).

These measured 20 -60 µm in diameter, were round or oval and consisted of a dark-stained core with delicate radiating periphery surrounded by a pale "halo". Kuru plaques were most numerous in the granular cell layer of the cerebellum, basal ganglia, thalamus, and cerebral cortex in that order of frequency. It is noteworthy that in Gerstmann-Sträussler-Scheinker disease (GSS) plaques are located in both the granular cell and molecular layers, whereas in CJD plaques are confined to granular cell layer. Kuru plaques are metachromatic and stain with PAS, Alcian blue, and Congo-red, and a proportion of them are weakly argentophilic when impregnated according to Belschowsky or von Braunmühl techniques. Klatzo and Gajdusek (1959) reported that plaques were most readily visualized by Holmes' silver impregnation method. Of historical interest, another unique disease reported by [START_REF] Seitelberger | Eigenartige familiar-hereditare Krankheit des Zetralnervensystems in einer niederosterreichischen Sippe[END_REF] as "A peculiar hereditary disease of the central nervous system in a family from lower Austria" (germ. Eigenartige familiar-hereditare Krankenheit des Zentralnervensystems in einer niederoosterreichen Sippe) was mentioned by Neumann et al., (1964) who was thus the first person to suggest a connection between kuru and GSS and kuru.

Renewed interest in kuru pathology has been provoked by the recent appearance of a variant form of CJD (vCJD) resulting from infection by the agent of BSE, also characterized by numerous plaques, including "florid" or "daisy" plaques -a kuru plaque surrounded by a corona of spongiform vacuoles. [START_REF] Hainfellner | Pathology and immunohistochemistry of a kuru brain[END_REF] Immunohistochemical studies revealed that misfolded PrP was present not only in kuru plaques. PrP Sc but also in synaptic and perineuronal sites, and in the spinal cord the substantia gelatinosa was particularly affected, as in iatrogenic CJD cases following peripheral inoculation.

Conclusions and speculations

Kuru, a nearly extinct exotic disease of a cannibalistic Stone Age tribe in a remote Papua New Guinea, still exerts an influence on many aspects of neurodegeneration research.

First, it showed that a human neurodegenerative disease can result from an infection with an infectious agent, then called then a "slow virus". This discovery opened a window into the new class of human diseases including Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease (Fig. 12) and, recently, fatal familial insomnia. Parenthetically, CJD was pointed out as a possible analogue of kuru based of non specific neuropathological findings (Klatzo and Gajdusek, 1959) but Gerstmann-Sträussler-Scheinker disease was identified as linked because of the presence of numerous amyloid plaques not unlike of kuru plaques [START_REF] Seitelberger | Eigenartige familiar-hereditare Krankheit des Zetralnervensystems in einer niederosterreichischen Sippe[END_REF][START_REF] Masters | Creutzfeldt-Jakob disease virus isolations from the Gerstmann-Sträussler syndrome. With an analysis of the various forms of amyloid plaque deposition in the virus induced spongiform encephalopathies[END_REF]. The kuru plaque became a link to Alzheimer disease and, as Gajdusek suggested (1988), all amyloidoses share a common pathogenetic mechanism -processing of a normal protein into an amyloid deposit. This event underlies all "conformational disorders", including pathogenetically novel classes of neurodegenerations like alpha-synucleinopathies, tauopathies and expanded triplet disorders.

We may also speculate what would happen if kuru had not been discovered or did not exist. The infectious nature of Creutzfeldt-Jakob disease would probably not have been suspected until the beginning of the vCJD outbreak in the UK. Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker disease would have remained for decades as obscure neurodegenerations of merely academic interest. The familial forms of Creutzfeldt-Jakob disease would not have benefited from PRNP gene analysis, but only later would have been studied by linkage analysis and reverse genetics probably. The study of scrapie and kuru would have probably been of minimal interest to veterinarians and anthropologistsuntil the BSE epidemic began to exert its devastating effect. The discovery of vCJD would have been delayed, as no surveillance would have been initiated for Creutzfeldt-Jakob disease. And perhaps most importantly, the realization that has led to the conception of 'protein-misfolding diseases", including not only the neurodegenerative but also an increasing number of nonneurological disorders, would have been delayed by decades. France. The immunohistochemical study was performed courtes by Prof. Herbert Budka, Vienna, Austria [START_REF] Hainfellner | Pathology and immunohistochemistry of a kuru brain[END_REF] Fig. 9. A microphotograph showing a specimen of cerebellar cortex stained against prion protein (antibodies 3F4) to visualize "kuru" plaques (arrows). Specimen courtesy of Dr. D.C. Gajdusek, Paris, France. The immunohistochemical study was performed courtesy by Prof. Herbert Budka, Vienna, Austria [START_REF] Hainfellner | Pathology and immunohistochemistry of a kuru brain[END_REF]. Fig. 10. A microphotograph showing a specimen of cerebellar cortex stained against prion protein (antibodies 3F4) to visualize "kuru" numerous plaques. Specimen courtesy of Dr. D.C. Gajdusek, Paris, France. The immunohistochemical study was performed courtesy by Prof. Herbert Budka, Vienna, Austria [START_REF] Hainfellner | Pathology and immunohistochemistry of a kuru brain[END_REF].

  Finally, in 1965, in a monograph "Slow, Latent and Temperate Virus Infections" Gibbs and Gajdusek included this statement"although several of the inoculated primates died of acute infection during the period of observation, [..] none has developed signs suggestive of chronic neurological disease until the recent onset in two chimpanzees. The first of these, inoculated 20 months previously with a suspension of frozen brain material from a kuru patient, has developed progressive incapacitating cerebellar signs with ataxia and tremor; the second, similarly inoculated with a suspension of brain material from another kuru patient, has developed, 21 months after inoculation, slight wasting lassitude, and some tremor which appeared to be progressive. Whether these syndromes are spontaneous or related to the inoculation remains to be determined."

  using modern immunohistochemistry the case of a young male kuru victim from the South Fore region whose brain tissue had transmitted disease to chimpanzees, and McLean et al. (1998) examined a series of 11 cases of kuru still in the archives of the University of Melbourne. In contrast to the classical studies described above, both papers stressed the presence of typical spongiform change (Fig. 8) present in deep layers (III -V) of the cingulate, occipital, enthorrinal and insular cortices, and in the subiculum. Spongiform change was also observed in the putamen and caudate, and some putaminal neurons contained intraneuronal vacuoles. Spongiform change was prominent in the molecular layer of the cerebellum, in peraqueductal gray matter, basal pontis, central tegmental area, and inferior olivary nucleus. The spinal cord showed only minimal spongiform change.

  Figures legendFig.1. Dr. D. Carleton Gajdusek, a photograph taken by P.P. Liberski at the symposium "The

Fig. 2 .

 2 Fig. 2. A map of kuru prevalence in early fifties. Courtesy of Dr. D.C.Gajdusek, Paris, France.

Fig. 3 .

 3 Fig. 3. A kuru expedition in the Papua New Guinea. A photograph dcg-57-ng-857-b courtesy

Fig. 4 .

 4 Fig. 4. A body of the young kuru victim transported by his kinsmen. A photograph kuru-dcg-

Fig. 5 .

 5 Fig. 5. A chimpanzee brain with experimental kuru. A photograph 67-10825-2-1261214,

Fig. 6 .

 6 Fig. 6. A young kuru victim affected with kuru. Note the abnormal posture of the upper

Fig. 7 .

 7 Fig. 7. A group of kuru victims. Courtesy of D.C.Gajdusek, Paris, France.

Fig. 8 .

 8 Fig. 8. A microphotograph showing a specimen of cerebral cortex stained against glial

Fig. 11 .

 11 Fig. 11. A kuru plaque. A specimen reversed from a paraffin block to transmission electron

Fig. 12 .

 12 Fig.12. A specimen from a GSS case stained against PrP. Note innumerable plaques

Figure

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Acknowledgements: this study was supported in part by grants from Ministry of research, Poland and OEAD, Austria and it is a part of "Neuroprion".


Christoph Zenzmaier

Lilian Sklepos

Ph.D Peter Berger
email: peter.berger@oeaw.ac.at

Increase of Dkk-3 Blood Plasma Levels in the Elderly

Keywords: Dickkopf, Blood Plasma, IEMA, Aging BPH, benign prostatic hyperplasia, Dkk, Dickkopf, IEMA, immunoenzymometric assay, FSH, follicle stimulating hormone, HRP, horse radish peroxidase, LH, luteinizing hormone, mAb, monoclonal antibody, NSB, non-specific binding, PCa, prostate carcinoma, SAGE, serial analysis of gene expression

come

Introduction

The secreted glycoprotein Dkk-3 is the most divergent member of the human Dickkopf family [START_REF] Krupnik | Functional and structural diversity of the human Dickkopf gene family[END_REF][START_REF] Niehrs | Function and biological roles of the Dickkopf family of Wnt modulators[END_REF] and in contrast to other family members does not modulate Wnt signaling [START_REF] Mao | LDLreceptor-related protein 6 is a receptor for Dickkopf proteins[END_REF][START_REF] Wu | Mutual antagonism between dickkopf1 and dickkopf2 regulates Wnt/beta-catenin signalling[END_REF]. Our previous results indicated a possible link between Dkk-3 expression and age associated processes in the human prostate. Serial analysis of gene expression (SAGE) of human prostate basal epithelial cells revealed specific induction of DKK3 gene during cellular senescence [START_REF] Untergasser | Characterization of epithelial senescence by serial analysis of gene expression: identification of genes potentially involved in prostate cancer[END_REF] in vitro. In a recent immunohistochemical study [START_REF] Zenzmaier | Dysregulation of Dkk-3 expression in benign and malignant prostatic tissue[END_REF] we demonstrated, that Dkk-3 is downregulated in vivo in prostate epithelium of patients suffering from age-related prostate diseases (benign prostatic hyperplasia, BPH and prostate carcinoma, PCa). This downregulation is counterbalanced by increased protein expression in the endothelial cells of the blood vessels supplying the diseased tissue.

To investigate if the identified cellular senescence-related changes in expression became manifest in body fluids, particularly in human blood plasma, we developed a sensitive indirect immunoenzymometric assay (IEMA).

Materials and Methods

Generation and characterization of monoclonal antibodies

A series of mouse monoclonal antibodies (mAbs) against Dkk-3 was raised according to methods published for mAbs against FSH [START_REF] Berger | Antigenic features of human follicle stimulating hormone delineated by monoclonal antibodies and construction of an immunoradiomometric assay[END_REF]. To produce recombinant protein for the immunization, the DKK3 ORF was cloned in-frame upstream of the E epitope tag (GAPVPYPDPLEPR) into the expression vector pcDNA3.1 (Invitrogen). COS7 cells were transiently transfected with the construct and the protein was purified from the conditioned media via affinity chromatography using a column packed with Anti-E Tag Sepharose (GE Healthcare).

Radioimmunoassays

Antibodies were characterized according to affinity and specificity with native (untagged) Dkk-3 that was purified via HPLC (anion exchange followed by size exclusion chromatography) from conditioned media of stably transfected LNCaP cells [START_REF] Zenzmaier | Dysregulation of Dkk-3 expression in benign and malignant prostatic tissue[END_REF] and the commercially available homologous proteins Dkk-1, Dkk-4 and Soggy-1 (R&D systems). The proteins were radiolabelled with 125 I using chloramine T. Antibodies were incubated over night at 4°C with radiolabelled protein (approximately 25 000 cpm in 100 µL 0.3% BSA in PBS). Separation of bound from free tracer was achieved by 2 h incubation at room temperature on a shaker with 100 µL immunoabsorbent (donkey anti-mouse IgG or donkey anti-goat IgG; Guildhay, respectively, coupled to bromide-activated Sepharose CL-4B beads; GE Healthcare).

After 3 times washing with 0.5% Tween-80 in PBS bound radioactivity was determined in 1470 WIZARD automatic gamma counter (Wallac).

Sensitive IEMA protocol

An indirect IEMA was established using mAb INN(sbruck)-Dkk3-1 at a concentration of 4 µg/mL in coating buffer (200 mM NaHCO 3 ; pH 9.5; incubation for 1 h at 37°C) to coat 96-well plates. After a 30 min blocking step with 1% BSA/PBS, wells were incubated with antigen diluted in 1% BSA/PBS over night at 4°C. After 5 times washing with washing buffer (PBS containing 0.05% Tween20 and 0.01% Thiomersal) plates were incubated with the biotinylated polyclonal anti Dkk-3 antibody (R & D Systems) at a concentration of 200 ng/mL in 1% BSA/PBS for 2 h at room temperature. Signals were recorded after incubation with streptavidin / horse radish peroxidase (HRP; DAKO; 1:500 in 1% BSA/PBS) and the substrate tetramethylbenzidine / H 2 O 2 (Substrate Reagent Pack, R & D Systems), each for 30 min at room temperature, by measuring the absorbance at 450 nm (Victor 2 1420 multilabel counter, Wallac). All samples were run in duplicate and results were calculated from specific mean signal (mean -zero standard). Native i.e. untagged Dkk-3 that was purified via HPLC (anion exchange followed by size exclusion chromatography) from conditioned media of stably transfected LNCaP cells [START_REF] Zenzmaier | Dysregulation of Dkk-3 expression in benign and malignant prostatic tissue[END_REF] was used as a standard. The concentration of the standard was quantified via the absorbance at 280 nm in a HITACHI U-2000 spectrophotometer using the extinction coefficient of 19940 calculated with the ProtParam tool (www.expasy.ch/tools/protparam.html).

Probands

Blood plasma samples were obtained from 63 healthy donors. The local ethical committee approved the bleeding protocol and all participants provided informed consent. A medical history was obtained and individuals with malignancies, acute diseases or advanced stages of severe chronic diseases were excluded from the study. Persons who were under immunosuppressive therapy were also excluded as well as persons under medication for type II diabetes.

Results

A series of mouse monoclonal antibodies (mAbs) against Dkk-3 was raised. Six of Given the specific induction of Dkk-3 during in vitro cellular senescence of human prostate basal epithelial cells [START_REF] Untergasser | Characterization of epithelial senescence by serial analysis of gene expression: identification of genes potentially involved in prostate cancer[END_REF], protein expression during aging was analyzed in vivo. Blood plasma samples from 63 healthy individuals from two age cohorts were investigated (for proband characteristics see Table 1). A mean concentration of 1.42 ± 0.56 nM Dkk-3 (n = 63) was determined, corresponding to 51.3 ± 20.3 ng/mL of the non-glycosylated protein. There were no significant sexspecific differences in the obtained protein levels (female: n=33, mean±SD: 1.42 ± 0.62 nM; male: n=30, mean±SD: 1.41 ± 0.49). When the data were analyzed according to probands´ ages, a significant increase (37.6%; p < 0.01; Figure 2A) of the mean blood Dkk-3 levels was observed. The age cohorts were further subdivided and the age-related increase of the mean plasma protein concentration was confirmed for both sexes (female: 32.5%, p = 0.03; male: 44.2%, p < 0.01; Figure 2B). The mean values obtained from the single groups are listed in Table 1, a scatter plot of all individual values is depicted in Figure 2C.

Discussion

To our knowledge this is the first quantitative determination of secreted Dkk-3 protein levels. The age-correlated increase of Dkk-3 confirmed our previous in vitro cellular senescence data and indicate a non negligible physiological role and its use as a marker for senescence. However, the precise physiological function of the nongender-specific constitutive presence of high amounts of Dkk-3 in the blood plasma of young and elderly individuals (1.17 ± 0.36 nM and 1.61 ± 0.61 nM corresponding to 42.2 ± 13.0 ng/mL and 58.1 ± 22.1 ng/mL of the non-glycosylated protein) remains to be clarified. Its levels e.g. are significantly higher than those of other important glycoprotein hormones like FSH (physiological range 0.18 -21.3 ng/mL; Madersbacher et al.,1993) or LH (physiological range 0 -3.23 ng/mL) in young and elderly probands.

Despite its homology to the Wnt antagonist Dkk-1, Dkk-3 does not modulate Wnt signaling [START_REF] Mao | LDLreceptor-related protein 6 is a receptor for Dickkopf proteins[END_REF][START_REF] Wu | Mutual antagonism between dickkopf1 and dickkopf2 regulates Wnt/beta-catenin signalling[END_REF]. Due to its downreguation in a number of cancer cells [START_REF] Hsieh | Dickkopf-3/REIC functions as a suppressor gene of tumor growth[END_REF][START_REF] Kurose | Decreased expression of REIC/Dkk-3 in human renal clear cell carcinoma[END_REF][START_REF] Tsuji | A REIC gene shows down-regulation in human immortalized cells and human tumor-derived cell lines[END_REF][START_REF] Tsuji | Antiproliferative activity of REIC/Dkk-3 and its significant down-regulation in non-small-cell lung carcinomas[END_REF] Dkk-3 was considered to function as a tumor suppressor but when studying the effect of Dkk-3 on cell growth of either primary prostate cells or BPH and PCa cell lines it appeared that proliferation rates were neither impaired by overexpression nor addition of recombinant protein [START_REF] Zenzmaier | Dysregulation of Dkk-3 expression in benign and malignant prostatic tissue[END_REF]. Thus the biological role of the protein remains unclear.

The origin of the Dkk-3 in blood plasma is not resolved either. One source could be The blood Dkk-3 content is significantly elevated in elderly persons (A; young: n=27, old n=36) of both genders (B). A scatter plot of all values with linear regression curves for the gender cohorts demonstrates, that the slope of the age-correlated increase is similar for females and males (C).

  these antibodies and an affinity purified goat polyclonal antibody (R & D Systems) were then characterized according to affinity and specificity with recombinant Dkk-3 and the homologous proteins Dkk-1, Dkk-4 and Soggy-1 (R&D systems). None of the tested antibodies had significant affinities towards the homologous proteins. The antibodies wih the highest affinity towards Dkk-3 (mAb INN(sbruck)-Dkk3-1 and the goat polyclonal antibody) were then used to establish a sensitive IEMA protocol. To quantify the signals obtained in the IEMA recombinant untagged Dkk-3 was used as a standard. Crossreactivities in the IEMA towards the homologous proteins Dkk-1, Dkk-4 and Soggy-1 (R&D systems) were determined to be << 0.1%. The recovery was calculated by spiking with recombinant protein and was approximately 92%. Assay sensitivity was defined as the mean signal of non-specific binding (NSB = zero standard) plus three standard deviations (NSB + 3SD) and was about 1 pM. The intra-and inter-assay variances (CV%) at a protein concentration of 40 pM were 11% and 13%, respectively. A typical standard curve is shown in Figure 1. The standard was compared to commercially available recombinant Dkk-3 (R&D systems). Although the commercially available protein has a 10X histidine tag at the C-terminus and was expressed in the mouse myeloma cell line NS0, it yielded signals in the IEMA in approximately the same range as the native (untagged) recombinant protein isolated from a human cell line (data not shown).

Figure 1 .

 1 Figure 1. Typical standard curve for the Dkk-3 IEMA. Specific signals (signal -nonspecific binding (NSB; zero standard) from doubling dilutions of recombinant protein from 140 pM to 0.5 pM are depicted. The sensitivity limit of the assay (specific signal > three times the standard deviation of NSB) is approximately 1 pM. Unknown samples were diluted in ELISA buffer to Dkk-3 concentrations between 10 and 100 pM.

Figure 2 .

 2 Figure2. Age-correlated increase of blood plasma Dkk-3 levels. Dkk-3 concentration in blood plasma was determined from 63 individuals (33 females / 30 males).

Table 1 :

 1 Study participant age characteristics and Dkk-3 ELISA results

		All Individuals	Female		Male
		Young	Old	Young	Old	Young	Old
	Number of	27	36	14	19	13	17
	individuals
	Age (years)
	mean±SD 25.1±3.7 73.0±7.1 23.4±3.6 70.9±7.0 27.0±2.8 75.4±6.6
	Dkk-3
	(nmol/L)
	mean±SD 1.17±0.36 1.61±0.61 1.20±0.31 1.59±0.74 1.13±0.42 1.63±0.44
	p	< 0.01*		0.03**		<0.01**
	Median	1.09	1.54	1.16	1.38	0.96	1.68
	Range 0.49-1.79 0.57-3.42 0.79-1.76 0.57-3.42 0.49-1.79 0.95-2.66
	SD: standard deviation
	p values are calculated as two-(*) or one-sided(**) student´s t-test

Acknowledgments

The authors wish to thank Michael Keller and Katharina Gapp for their excellent technical support.

endothelial cells, were Dkk-3 is reported to be expressed [START_REF] Goodwin | Cultured endothelial cells display endogenous activation of the canonical Wnt signaling pathway and express multiple ligands, receptors, and secreted modulators of Wnt signaling[END_REF][START_REF] Kupatt | Embryonic endothelial progenitor cells expressing a broad range of proangiogenic and remodeling factors enhance vascularization and tissue recovery in acute and chronic ischemia[END_REF]. Moreover, we detected high expression of Dkk-3 in vascular endothelial cells BPH and PCa tissue sections [START_REF] Zenzmaier | Dysregulation of Dkk-3 expression in benign and malignant prostatic tissue[END_REF].

Upregulation of the protein in tumor endothelium has also been demonstrated in colorectal carcinoma, glioma, high-grade NHL and melanoma [START_REF] St Croix | Genes expressed in human tumor endothelium[END_REF][START_REF] Untergasser | The Dickkopf-homolog 3 is expressed in tumor endothelial cells and supports capillary formation[END_REF]. Therefore Dkk-3 can be considered a pro-angiogenic protein in neovascularisation. High expression of the protein was also seen in a subset of adult human pancreatic beta cells [START_REF] Hermann | Dickkopf-3 is expressed in a subset of adult human pancreatic beta cells[END_REF]. These cells could also contribute to the Dkk-3 levels determined in human blood plasma.

The established assay may help to elucidate the mechanism by which the protein acts, as it gives us the opportunity to quantify differences in Dkk-3 secretion in in vivo and in vitro models of aging and disease.


Christoph Zenzmaier

Gerold Untergasser

Ph.D Peter Berger
email: peter.berger@oeaw.ac.at

Aging of the prostate epithelial stem/progenitor cell

Keywords: Prostate, epithelial stem cells, aging, prostate cancer, tissue regeneration

HAL is

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Abstract

Maintenance of the prostatic epithelial cell compartment is ensured by proliferation of adult epithelial progenitor or stem cells. These cells are characterized by an undifferentiated state, high proliferative capacity and long life span. Prostate progenitor/stem cells are localized in their stem cell-niche in the basal cell compartment in close contact to the basement membrane and the stromal cell compartment and are characterized by expression of the basal cytokeratins 5 and 14, high levels of integrins, CD44, the stem cell markers CD133 and ABCG2, and AR negativity. They give rise to secretory luminal (cytokeratins 8/18, CD57, AR, p27, PSA, PAP) and neuroendocrine cells (cytokeratins 8/18, CD57, CgA, NSE, NEPs), the two major cell types observed in the glandular epithelium.

A growing body of experimental evidence has identified the amplifying progenitor/stem cell (CD44 + , α 2 β 1 hi , CD133 + ), as a putative origin of prostate cancer.

Differentiation of this cell type can be affected by mutations in the intrinsic genetic program, by age-related changes in stromal-epithelial interactions or in the basement membrane /ECM composition. All these stochastic events occur during aging and can transform a normal prostate progenitor/stem cell into a cancer stem cells, a source of androgen-dependent and independent tumor cell clones. Thus, the heterogeneous and multifocal nature of prostatic cancer with a pleora of different tumor cell clones clearly reflects the differentiation capacity of the prostatic epithelial progenitor cells.

Characterization of prostate epithelial stem/progenitor cells

Stem and progenitor cells act as a regeneration system replenishing specialized cells, and also enabling the normal turnover and functional regeneration of organs in the adult organism. Stem /progenitor cells are defined by their ability to self-renew and their capacity to differentiate into specialized cell types. Toti-and pluripotent stem cells can give rise to any mature cell type, whereas multipotent stem/progenitor cells can differentiate into cells of a closely related cell family, e.g. epithelial cells.

Stem/progenitor cells responsible for tissue homeostasis of various epithelial tissues with diverse architectural design and physiology like intestine, epidermis, mammary gland or cornea have been described and characterized [START_REF] Blanpain | Epithelial stem cells: turning over new leaves[END_REF].

The human prostate, an androgen-dependent walnut-sized sex accessory gland surrounding the urethra at the base of the bladder, serves a crucial function in the male reproductive tract by producing approximately 30% of seminal fluid providing nutrients and optimal ionic milieu and pH for sperm.

Prostate epithelial stem/progenitor cells (PESCs) have long been hypothesized to be present in the basal cell layer of the stratified prostate epithelium [START_REF] Isaacs | Etiology and disease process of benign prostatic hyperplasia[END_REF], although only in recent years several markers for their characterization and localization have been investigated. In the murine prostate the actively proliferating cells were found to be located in the distal region of the prostatic ducts, whereas in the proximal ductal region cells with stem cell features (quiescent, high proliferation potential) were concentrated [START_REF] Tsujimura | Proximal location of mouse prostate epithelial stem cells: a model of prostatic homeostasis[END_REF]. These cells were further characterized by high expression of the cell surface protein stem cell antigen 1 (Sca-1), coexpression of α6 integrin and the antiapoptotic factor Bcl-2, and showed a higher efficiency to generate prostatic tissue in in vivo reconstitution assays when compared with Sca-1 -cells or Sca-1 expressing cells from remaining regions of the duct [START_REF] Burger | Sca-1 expression identifies stem cells in the proximal region of prostatic ducts with high capacity to reconstitute prostatic tissue[END_REF]. Bcl-2 is supposed to protect the PESCs from the apoptotic effects of high levels of active TGF-β produced by cells in the proximal niche to maintain the PESCs in a dormant state [START_REF] Salm | TGF-{beta} maintains dormancy of prostatic stem cells in the proximal region of ducts[END_REF].

High levels of α6 integrin has also been demonstrated for other stem/progenitor cells like basal keratinocytes in the epidermis and spermatogonial stem cells in the testis [START_REF] Jones | No place like home: anatomy and function of the stem cell niche[END_REF]. Expression of adhesion molecules like cadherins or integrins is important to maintain stem/progenitor cells in their niche in close proximity to supporting stromal cells, the basal lamina, extra-cellular matrix proteins and selfrenewal signals [START_REF] Jones | No place like home: anatomy and function of the stem cell niche[END_REF]. Cadherins establish adherens junctions with the neighboring cells, while integrins are receptors for components of the extracellular matrix and major components of the basal lamina (laminin, collagen, fibronectin) and attach the cells to the latter.

Based on high expression of α 2 β 1 integrin Collins and colleagues identified PESCs in the basal layer of the human prostate. After immunomagnetic separation of the basal cells via their expression of the hyaluronan-binding surface glycoprotein CD44 clonogenic cells were enriched by rapid attachment to extra-cellular matrix proteins [START_REF] Collins | Identification and isolation of human prostate epithelial stem cells based on alpha(2)beta(1)-integrin expression[END_REF].

The CD133 (prominin-1) antigen was originally classified as a marker of primitive haematopoietic and neural stem cells monoclonal antibodies against this antigen were successfully used to enrich for haematopoietic stem cells, endothelial cells, neurons and glial cells. Similar to other tissues, in the human prostate a small number of cells (approximately 0.75% of the cells in the basal layer) express CD133 [START_REF] Richardson | CD133, a novel marker for human prostatic epithelial stem cells[END_REF]. The clonogenic cells of the rapidly adhering α 2 β 1 hi cell population could be further enriched by CD133 selection cells and the proportion of CD133 + cells within the α 2 β 1 hi cell population was approximately 1 in 4.

Expression of the ATP-binding cassette transporter family membrane efflux pump ABCG2 (Breast cancer resistance protein 1, BCRP1) is a conserved feature of stem cells from a wide variety of sources [START_REF] Zhou | The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype[END_REF]. ABCG2 was first identified in the MCF-7 breast cancer cell line and is associated with multi-drug resistence. The ATP-binding cassette transporter family proteins are responsible for the active efflux of the vital dye Hoechst 33342 resulting in a Hoechst low cell subpopulation termed side population (SP). Cells of the SP phenotype have been identified in a variety of tissues among them prostatic epithelium, where this population constitutes 1.38% of the cells [START_REF] Bhatt | Novel method for the isolation and characterisation of the putative prostatic stem cell[END_REF]. ABCG2-mediated efflux of androgen has been proposed

to contribute to the maintenance of the PESC phenotype, which among other features is characterized by lack of androgen receptor (AR) protein [START_REF] Huss | Breast cancer resistance protein-mediated efflux of androgen in putative benign and malignant prostate stem cells[END_REF].

PESC function: tissue homeostatsis

The main function of the PESCs is to preserve tissue homeostasis of the gland by giving rise to differentiated specialized progeny as there are secretory and neuroendocrine cells. The prostate is an androgen-dependent organ, that reduces its size dramatically upon androgen deprivation. This is mainly caused by apoptosis of luminal cells, while basal epithelial cells stay intact. Early experiments in rodents demonstrated, that administration of androgens renewed the prostate epithelium in castrated rats. The fact that basal cells remained intact upon androgen deprivation and gave rise to fully functional luminal cells after re-administration led to the stem cell theory of prostate growth proposing androgen independent stem/progenitor cells [START_REF] Isaacs | Etiology and disease process of benign prostatic hyperplasia[END_REF].

Epithelial cell differentiation is a complex process governed by the expression of intrinsic genes that mediate differentiation and growth processes. Mesenchymalepithelial interactions have an instructive role during the androgen-induced development of the prostate. Freshly isolated mouse prostate epithelial cells can regenerate fully differentiated prostate tissue when combined with embryonic urogenital sinus mesenchyme and grafted in vivo [START_REF] Cunha | The possible influence of temporal factors in androgenic responsiveness of urogenital tissue recombinants from wild-type and androgen-insensitive (Tfm) mice[END_REF] but not when transplanted without supporting stromal cells. Stromal-epithelial interactions play also a key role in the maintenance of the adult prostatic epithelium (Sampson et al., 2007;[START_REF] Untergasser | Benign prostatic hyperplasia: age-related tissue-remodeling[END_REF]. The local extracellular matrix, stromal growth factors and androgens are essential for functional and morphological differentiation of the prostatic epithelium [START_REF] Hayward | A functional model of adult human prostate epithelium. The role of androgens and stroma in architectural organisation and the maintenance of differentiated secretory function[END_REF]. [START_REF] Hudson | Epithelial cell differentiation pathways in the human prostate: identification of intermediate phenotypes by keratin expression[END_REF] and prostate stem cell antigen [START_REF] Tran | Prostate stem cell antigen is a marker of late intermediate prostate epithelial cells[END_REF]. Moreover, they still retain basal cytokeratins 5/14 and acquire luminal cytokeratins 8/18. Depending whether they differentiate into secretory or neuroendocrine cell lineages they express androgen receptors (AR) [START_REF] Bonkhoff | Widespread distribution of nuclear androgen receptors in the basal cell layer of the normal and hyperplastic human prostate[END_REF] or Chromogranin A (CgA), neuron specific enolase (NSE) and human chorionic gonadotropin alpha (hCGα) [START_REF] Berger | Human chorionic gonadotropin (hCG) in the male reproductive tract[END_REF][START_REF] Bonkhoff | Multidirectional differentiation in the normal, hyperplastic, and neoplastic human prostate: simultaneous demonstration of cell-specific epithelial markers[END_REF][START_REF] Rumpold | Neuroendocrine differentiation of human prostatic primary epithelial cells in vitro[END_REF]. Prostate epithelial cell differentiation is schematically depicted in Figure 1.

Normal

The aged PESC and prostate cancer development

Prostate cancer (PCa) is the most common diagnosed male malignancy in Western societies and the second most common cause of male cancer-related death with an estimated incidence of 232 090 new cases and 30 350 deaths in the United States in 2005 [START_REF] Jemal | Cancer statistics[END_REF]. PCa is a multifocal, nonclonal and heterogeneous tumor mainly localized in the peripheral zone (over 70%) of the prostate (Abate-Shen and Shen, 2000).

Among the risk factors for the development of PCa age and the presence of androgens are most important followed by dietary factors, familiar predisposition and prostatic inflammation processes. Within the last years hereditary components, so called inherited PCa susceptibility genes like the interferon-induced RNAse (RNASEL), macrophage-scavenger receptor (MSR1), 5-α-reductase type2 (ARSRD5A2) and cytochrome P-450 17A1 (CYP17A), have been identified that correlate with a high risk for development of PCa [START_REF] Nelson | Prostate cancer[END_REF]. Interestingly, most of these predisposition genes are involved in host responses to infections or in metabolism of sex-steroid hormones.

Somatic mutations of stem/precursor cells, accumulating over a period of several decades can affect genes involved in growth regulation (p27; NKX3.1), cell signaling (PTEN), terminal differentiation (AR), DNA-protection (GSTP1) and repair [START_REF] Bell | Stem cells, aging, and cancer: inevitabilities and outcomes[END_REF][START_REF] Reya | Stem cells, cancer, and cancer stem cells[END_REF]. The frequency of somatic mutations are dramatically increased by inflammatory processes. Prostatic inflammation is a common event observed in prostate tumorigenesis glandular atrophy [START_REF] De Marzo | Proliferative inflammatory atrophy of the prostate: implications for prostatic carcinogenesis[END_REF]. Moreover, proliferative inflammatory atrophy (PIA) shares molecular traits with prostate intraepithelial neoplasia (PIN), that is accepted as the most likely preinvasive stage of adenocarcinoma of the prostate [START_REF] Bostwick | High-grade prostatic intraepithelial neoplasia[END_REF][START_REF] Nelson | Prostate cancer[END_REF]. The most frequent tumor-associated genetic event identified to date is fusion of the 5' untranslated region of the androgen-regulated gene TMPRSS2 to the oncogenic ETS transcription factor family members, ERG and ETV1 that are commonly overexpressed in PCa [START_REF] Tomlins | Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer[END_REF].

Mutations in these genes responsible for normal prostate development, differentiation and growth regulation are thought to trigger initiation and then progression and metastasis of PCa [START_REF] Isaacs | Molecular biology of prostate cancer progression[END_REF]. As tumor cells express luminal characteristics, classically mutated luminal cells were thought to be the origin of PCa.

However, after initially successful androgen ablation therapy of the strongly androgen-dependent primary adenocarcinomas in most cases highly aggressive androgen independent tumors recur, that may express some basal cell characteristics. Therefore it is hypothesized that prostate cancer cells may be derived from stem cells or intermediate transit amplifying cells (Figure 1) as stem cells possess many features in common with those of the tumor phenotype, including selfrenewal, pluripotency in differentiation and high replicative potential. Furthermore mutations that initiate tumor formation seem to accumulate in cells that persist throughout life, as suggested by the exponential increase of cancer incidence with age, while non-stem/precursor cells are generally destined for terminal differentiation within a window of time too short for acquisition of sequential mutations that must affect both copies of a wild-type tumor suppressor [START_REF] Hanahan | The hallmarks of cancer[END_REF].

Despite a variety of genetic and environmental factors, age is the most significant risk factor for the development of the malignancy. Within aging precursor/stem cells accumulate several mutations that lead to neoplastic transformation, when mutations affect genes involved in terminal growth arrest or differentiation [START_REF] Bell | Stem cells, aging, and cancer: inevitabilities and outcomes[END_REF][START_REF] Reya | Stem cells, cancer, and cancer stem cells[END_REF]. These transformed cells give rise to transit amplifying daughter cells that in turn are not able to differentiate appropriate into columnar secretory cells and form precancerous proliferative lesions associated with chronic inflammation (PIA) [START_REF] De Marzo | Proliferative inflammatory atrophy of the prostate: implications for prostatic carcinogenesis[END_REF][START_REF] Van Leenders | Intermediate cells in human prostate epithelium are enriched in proliferative inflammatory atrophy[END_REF]. Acquisition of further mutations and genomic instabilities in turn lead to transition of inflammatory lesions into low and high grade prostatic intraepithelial neoplasms (PIN), that are heterogeneous, non-clonal and accumulate age-dependently [START_REF] De Marzo | Human prostate cancer precursors and pathobiology[END_REF].

Most of these damaged transit-amplifying cells will differentiate into the intermediate/luminal phenotype that utilizes androgens and stroma-derived andromedins for proliferation, but not for terminal differentiation. These primary adenocarcinomas are strongly androgen-dependent and express luminal (PAP, PSA, AR, K8/18, as well as intermediate cell markers (PSCA, pp32). To some lower extent transit-amplifying cells will also differentiate into intermediate/ neuroendocrine cells that express CgA, NSE, K5, and Bcl-2 [START_REF] Schalken | Cellular and molecular biology of the prostate: stem cell biology[END_REF] and focally build subpopulations in the adenocarcinoma [START_REF] Bonkhoff | Neuroendocrine differentiation in human prostate cancer. Morphogenesis, proliferation and androgen receptor status[END_REF].

This primary androgen-sensitive adenocarcinomas contain predominantly intermediate /luminal phenotypes and can be effectively treated by androgen-ablation therapy. Nevertheless, many tumor patients fail this therapy and die of recurrent androgen-independent PCa (AIPC). Several pathways of selection of androgen independent tumor phenotypes by which AIPC can develop have been proposed, all of which may be operative [START_REF] Craft | Evidence for clonal outgrowth of androgenindependent prostate cancer cells from androgen-dependent tumors through a two-step process[END_REF][START_REF] Feldman | The development of androgen-independent prostate cancer[END_REF]. These pathways include selection of antiapoptotic basal/intermediate phenotypes either

ACCEPTED MANUSCRIPT

-10 -from transformed precursor cells or transdifferentiation from intermediate/ luminal cells under androgen-ablation [START_REF] Long | Prostate epithelial cell differentiation and its relevance to the understanding of prostate cancer therapies[END_REF], selection for androgenindependent neuroendocrine/intermediate phenotypes either from malignant precursor cells or transdifferentiation from intermediate/ luminal cells under androgen ablation therapy [START_REF] Bonkhoff | Neuroendocrine differentiation in human prostate cancer. Morphogenesis, proliferation and androgen receptor status[END_REF] and selection for luminal/intermediate phenotypes with mutated AR signaling [START_REF] Feldman | The development of androgen-independent prostate cancer[END_REF]. These mechanisms are referred to in the literature [START_REF] Feldman | The development of androgen-independent prostate cancer[END_REF] as the hypersensitive pathway (overexpression of the AR), the promiscuous pathway (mutations of the AR decrease its specificity), the outlaw pathway (ligand-independent AR activation) and the bypass pathway (overexpression of antiapoptotic genes, differentiation to androgen-independent neuroendocrine phenotypes).

The presence of prostate cancer stem cells that continously resupply the tumor cell

  prostate epithelium is composed of at least five cell types (PESC, basal cells, transit amplifying cells, luminal secretory cells, neuroendocrine cells) which are supposed to be derived from PESC that are scattered among the basal cell compartment. There they undergo mitosis and give rise to basal cells, which are characterized by the basal cell markers cytokeratin 5 and 14 and CD44, androgen independence, a high proliferative capacity and a low apoptotic index (Schalken and van Leenders, 2003). The progeny of the basal compartment are intermediate transit amplifying cells that in turn give rise to heterogeneous subpopulations of intermediate cells that subsequently differentiate to luminal secretory and neuroendocrine cells (Hudson et al., 2001). The intermediate cell population is characterized by the expression of cytokeratin 19

  Under the influence of androgens intermediate cells differentiate into luminal secretory cells and express luminal cytokeratins 8/18, CD-57 (HNK-1 carbohydrate epitope of cell adhesion molecules), AR, the cycline-dependent kinase inhibitor p27[START_REF] Waltregny | Androgen-driven prostate epithelial cell proliferation and differentiation in vivo involve the regulation of p27[END_REF], prostate specific antigen (PSA) and prostatic acidic phosphatase (PAP). Independent of the action of androgens subpopulations of intermediate cells differentiate into terminally growth arrested neuroendocrine cells, expressing a variety of neuroendocrine peptides (NEPs)[START_REF] Sant'agnese | Endocrine-paracrine (APUD, neuroendocrine) cells in the normal and pathologic prostate[END_REF].

  population and are not affected by androgen-depletion therapy also represents a potential mechanism for tumor survival in the androgen-depleted environment. In recent years evidence for the existence of prostate cancer stem cells with similar properties similar to prostate epithelial stem cells emerged. By the use of markers established for normal prostate epithelial stem cells a cancer stem cell population out of tumor tissue was identified (Collins et al., 2005). The prostate cancer stem cells were isolated by their expression of the surface marker profile CD44 + /α 2 β 1 hi /CD133 + , that was expressed in approximately 0.1% of the tumor cells independent of the tumor's Gleason grade or metastatic state. These cells were able to regenerate phenotypically mixed populations of nonclonogenic cells and were invasive in Matrigel, indicating their tumor cell origin. Conclusions During the past few years our knowledge of the PESC increased considerably. Their regeneration and differentiation potential was investigated and various stem cell markers were identified. Furthermore the presence of androgen-independent prostate cancer stem cells with the ability to give rise to androgen dependent differentiated tumor cells was demonstrated. The similarity with PESC of the healthy prostate supports the stem cell origin theory of the prostate cancer stem cells. Mutations accumulated during lifetime and / or age related changes (e.g. inflammation; PIA) of PESC or their niche lead to changes in the balance between cell division and differentiation which manifests in the development of PINs. Subsequently mutations and autocrine / paracrine mechanisms further amplify this imbalance which proceeds to PINs and finally PCa. Androgen-ablation therapy then leads to the selection of AIPC phenotypes. A better understanding of the role of aged / mutated PESC in the processes leading to the development of androgen-dependent and -independent PCa will hopefully lead to new preventive and therapeutic treatments for these malignancies.

Figure legend Figure 1 .

 legend1 Figure legend

Acknowledgement P.B. is supported by the Austrian Science Fund (FWF, NRN S9307-B05).