Their deleterious action has been demonstrated for peripheral organs. We postulate that antibodies to the α1-adrenergig receptor are relevant pathomolecules in diseases of the central nervous system associated with vascular impairments.Circulating agonistic autoantibodies acting at G protein-coupled receptors have been associated with numerous sever pathologies in humans. Antibodies directed predominantly against the α1-adrenergig receptor either induced by immunization with a receptor peptide or applied by intravenous injection. The vasculature in the rat brains was investigated by time-of-flight magnetic resonance angiography using a 9.4 Tesla small animal MR imaging system. Visual examination of maximum-intensity-projections (MIPs) of brain angiographs revealed the development of vascular defects in antibody- exposed animals between three and eight months of treatment. Relative vascular areas were derived from representative MIP image sections by grayscale analysis and used to form an index of vascular circulation. Animals exposed to the action of α1-adrenergig receptor antibodies showed significantly reduced vascular areas (p<0.05). Calculated index values indicated attenuated blood flow in both antibody-treated cohorts compared to their respective controls reaching with  0.839±0.026 versus 0.919±0.026 statistical significance (p<0.05) for peptide-immunized rats.Using a rat model we studied the long-term action of antibodies against the α1-adrenergig receptor cause cerebrovascular impairments in the rat. Our findings suggest the pathological significance of these antibodies in pathologies of the human central nervous system linked to impairments of brain vasculature such as stroke and dementia.We present evidence that antibodies to the α Structural and functional impairments of the vasculature are causally linked or significantly contribute to different pathologies in humans, among them numerous widespread diseases. In the cardiovascular system hypertension, angina pectoris and cardiac infarction involve vascular impairments. Patients suffering from diabetes develop vascular injuries. Severe defects of blood supply to and circulation in the brain are the acute cause of stroke. Brain vasculature is critical for the development of different types of dementia such as Alzheimer’s and vascular dementia. There is evidence that dementia of the Alzheimer’s type may be primary a vascular disease 1-adrenergic receptor (α1-AR) were found to be associated with widespread diseases such as different types of hypertension and type 2 diabetes 1-AR were shown to cause cardiomyocyte hypertrophy and diastolic dysfunction in rats 1-AR by immunoadsorption resulted in a significant and long-lasting decline of the mean arterial blood pressure 1-AR in the regulation of blood vessels, the occurrence of antibodies acting at this receptor in diseases with significant vascular involvements suggests their importance in vascular pathology 1-AR peptides developed receptor-specific antibodies and damages in the aorta and mesenteric artery Agonistic autoantibodies acting at G protein-coupled receptors (GPCR) have been detected in the circulation of patients with different, mainly cardiovascular diseases 1-AR antibodies to cause damages in the vasculature of the central nervous system. We therefore studied the long-term effects of α1-AR antibodies in vital rats by time-of-flight magnetic resonance angiography (TOF-MRA) using a 9.4 Tesla small animal magnetic resonance imaging system. We observed substantial attenuations of vascular blood flow in the brain after long-term exposure to the α1-AR antibody.The present investigation aimed at shedding light on the potential of αLandesamt für Gesundheit und Soziales Berlin . Taking blood samples and imaging experiments were performed under isoflurane anesthesia. All manipulations of animals were performed by authorized personnel, and all efforts were made to minimize suffering of animals.Animal experiments were carried out in accordance with the guidelines provided and approved by the animal welfare department of the Forty male Wistar rats  were obtained from Charles River Laboratories, Sulzfeld, Germany. Animals were housed in one acclimatized windowless indoor room in standard IVC cages Type 1500  with a wire mesh top in groups of maximum 3 animals dependent on weight. The animals had free access to commercial rodent food, and tap water was available ad libitum. The environmental conditions were held constant, room temperature 20+2°C and 50–60% relative humidity. The animals were allowed to acclimatize before studies took place.1-AR isoform A was coupled to bovine serum albumin (BSA) by the glutaraldehyde method. The peptide-BSA reaction product was desalted and pre-buffered to phosphate buffered saline (PBS) using Sephadex-G25 columns . For controls, BSA fractions were prepared the same way except the peptide was absent. Antiserum to the α1-AR peptide was produced in goat . The antiserum was affinity purified by column chromatography using the peptide immobilized to an agarose matrix (α1-AR antibody). Goat control immune gamma globulin was obtained from Dianova, Hamburg, Germany (control IgG).The synthetic peptide PAPEDETICQINEE  corresponding to the second extracellular loop of the human α1-AR antibody (700 µg/kg body weigh). The corresponding control group (C-AB) was injected with the same dose of control IgG. The injections were repeated monthly. A third cohort of animals (PEP) was immunized by subcutaneous injection of 300 µg α1-AR-peptide coupled to BSA and emulsified in incomplete Freund’s adjuvance at 0, 2 and 4 weeks. Then the injections were repeated monthly. The respective control animals (C-PEP) were subcutaneously injected with BSA. Blood aliquots were taken from anesthetized animals by retro-orbital sampling. The obtained sera were analyzed for the presence of α1-AR antibodies by standard ELISA techniques. For detecting the systemic immune response and the applied goat α1-AR antibody (cohorts AB and C-AB), aliquots of rat serum were bound to 96-well plates  and probed with anti-rat IgG and anti-goat IgG, respectively. To specifically detect α1-AR antibodies generated in immunized rats (cohorts PEP and C-PEP), the immobilized α1-AR peptide was used as capture antigen. As secondary antibody horseradish peroxidase conjugated anti-rat IgG or anti-goat IgG was used as appropriate. Antibody binding was visualized by the 1-Step Ultra TMB ELISA . The absorbance was measured at 450 nm with a SLT Spectra multiplate reader .To four experimental cohorts 10 rats each were allocated at random. One cohort (AB) obtained intravenous injections of αFor the imaging experiments animals were anesthetized in a warmed anesthetic chamber using 3.5% isoflurane in an oxygen/air mixture (300∶700) with a flow rate of 1000 ml/min. After approximately one minute the isoflurane was reduced to 2% to 3%. A body temperature of 37°C was maintained throughout the experiments by warming the animal bed using a circulating heated water system  and monitoring by a rectal temperature probe. Respiratory signals were continuously monitored using a commercial monitoring and gating system  and kept between 50–70 respiration cycles per minute by regulating the isoflurane dose.All imaging experiments were carried out on a 9.4T small animal MR system . A curved four channel receive only rat brain coil array  was used in conjunction with a linear polarized birdcage resonator for transmission . Due to the large increase in body size with age a shorter head-only birdcage resonator  was used for transmission at 11 months.2-weighted RARE sequence  was acquired in axial orientation. Time-of-flight angiograms were acquired using a 2D FLASH sequence  in coronal orientation.For anatomical reference and to screen for possible pathologies  a high resolution TTo analyze TOF-MRA measurements maximum-intensity-projections (MIPs) were produced using the OSIRIS software . In the two-dimensional viewer mode images were virtually screened using coronal MIP-slabs of three millimeter thickness to find the appropriate image section for visual evaluation. This interactive approach aimed at defining the corresponding section plane of TOF-MRA measurements of each animal at 3 months and 11 month for comparison. All efforts were made to select the same brain section and angle of view for the two-dimensional MIPs at both time points for each animal. The respective two-dimensional MIP image, which was subsequently used for grayscale analysis, was checked against the three-dimensional MIP view of the entire volume covered by the TOF-MRA to verify the visually observed alterations and to exclude artifacts due to spatial variations in brain vascular architecture.Images were analyzed using the ImageJ software . Edge-preserving median filtering was performed for smoothing and to remove background noise. For each two-dimensional image section basic statistical parameters were calculated. The vascular fractions of images were differentiated based on the assumption that the most frequent and highest ranked median gray value represents the image background consisting of tissue not perfused with blood. Grey values above median plus two standard deviations were considered to correspond to the vascular fraction of the image. A threshold operation was performed to obtain black and white images. White areas representing the vascular fractions were estimated using the histogram. Changes in brain vascular circulation are expressed as ratio of the relative vascular areas of the last and the first TOF-MRA.Data were analyzed using GraphPad Prism version 3.00 for Windows . Statistical significances were calculated with the unpaired Student’s t-test after data had been checked for normal distribution. Differences with p-values<0.05 were considered statistically significant.1-AR-antibodies produced in goat (cohort AB) showed a fast clearance of these antibodies from the circulation. Six hours after injection of α1-AR-antibodies there was still a high level of the antibody detectable. Then the α1-AR-antibody level declined to less than 10% of the 6-hour value at day 1 and was not detectable in most animals after day 3. The peptide used for immunizing rats (cohort PEP) corresponds to the cell surface epitope of the rat α1-AR which is permanently exposed to the immune system. To ensure that there is an immune reaction to the application of the autoantigen, the IgG status of the animals was measured. Compared to the pre-immune situation, there was an increase in IgG. Thus, the immunization procedure was efficient and resulted in a systemic immune response. Analysis of the specific immune response to the peptide antigen demonstrated low levels of α1-AR-specific antibodies in serum samples of the animals. 1-AR antibody formation in peptide- (PEP) and vehicle-immunized rats (C-PEP) one month before the final TOF-MRA measurement.Animals receiving intravenously αThe total period of observation was 11 months. During this time span there was a massive growth of the rats resulting in more than doubling of their body mass. The increase within the first three months of the observation was with 60% much higher than between month three and month 11 with 26%. Considering previous experiences and data from comparable animal studies of other groups, vascular alterations were unlikely to be visible already after three months of treatment Anatomical magnetic resonance images of the rat brain and maximum-intensity-projections (MIP) of corresponding TOF-MRA data are shown in 1-AR antibody action within eight months of observation. In the cohort of rats immunized with the α1-AR peptide (PEP) 8 out of 10 animals showed visible vascular defects which were abundantly clear in four individuals. Notably, larger vessels in the right part of the brain appeared to be preferentially affected.  A, B exemplifies coronal two-dimensional MIP sections approximately at the level of the hypothalamus taken at the early stage of immunization with the α1-AR -peptide (A) and at the end of the treatment eight months later (B). When comparing the images regional damages in blood flow were obvious which developed within eight months of repeated immunizations with the α1-AR -peptide (B versus A).  C, D shows an example of comparable two-dimensional views of a brain from a control animal. In this case, no defects were detectable at the end of the observation period. However, 3 out of 10 animals in the control group (C-PEP) also showed brain regions with impaired blood flow. Although they appeared less pronounced when judged visually, quantifiable measures are required to verify these observations.Visual screening of TOF-MRA MIP images revealed impairments of vascular blood flow in the anterior part of the brain in most of the animals of both cohorts exposed to α1-AR antibodies (cohort AB) showed a similar situation as obtained for the α1-AR peptide-treated animals. Comparing the two-dimensional MIP sections of the same individual, there were clearly vascular defects visible at 11 months after the beginning of the treatment in 9 out of 10 animals with 4 rats showing only slightly affected blood flow. As in peptide-treated rats, the impairment of different, predominantly larger brain vessels was obvious. In the respective control group (C-AB) which received control IgG, 3 out of 9 animals developed clearly visible defects. Three-dimensional MIPs of brain TOF-MRA as depicted in Visual evaluation of TOF-MRA MIP sections of rats injected with α1-AR antibody  and a control animal . The white areas correspond to blood perfused vessels. Their quantification produces a relative measure for the area of the functionally intact vasculature. 1-AR antibody (AB) and rats immunized with the α1-AR peptide (PEP). The respective control animals  showed slightly, but not significantly decreased mean vascular areas at the final measurement. To compensate for differences between the experimental groups already obvious at the beginning of the observation, the ratio of relative vascular areas of the final measurement 2 and of the initial measurement 1 was calculated for each animal. This ratio served as an index for changes of vascular blood flow and was used to compare the animal cohorts exposed to α1-AR antibody action  with their respective control cohorts . Values <1 indicate impaired blood flow. The box plot in 1-AR antibody (AB) and rats immunized with the α1-AR peptide (PEP) showed larger decrements compared to the respective control animals . The decline in the α1-AR peptide- treated cohort (PEP) was with (relative units ± standard error) 0.839±0.026 versus 0.919±0.026 of the C-PEP control cohort statistically significant (p<0.05). There was a clear tendency towards attenuated brain blood circulation also in the α1-AR antibody- treated animals which, however, did not reach statistical significance (0.822±0.021 for AB versus 0.877±0.029 for C-AB). Both control cohorts showed values of relative vascular circulation slightly below 1 indicating some worsening of blood supply in the brain during the observation period.Gray scale analysis of two-dimensional TOF-MRA MIP sections was performed to approximate relative values of vascular areas. An essential step of the employed procedure is the generation of black and white images. An example is given in 1-AR in the brain vasculature of rats by TOF-MRA. Two parallel approaches, immunization of animals with the α1-AR peptide and injection of the α1-AR antibody, resulted in impairments of brain blood flow.We demonstrate first evidence of the pathogenic significance of antibodies to the α1-AR which contains the antibody epitope. This approach differs from previous studies with regard to the peptides used for immunization. In these studies longer α1-AR peptides were used containing amino acid stretches of transmembrane regions of the receptor 1-AR antibody levels of the animals observed in the present study although the generalized immune response demonstrated the efficiency of the immunization procedure. In a parallel approach, rats were treated by intravenous injection of an antibody raised against the α1-AR peptide. This antibody we previously characterized to be agonistic and to act comparable to α1-AR antibodies from hypertensive patients 1-AR antibody raised in goat disappeared fast from the circulation of the rats. Human IgG applied to mice was reported to persist in the circulation and was found with 25% recovery at four days after injection 1-adrenergic receptor domain raised in rats and then transferred into naive rats, a half-life time in the circulation of nine days was reported 1-AR antibody applied to the rats was generated in a different animal species, and a fast clearance of the antibody by the immune system must be expected. However, the serum concentration of the antibody does not necessarily reflect the situation at its binding sites. Low levels of the antibody were detectable up to two days in the circulation of the animals and were obviously sufficient to act at the α1-AR of the target tissue. Data on retention time at the receptor and stability of the receptor bound antibody in vivo are not available yet.For immunizing animals we used a peptide exactly corresponding to the extracellular region of the α1, measurement protocol specific MR parameters , and geometrical parameters like image slice thickness and image orientation, or blood flow velocity. Assuming the T1 of blood and all MR acquisition parameters being the same in all experiments, the enhancement of blood is primarily flow-related and allows for the assessment of vascular impairments, such as reduced flow velocity or turbulences.TOF-MRA allows the depiction of the vascular tree by producing images, in which flowing blood appears bright, while the surrounding stationary tissue is suppressed and appears dark. The amount of flow-related enhancement depends on several factors, including tissue specific magnetic resonance (MR) parameters like the spin-lattice relaxation time T1-AR antibodies concerned larger vessels. Endothelial cells and smooth muscle cells express α1-AR and thus present binding sites for agonistic autoantibodies in small and large vessels. The unphysiological stimulation of α1-AR pathways by agonistic autoantibodies results in pathological changes of cell functions and eventually may lead to substantial cell death and thus to severe damages of vessel structure. Agonistic autoantibodies to the α1-AR were demonstrated to induce hyperplasia of smooth muscle in large vessels such as aorta and mesenteric arteries accompanied by the reduction of the vessel lumen The most striking defects visible in TOF-MRA MIP sections from animals exposed to the action of α1-AR antibodies, relative correlates of vascular circulation were obtained by grayscale analysis of TOF-MRA MIPs. A crucial and critical step to differentiate vessels from surrounding brain tissue was the threshold operation using the median plus two standard deviations. This operation robustly eliminated noise and tissue background, but implied the loss of some smaller or less perfused vessels. The rat is known for the large potential to develop collateral vessels 1-AR antibody. Index values indicated significant attenuation of vascular circulation in α1-AR peptide-immunized rats compared to controls. Considering the loss of a fraction of smaller vessels due to the stringent threshold operation, the observed deleterious effects on brain vasculature may be still underestimated. Furthermore, hemodynamic changes as result of the reduction of vessel lumen, which was reported to be part of α1-AR antibody action, are not addressed by the techniques employed in the present study To affirm the visually detected defects in brain vessels of animals exposed to the action of the αData also clearly demonstrate the tendency for impaired circulation in the brain of both control cohorts at the end of the treatment period. This is in line with the visual observations of vascular defects in some of control animals. Worsening of blood supply in the brain may be attributed to degenerative processes known to typically occur with aging. At the final TOF-MRA measurement the animals were with 17 months relatively old given the average live span of rats.1-AR showed signs of cardiac remodeling, developed left ventricular damages with diastolic dysfunction and vascular damages in aorta and mesenteric artery 1-AR isolated from hypertensive patients and α1-AR antibodies raised in rabbits induced signaling pathways involved in cardiac and vascular remodeling The defects observed by TOF-MRA indicate the loss or the reduction of vascular blood flow in the respective brain region most likely caused by pathological alterations of vessel morphology. Damages of target organs caused by antibodies directed at G protein-coupled receptors were reported in numerous animal studies 1-AR in vascular pathology was shown for peripheral vessels such as aorta and mesenteric artery 1-AR antibodies in the vasculature of the central nervous system of rats. Agonistic antibodies to the α1-AR were found to be associated with different forms of hypertension and type 2 diabetes, widespread diseases with a crucial role of vascular impairments 1-AR to diseases which directly affect the central nervous system such as stroke and dementia. They may be part of the mechanism of age-related cerebral small vessel diseases (CSVD) affecting blood brain barrier (BBB) integrity and known to be a major cause for stroke-like symptoms and pre-stages of vascular dementia 1-AR in the circulation of patients with Alzheimer’s/vascular dementia The importance of antibodies directed at the α1-AR antibodies to cause damages of brain vessels in a rat model. Our data suggest the pathogenic significance of autoimmunity to the α1-AR for diseases of the central nervous system such as stroke and dementia. They further affirm previous evidences that α1-AR autoantibodies are important players in vascular pathology.Taken together, in the present study we provide evidence for the potential of α


AbstractAdephaga and the comparative anatomy of carabid coxal cavities. Ross and Joyce inspired several generations of students at UVM to take up advanced work in entomology and natural history.The lives and contributions of Ross and Joyce Bell are described with particular attention to studies of invertebrate natural history in the state of Vermont and carabid beetles of several groups, including the world rhysodine fauna. Their work, all done at the University of Vermont, was mainly taxonomic in nature and included aspects of the biology of the species considered. During their careers they described more than 75% of the c. 340 rhysodine species known to science. Ross Bell also wrote a number of seminal papers about the basal relationships of the  PageBreakJune 2010, to honor them and recognize their contributions to local natural history in particular, to systematic entomology in general. In the printed Festschrift it is appropriate to provide a brief summary of their lives and their work in entomology. Ross Taylor Bell was born in Champaign, Illinois, on 23 April 1929. His father, Alfred Hannam Bell, was from Ontario, Canada, and studied Geology at the Universities of Toronto (B.Sc.) and Chicago (Ph.D.). He was an influential and productive petroleum geologist with the Illinois Geological Survey in Urbana. Alfred Bell’s second wife, Dorothy Becker of Cincinnati, Ohio, was also a geologist employed at the Survey, having received her B.S. in Geology at the University of Cincinnati. Ross’ parents met after the early death of Alfred’s first wife from tuberculosis. They had three children, Ross and his two sisters Martha and Enid. By all accounts they were a family of enthusiastic naturalists. Both Martha and Enid married students who were contemporaries of Ross in the Department of Zoology at the University of Illinois and, furthermore, Martha became a professional botanist. Family vacations, which included long drives to various points in North America, were invariably a mix of botany, zoology and geology, including all the ‘stuff’ of classical natural history.Preceding production of this Festschrift, long-time friends and associates of Joyce and Ross Bell assembled at the University of Vermont (UVM) in Burlington during 10-13 Ross Bell’s interest in insects began with the childhood gift of an insect collecting kit from his parents. This interest was exacerbated when someone gave him a jar full of silk worm larvae, which he reared on mulberry leaves, incidentally he claims developing his skills as a tree climber. At age 14, Ross got a job at the Natural History Survey at the University of Illinois. In this capacity he trapped flies and rose to the challenge of sorting sarcophagids, muscids and calliphorids and identifying them. The next summer he accepted a position with the taxonomic survey that he thought would take him toward his new found passion for insect identification, but instead he ended up washing glassware, installing moth balls in the collection and gathering box elder leaves for some reason that can no longer be recollected. Because this was during WW II, Ross posits that his eclectic roster of tasks resulted from a shortage of grad students for ‘slave labor’. Very likely these experiences had something to do with the respectful way that he subsequently treated his own students, always encouraging and supporting them to follow their own noses forward, knowing all along that a professor was interested in both them and their scientific findings.PageBreakRoss went to High School at the University Laboratory High School in Champaign, which he recalls as a magnificent place -- and so it must have been because it produced three Nobel Prize Laureates, two of them in science. Ross’s mother always thought that he should’ve done the same, but alas, as Ross pointed out in self-defense, no such prize was awarded in the category of ‘carabidology’. Ross spent the halcyon summers of his youth at his Aunt and Uncle’s farm in West Alexandria, Ohio, helping with farm chores, and at every chance collecting and attempting to name insects from the fields and a stream which bordered the east boundary of the farm. This was his first bash with aquatic insects, something that remained a side interest throughout his career. In his last year of high school he rode his balloon-tired bicycle all the way to West Alexandria. It required two days of pedaling and an overnight at a hotel in Noblesville, Indiana. This 215 mile bicycle ride was an achievement that gave Ross much pleasure, both in the doing and in the subsequent telling.Carabidae (Simplicia) of Illinois. Between 1950-53 he completed a doctoral dissertation about the comparative morphology and phylogeny of Adephaga, under the sponsorship of the well-known entomologist, W. V. Balduf, whose early pioneering work in ‘bionomics’ helped establish the scientific basis for insect ecology in North America. Balduf introduced Ross to Blatchley’s Coleoptera of Indiana in exchange for Ross’s efforts in sorting and counting rose hips for one of Balduf’s studies. Ross became much interested in the proper application of names through exposure to the plant taxonomist E. Neville Jones. With this motivation, Blatchley’s useful keys allowed Ross to confidently name his catches and lured him into the world of beetles . His resulting thesis work on the Adephaga is recognized as a classic among carabidologists and it led to a host of publications on the topic . One of Ross’s best friends during his time at Illinois was “Butterfly” Bob Snetsinger, who became an economic entomologist at Penn State. Spurred on by interactions with Bob, Ross developed interests in life history and larval biology , and passed them on enthusiastically to his students . While at Illinois, Ross also became interested in ecology, was inspired by Professors Victor Shelford, S. Charles Kendeigh and Arthur Vestal, and served as President of the department’s Ecology Club.Ross spent the immediate post-war years (1946-49) at the University of Illinois where he earned a B.S. in Zoology. In one additional year, he earned an M.S. with a thesis about the At graduation and with Ph.D. in hand, Ross was awarded a Fulbright Fellowship to go to India. After receiving his boat ticket he was packing to sail in two weeks when he received a letter from the Selective Service Board ordering him to appear instead for military duty. Thus he got an unexpected all-expense paid trip to Fort Leonard Wood in the winter for basic training. After his basic training he was sent to Fort Dietrich, MD, then known as the nation’s ‘Germ Warfare Center’. Here he spent two years in the company of fleas. He regards his main accomplishment as discovery of a fast way of distinguishing males from females during the sorting of samples; males wear pinstripes, females do not. Upon discharge from the US Army’s flea sorting brigade in 1955, Ross was hired by the Department of Zoology at the University of Vermont to fill a one-year position. The University realized that they had a ‘catch’ and offered him a permanent tenured position a year later. He spent his entire career there as a popular professor contributing excellent service to the University and teaching inspirational courses in Field Zoology, Invertebrate Zoology, Entomology and a summer course for graduate students in Mountain Ecology. During this period he inspired numerous undergraduate students, guided seven graduate students over the course, pursued taxonomic and natural history research on the New England and Vermont faunas and studied rhysodine beetles on a worldwide basis. For most of his career Ross was the Zoology Department’s main strength in systematics and natural history as it connected to ecology and evolutionary biology.PageBreakcompanion at the University of Illinois, who had gone on to teach entomology at the University of Colorado. It was on this trip that Ross found his first undescribed rhysodine beetle species, and so began a thread of passionate discovery that established him as the world’s expert on these interesting beetles . In the following year Ross collected a new entomological companion through marriage to Joyce Elaine Rockenbach of Whitestone, Queens, New York City. Joyce, who had completed her B.S. at Queen’s College, had come to UVM to pursue a Master’s degree after eight years of being a research assistant at the Columbia College of Physicians and Surgeons. Interestingly, Joyce’s graduate advisor at UVM was Reuben Torch, who had been Ross’ assistant lab instructor at the University of Illinois. After ten years of teaching nurses in the UVM School of Nursing, Joyce turned her fulltime attention to entomological pursuits, jointly with Ross. They became inseparable companions in the pursuit of entomology -- especially of carabid lore – and through these endeavors also became well-known pillars of the Vermont naturalists community. As Ross’ eyesight waned, Joyce became his eyes and illustrator for the taxonomic work. Her skills as an excellent microscopist were much appreciated by Ross and his students. Although her grandfather had told her that ‘a woman can either teach or be a nurse’, Joyce Bell became an excellent entomologist in her own right.Shortly after joining UVM, Ross began a program of summer collecting followed by taxonomic work that would characterize his research career. He spent the summer of 1956 collecting insects in Mexico with Don Van Horn, a graduate student The newlyweds went on a honeymoon ‘collecting’ trip to Bar Harbor, during which they recall that they spent more time making car repairs than collecting beetles. The following summer they collected on Cape Breton, Nova Scotia, and also in the Great Smoky Mountains, where they met career-long ‘beetle buddies’ Tom Barr and Willy Rosenberg. He and Joyce returned to Mexico in the summer of 1959, driving from Burlington to Chiapas where they stayed with Franz Blom in San Cristobal. Franz was an archeologist-anthropologist, who worked with the indigenous people of the area, and his wife, Trudy, was a Swiss photographer studying the Lacondon Indians of the forests in the area. Franz had purchased a monastery and used it as his home and research base. He generously took in grad students, mainly in anthropology, and mainly from the University of Michigan, but also opened his doors to other people who wanted to study in the region. A stay in the monastery came with room and board and free evening fireside chats. The food, fondly recalled as ‘tasty Mexican dishes, including memorable stuffed squash blossoms’, came mainly from the Blom garden. During their return to Vermont, Ross and Joyce stopped in Arizona near Nogales in the Chiricahua Mountains where they met for the first time George and Kay Ball, their 2 sons and Ron Madge. So began another career-long association and beetle intensive interaction.PageBreakrmont. During three summers spent in Stowe, they collected extensively on and around Mt. Mansfield, the state’s highest mountain. They also spent summers collecting in northeastern Vermont around Lake Willoughby, in southern Vermont based near Manchester and at other locations in the state. Through this work they built the UVM Entomological Collection into a significant resource for northern New England. In Victory Bog on the Moose River near Lake Willoughby, Andy Moldenke and the Bells first collected Bembidion bellorum, eventually described and named in their honor by David Maddison.During the 1960s the Bells began a program to learn the fauna of Vermont and to compile extensive records of natural history information. Their goal, as explained to us, was to provide a scientific basis for the study of arthropods in Vermont and to facilitate proper identification of insect specimens. Ross and Joyce developed a system of establishing a base camp of operation in an area and then collecting extensively in that general locale. For example, over a span of eight years they rented ‘summer houses’ around VePlatypatrobus lacustris. This was an exciting find because the beetle was known from only two puzzling specimens  and its habits were virtually unknown. Once advised of this capture, Phil Darlington and his wife, Libby, drove immediately from Harvard up to Vermont and they all invested much energy in searching for the habitat of this rare catch. Unfortunately they were not successful; however, the story did not end there, but instead the mystery was solved through the web of carabidological connections in which Ross and Joyce were enmeshed, as elaborated below.The Bells frequently ran light traps during these summer evenings, and this activity contributed much to the excitement of the chase. One night, for example, the light trapping garnered three specimens of Platypatrobus lacustris lived in beaver houses. Despite having extensively searched the beaver meadow near the light trap location, the Bells and Darlingtons had not thought to disassemble the house itself and so the discovery eluded them. During the Stowe summers Ross and Joyce also met Ken Cooper, then at Dartmouth. Although a wasp specialist, Ken was also interested in carabids, and a number of techniques that Ken developed, including methodology for everting the internal sac of carabid males, made their way into the playbooks used by Ross and his students. During a summer spent in southern Vermont, they met Carl Parsons, an ex-UVM professor who had abandoned academe to open a bookstore in Manchester. Parsons was an avid collector who willed his excellent Vermont collection to the University, and thus the room that houses the entomological collections in historic Torrey Hall at UVM was named in his honor.Pursuit of their ‘naturalist’ activities led to numerous intersections between the Bells and others of entomological persuasion, especially those interested in ground beetles. These connections were of great benefit, interest and inspiration to Ross and his students. For example, in 1967 Carl Lindroth, the famous Swedish entomologist and carabid aficionado, visited the Bells and gave a talk at UVM about his work on the faunal development of Surtsey Island, Iceland, after its then recent volcanic uprising from the ocean floor. This drew three Quebec entomologists to Vermont and provided the first connection between the Bells and André Larochelle and Henri Goulet. Only later, however, did Henri Goulet discover that PageBreaksociety’s summer field trips and provided much input for the VES Newsletter. This newsletter has become a heartbeat for entomology in Vermont and it is edited by Trish Hanson, one of Ross’ graduate students and who is now an entomologist with the Vermont Forest, Parks and Recreation Department.Although this valuable aspect of the careers of academics is frequently underestimated, interested people with active research programs become the nodes of scholarly interaction that link and encourage development of research communities. Ross and Joyce Bell were for many years a significant link in North America’s carabidological community, and a central node for entomology in the State of Vermont. They have been mainstays of the Vermont Entomological Society (VES) founded in 1992. Ross served as one of the first Presidents of the VES, and the Bells participated in the Carabidae of the area (Bell 1971) as well as a key to the darkling beetles of the area. In 1974 the Bells joined newly-hired UVM colleague and mammalogist Charles Woods on a trip to Haiti. While Charles looked for mammals, the Bells enriched their rhysodine collection and were drawn further into the mysterious tunnels of rhysodine biology. In 1982 Ross had a sabbatical which took Joyce and he to New Zealand and Papua New Guinea, where they worked out of the Wau Ecological Institute in the northeastern New Guinea Highlands. They were introduced to the Institute by Lindsay and Peg Gressitt of the Bernice P. Bishop Museum, who were killed tragically later that year in a plane crash in China. In addition to fending off burglaries by natives, they spent many days climbing Mt. Missim searching for rhysodines and other entomological treasures. One of those rhysodines was an undescribed species, named by Ross as Omoglymmius (s. str.) gressitti in honor of Gressitt. Their ‘guard’ at Wau was a reformed cannibal who sported a bone through his nose and carried a spear, and Joyce spent many a sleepless night fearing that the guard might have a relapse in habit.The 1970’s and 1980’s were very busy but stimulating years for Ross and Joyce, partly because they extended the geographical boundaries for their entomological work beyond Vermont. For example, in 1970 Ross was hired for the summer to work on the NSF ‘Biome Project’ on the short grass prairie of Pawnee, Colorado. Ross produced keys to  In 1989-90 Ross took a second sabbatical during which he and Joyce were ‘down under’ in Australia, this time headquartered in Canberra with the CSIRO . During this trip they undertook two extensive collecting trips in search of rhysodines, one to Tasmania and the other to Queensland. The brilliant carabidologist, Barry Moore, was their host, friend and tour guide for the year. Ross and Joyce fondly recall his enormous contributions to their understanding of Australian natural history. Upon their return from the last trip to Australia, Jonathan Leonard proposed that he and Ross should write a book on Tiger beetles of the Northeastern United States. Thus, the “Northeastern Tiger Beetles” (Leonard and Bell 1999) was born as the definitive reference to these beetles, with color plates of all the species and including keys for third instar larval identification.PageBreak married and immigrated to Tasmania where Mike took a job teaching High School Biology in Huonville. Mike was recently awarded Australia’s highest award for high school teaching and remains active as a buprestid enthusiast and collector, contributing among other things Tasmanian carabids of interest to the work of Bob Davidson at CMNH – a further example of continuing entomological threads starting with the Bells. Students fortunate to be part of the summer collecting blitzes learned the fascination of finding new things and learning to put names on them effectively. In the big picture they also learned about how such activities form the basis for zoological research that connects to what people can observe directly in their own backyards. There were ancillary lessons; e.g., that gin and tonic is not just some high-brow concoction, but also a great pre-dinner drink on a sultry July evening, or that Joyce Bell was an excellent and adventurous cook, in addition to having rare talents for making unusual entomological discoveries!Students at UVM were attracted to entomological work because of Ross’ informative teaching style, filled with anecdotes about the doing of natural history, and his clear philosophy of helping others learn how to construct and pursue their own dreams. He was among a handful of professors at UVM during those times whose research work connected to field biology and natural history, and thus was a lightning rod for many students interested in these areas. The Bells generously involved many students in their summer research adventures. For example, the late Bob Mills, who was an accomplished Biology teacher at Putney School, was an able, enthusiastic assistant during the summers in Stowe. Mike Bouffard also worked with the Bells as an undergraduate student. He and Reni Shangraw, another student who worked in Biology with Ross,Chlaenius also flowed downstream from work that Ross had done . Bob, who began as an English major at UVM, was coaxed into entomology through exposure to Ross’ course, and began to collect and study carabids as an undergraduate. Encouraged by the Bells, he continued to collect during his three years in the Peace Corps in Nepal (many new carabids subsequently described), and during this time was invited to return to UVM as Ross’ graduate student in entomology, which led to a successful career as a coleopterist and curator at the Carnegie Museum of Natural History. John Spence’s Master’s work on Nebria grew out of a discussion that linked Ross’ first publication (Bell 1955) to observations of the day on the rocky banks of Gleason Brook. This work and Denise Martin Leonard’s M.S. project about stoneflies involved periodic visits to many stations along the length of the stream at different elevations, and through active participation in these research projects, Ross and Joyce came to know virtually every inch of ‘the Brook’. In fact, Gleason Brook and the adjacent North-facing slopes of Camel’s Hump loom large in Ross’ life as a special area for both teaching and research. Other students, including Brian Farrell, Jonathan Leonard, John Strazanac, and Peter Wimmer participated in the ‘Bell natural history blitzes’ and went on to successful careers in entomology.Many of the things that Ross pursued were eventually connected to – or even expanded in – work done by his students. For example, Bart Chiolino, a promising Vermont student, worked as Ross’ assistant during the summer in Colorado, before completing an M.S. on wing-dimorphism in ground beetles. During the summer spent in the area of Lake Willoughby they were joined by Andy Moldenke, a summer research student on leave from Wesleyan College. Moldenke subsequently earned a doctorate in entomology at Stanford University and spent a productive career as a soil biologist at Oregon State University. Bob Davidson’s extensive treatments of PageBreakBrian Farrell undertook an all-taxon biotic inventory of the Boston Harbor Islands, the carabid portion of which was done in collaboration with Davidson and Bell. Other graduates of the Field Naturalist program who pursued entomological interests include Jeff Collins, Susan Morgan and Mark Ward.Ross’ Invertebrate Zoology course became especially popular with graduate students in the Field Naturalist Program at UVM in the 1990s, and this experience inspired Jessica Rykken, for example, to study the role of carabids as indicators of land types in the Green Mountains. She then followed the Bell network west to study carabids and other invertebrates in riparian areas with Andy Moldenke, and later with China and SE Asia. Most recently, Ross has recognized specimens that David Kavanaugh collected in Yunnan as belonging to three new species of three rhysodine genera (Bell and Bell 2011). Ross continues scientific activity as Research Associate with the Carnegie Museum in Pittsburgh. Ross has now turned his attention to the perplexing matters of rhysodine zoogeography, hoping to find some sensible patterns in the wealth of new data that he and Joyce have generated. They have more than quadrupled our knowledge of rhysodines by adding description of c. 260 new species to the list of about 80 known when they started.Ross retired from UVM in 2000 but spent four subsequent summers teaching a field course in entomology at UVM. He continues his work with rhysodine beetles and many collaborators in Europe still send him specimens mainly from expeditions to pallipes group of Nebria in eastern United States . Proceedings of the Entomological Society of Washington 57: 265-267.Bell RT (1955) Species of the Carabus auratus L.  in North America. Proceedings of the Entomological Society of Washington59: 254.Bell RT (1957) Chlaenius tomentosus (Say) . Annals of the Entomological Society of America 51: 432-435.Bell RT (1958) Intraspecific variation in Scaphinotus Dej., intermediate between Scaphinotus s. str. and Irichroa Newman . Proceedings of the Entomological Society of Washington 61: 11-13.Bell RT (1959) A new species of Chlaenius Bonelli  in North America. Miscellaneous Publications of the Entomological Society of America1: 97-166.Bell RT (1960) A revision of the genus Bell, RT, Bell JR (1962) The taxonomic position of the Rhysodidae (Coleoptera). The Coleopterists Bulletin 16: 99-106.Gehringia belong to the Isochaeta? (Coleoptera: Carabidae). The Coleopterists Bulletin 18: 59-61.Bell RT (1964) Does Platypatrobus lacustris Darlington in Vermont (Coleoptera: Carabidae). Proceedings of the Entomological Society of Washington 66: 100.Bell, RT, Bell JR (1964) Bell RT (1965) Coxal cavities and the phylogeny of the Adephaga. Proceedings of the XIIth International Congress of Entomology, pp. 80-81.Trachypachus and the origin of the Hydradephaga (Coleoptera). The Coleopterists Bulletin 20: 107-112.Bell RT (1966a) Chlaenius patruelis LeConte a valid species (Coleoptera: Carabidae). Proceedings of the Entomological Society of Washington 68: 321-322.PageBreakBell RT (1966b) Bell RT (1967) Coxal cavities and the classification of the Adephaga (Coleoptera). Annals of the Entomological Society of America60: 101-107.Bell RT (1970) The Rhysodini of North America, Central America, and the West Indies (Coleoptera: Carabidae or Rhysodidae). Miscellaneous Publications of the Entomological Society of America6: 289-324.Bell RT (1971a) Carabidae (ground beetles). Grassland Biome, United States International Biological Program, Technical Report  No. 66: 58 pages.Bell RT (1971b) Handbook of the Malacostraca of Vermont and neighboring regions. Privately printed. 65 pages.Clinidium from Guatemala . Proceedings of the Entomological Society of Washington 75: 279-282.Bell RT (1973) A new species of Omoglymmius Ganglbauer, a separate genus (Coleoptera: Carabidae or Rhysodidae). The Coleopterists Bulletin 29: 351-352.Bell RT (1975) Clinidium (Coleoptera: Rhysodidae or Carabidae) from eastern U. S., with a revised key to U. S. Clinidium. The Coleopterists Bulletin 29: 65-68.Bell RT Bell JR (1975) Two new taxa of Nebria lacustris (Casey) and Nebria pallipes (Say) . The Coleopterists Bulletin 30: 81-84.Spence JR, Bell RT , Bell JR (1976) The larvae of Bell RT (1977) Ergebnisse der Bhutan-Expedition 1972 des Naturhistorischen Museums in Basel Coleoptera: Fam. Rhysodidae. Entomologica Basiliensia 2: 151-158.Platynus opaculus LeConte (Coleoptera: Carabidae) in Vermont. Cordulia 3: 157-158.Davidson RL, Bell RT (1977) Note on the distribution and habitat of Nebria suturalis LeConte in Vermont (Coleoptera: Carabidae). Cordulia4: 82.Bell RT (1978) The habitat of Omoglymmius, subgenus Nitiglymmius, new subgenus (Coleoptera: Carabidae or Rhysodidae). Quaestiones Entomologicae 14: 43-88.Bell RT, Bell JR (1978) Rhysodini of the world. Part I. A new classification of the tribe, and a synopsis of Evarthrus sodalis sodalis LeConte in Vermont (Coleoptera: Carabidae). Cordulia 4:Bell RT, Nielsen GR (1978) Bell RT, Bell JR (1979) Rhysodini of the world. Part II. Revisions of the smaller genera (Coleoptera: Carabidae or Rhysodidae). Quaestiones Entomologicae 15: 377-446.Bell RT (1979) Zoogeography of Rhysodini--do beetles travel on driftwood? In: Erwin, TL, Ball GE, Whitehead DR, Halpern AL (Eds.). Carabid Beetles: their Evolution, Natural History and Classification. Proceedings of the First International Symposium of Carabidology, Smithsonian Institution, Washington, D. C., August 21, 23 and 25, 1976. Dr. W Junk bv Publishers, The Hague-Boston-London, 331-342.Bell RT, Bell JR (1981) Insects of Micronesia, Coleoptera, Rhysodidae. Bernice P. Bishop Museum Insects of Micronesia 15(2): 51-67.Omoglymmius Ganglbauer (Coleoptera: Carabidae or Rhysodidae) and substitutions for preoccupied generic names. Quaestiones Entomologicae 18: 127-259.Bell RT, Bell JR (1982) Rhysodini of the world. Part III. Revision of PageBreakBell RT (1985a) A catalog of the Coleoptera of America north of Mexico. Family: Rhysodidae. U. S. Department of Agriculture, Agriculture Handbook 529-4, fascicle 4: x + 4 pages.Bell RT (1985b) Zoogeography and ecology of New Guinea Rhysodini (Coleoptera: Carabidae). In: Ball, GE (Ed.). Taxonomy, Phylogeny and Zoogeography of Beetles and Ants. Dr. W. Junk Publishers, Dordrecht, 221-236.) Pentagonica of the West Indies (Coleoptera: Carabidae). The Coleopterists Bulletin 39: 321-327.Bell RT . Quaestiones Entomologicae 21: 1-172.Bell RT, Bell JR (1985) Rhysodini of the world. Part IV. Revisions of Pentagonica Schmidt-Goebel (Coleoptera: Carabidae) from the southwestern United States. The Coleopterists Bulletin 41: 373-376.Bell RT (1987) A new species of Clinidium Kirby (Coleoptera: Carabidae or Rhysodidae) from Mexico, and descriptions of the females of two Neotropical members of the genus. Annals of Carnegie Museum 56:193-196.Bell RT, Bell JR (1987a) A new species of Bell RT, Bell JR (1987b) A new subtribe, genus and species of Rhysodini from South Africa (Coleoptera: Carabidae or Rhysodidae). Journal of the Entomological Society of Southern Africa 50: 287-290.Yamatosa, and a major range extension for Omoglymmius sakuraii Nakane (Coleoptera: Carabidae or Rhysodidae). Revue suisse de Zoologie 94 : 683-686.Bell RT, Bell JR (1987c) Rhysodine beetles in the Geneva collection: a new species of Harpalus rubripes (Duftschmid), a European ground beetle new to North America (Coleoptera: Carabidae). The Coleopterists Bulletin 41: 56.Bell RT, Davidson RL (1987) Bell RT, Bell JR (1988) Rhysodini of Sulawesi and nearby islands (Coleoptera: Carabidae or Rhysodidae). Journal of the New York Entomological Society 96: 7-15.chimbu group of Scopodes (Coleoptera: Carabidae). The Coleopterists Bulletin 43: 157-161.Bell RT, Bell JR (1989a) Revision of the Yamatosa and Omoglymmius, descriptions of undescribed sexes in other species, and some major range extensions (Coleoptera: Carabidae or Rhysodidae). Revue suisse de Zoologie96 : 637-642.Bell RT, Bell JR (1989b) Rhysodine beetles in the Geneva collection II: new species of In: Dindal, DL (Ed.). Soil Biology Guide. John Wiley and Sons, New York. xx + 1,349 pages.Bell RT (1990) Chapter 35. Insecta: Coleoptera, Carabidae, Adults and Larvae. In: Stehr, FW (Ed.). Immature Insects. Volume 2. Kendall/Hunt Publishing Co., Dubuque, Iowa, 1053-1092.Bell RT (1991) Rhysodidae (Adephaga). Pages 304-305 Bell RT, Bell JR (1991) The Rhysodini of Australia (Insecta: Coleoptera: Carabidae or Rhysodidae). Annals of Carnegie Museum 60: 179-210.Bembidion obtusum (Coleoptera: Carabidae) in eastern North America. Entomological News102: 174-178.Bell RT, Hoebeke ER, Liebherr JK (1991) Revised distribution of the immigrant carabid In: Noonan, GR, GE Ball and NE Stork (Eds.). The Biogeography of Ground Beetles of Mountains and Islands. Intercept Ltd., Andover, United Kingdom, 43-52.PageBreakBell RT (1992) The carabid fauna of the New England mountains (Coleoptera: Carabidae). Omoglymmius (subgenera Omoglymmius and Pyxiglymmius). Annals of Carnegie Museum 62: 165-185.Bell RT, Bell JR (1993) Rhysodine beetles (Insecta: Coleoptera: Carabidae or Rhysodidae): new species, new data and revised keys to Bell RT (1994) Beetles than cannot bite: functional morphology of the head of adult rhysodines (Coleoptera: Carabidae or Rhysodidae). The Canadian Entomologist126: 667-672. doi: 10.4039/Ent126667-3Bell RT, Bell JR (1995) The Rhysodini (Insecta: Coleoptera: Carabidae) of Cuba. Annals of Carnegie Museum 64: 185-195.In: Ball, GE, A Casale and A Vigna Taglianti (Eds.) Phylogeny and Classification of Caraboidea (Coleoptera: Adephaga). Proceedings of a Symposium  XX International Congress of Entomology. Atti, Museo regionale di Scienze naturale, Torino 261-272.Bell RT (1998) Where do the Rhysodini belong? http://tolweb.org/Rhysodini/67/1999.12.14. In: The Tree of Life Web Project, http://tolweb.org/ Bell RT (1999) Rhysodini. Wrinkled Bark Beetles. Version 14 December 1999. Leonard JG, Bell RT (1999) Northeastern Tiger Beetles: a field guide to the tiger beetles of New England and eastern Canada. CRC Press, Boca Raton, Florida. 176 pages.Bell RT, Bell JR (2000) Rhysodine beetles (Insecta: Coleoptera: Carabidae or Rhysodidae): new species, new data II. Annals of Carnegie Museum 69:69-91.Yamatosa Bell and Bell and Omoglymmius (Pyxiglymmius) Bell and Bell. Stuttgarter Beiträge zur Naturkunde, Serie A (Biologie), Nr. 636:1-7.Bell RT, Bell JR (2002) Two new species of Rhysodini (Coleoptera: Carabidae) with revised keys to Bell RT (2003) Family Rhysodidae Laporte, 1840. In: Löbl, I, and A. Smetana (Eds.). Catalogue of Palaearctic Coleoptera. Volume I. Archostemata - Myxophaga - Adephaga. Apollo Books, Stenstrup, 78.Bell RT, Bell JR (2009) Rhysodine beetles (Insecta: Coleoptera: Carabidae): new species, new data III. Annals of Carnegie Museum 78: 45-77. doi: 10.2992/007.078.0104Bell, RT (2010) Les Rhysodini de Guyane . Suppl. Bulletin de liason d’ACOREP-France “Les Coleopteriste”, Tome II: 46-49.Grouvellina Bell & Bell and the mishmicus group of Rhyzodiastes Fairmaire. Stuttgarter Beiträge zur Naturkunde A, Neue Serie 4: 129-135.Bell RT, JR Bell (2011) Four new species of Rhysodini (Coleoptera: Carabidae) with revised keys to


Androgen receptors (AR) are frequently expressed in breast cancers, but their implication in cancer growth is still controversial. In the present study, we further investigated the role of the androgen/AR pathway in breast cancer development.AR expression was evaluated by immunochemistry in a cohort of 528 postmenopausal breast cancer patients previously examined for the association of serum testosterone levels with patient and tumor characteristics. AR expression was classified according to the percentage of stained cells: AR-absent (0%) and AR-poorly (1%-30%), AR-moderately (>30%-60%), and AR-highly (>60%) positive.P trend<0.001). Mean testosterone levels were significantly higher in the AR-high category than in the other categories combined (P=0.022), although a trend across the AR expression categories was not present. When women defined by ER status were analyzed separately, regression analysis in the ER-positive group showed a significant association of high testosterone levels with AR-highly-positive expression , but the association was essentially due to patients greater than or equal to 65 years . In ER-positive group, elevated testosterone levels appeared also associated with AR-absent expression, although the small number of patients in this category limited the appearance of significant effects : the association was present in both age groups (<65 and ≥65 years). In the ER-negative group, elevated testosterone levels were found associated (borderline significance) with AR-absent expression . In this ER-negative/AR-absent subset of tumors, elevated testosterone levels cannot stimulate cancer growth either directly or after conversion into estrogens, but they probably induce increased synthesis of some other substance that is responsible for cancer growth through binding to its specific receptor.Statistical analysis was performed in 451 patients who experienced natural menopause. AR-high expression was significantly related with low histologic grade and estrogen receptor (ER)- and progesterone receptor (PR)-positive status (The findings in the present study confirm that testosterone levels are a marker of hormone-dependent breast cancer and suggest that the contemporary evaluation of ER status, AR expression, and circulating testosterone levels may identify different subsets of cancers whose growth may be influenced by androgens. Androgen receptors (AR) are expressed in more than 70% of primary breast cancers -11, in uWe recently examined the relationships of serum testosterone levels with some tumor characteristics in a cohort of postmenopausal breast cancer patients and found that high levels of testosterone were significantly associated with the ER-positive status of tumors ,14, a fiIn the present study, we examined AR expression in tumors of the same cohort of postmenopausal patients in whom we had already measured testosterone levels. With the aim to further investigate the role of the androgen/AR pathway in breast cancer development, we evaluated the relationships of AR expression with testosterone levels, with age and body mass index (BMI) of patients and with selected tumor characteristics, both in the whole cohort and in age categories (<65 and ≥65 years).Of the 592 initial postmenopausal breast cancer women recruited in the TPM  cohort, for 538 we had information on AR expression. Inclusion and exclusion criteria of the TPM cohort were extensively presented elsewhere ,14 and aOf the 538 women with information on AR expression, 2 women aged 51–52 were excluded because they were not considered postmenopausal on the basis of their estradiol level . Eight women were excluded because they proved to fall outside the recruitment criteria. Specifically, one was excluded because the date of the last menstruation was less than 1 year before the date of recruitment, and the other seven women were excluded because they had stopped hormone replacement therapy only 3 months before recruitment. Thus, 528 women were available for the present analysis.Mean age at recruitment was 66.8 years . Four hundred and fifty-one women were going through natural  menopause ; of the remaining 77 women, 42 had undergone hysterectomy, 32 had undergone bilateral ovariectomy, and 3, with uncertain self-reported information, were classified as missing status for menopause type. This cross-sectional study is mainly addressed to those 451 naturally menopausal breast cancer patients. Analyses on all  postmenopausal breast cancer patients are included in the Additional file Testosterone assay, already described elsewhere , was perAR expression was evaluated using tissue microarrays. Slides and paraffin blocks of 538 consecutive invasive breast cancers were retrieved from the archives of the department of pathology of the INT. All slides hematoxylin and eosin stained were reviewed by two pathologists, and the slide with the most representative tumor section was selected for each case. Three representative core tissue samples (1.5 mm in diameter) were taken from different areas of the invasive tumor and an additional one was from benign breast tissue, when present, and were assembled in 45 tissue microarray paraffin blocks using a Galileo TMA CK3500 Tissue Microarrayer. From each tissue microarray block, 4-mm-thick sections were cut and immunostained for monoclonal mouse anti-human androgen receptor, Dako  using Dako Autostainer.ER and PR status were defined as negative when the percentage of stained tumor cells was <10% and as positive for ≥10% of stained tumor cells, in accordance with published guidelines . No guidDifferences in patient (age and BMI) and tumor characteristics  between AR expression categories were investigated by the chi-square test for trend.P values. Trends across AR expression categories were tested by the nonparametric Cuzick test.Testosterone circulating levels were square-root transformed, as the distribution of concentrations was not normal. Fisher’s test was used to assess overall differences in mean testosterone levels by categories of AR expression, and linear contrasts were used for post-hoc comparisons of the high AR category versus absent, poor and moderate AR combined categories and of the absent AR category versus poor and moderate AR combined categories. In case of multiple comparisons, the Bonferroni method was used to adjust Logistic regression analysis was used to assess the age-adjusted association between testosterone and AR expression, and the age-adjusted odds ratios (OR) of being in a given AR expression category rather than in the reference category were estimated across testosterone tertiles. The ER status resulted as a determinant of the relationship between testosterone and AR expression. In particular, (a) in the ER-positive group, the frequency of women with low AR expression did not differ from that of women with moderate AR expression, thus the poor and moderate AR expression categories were pooled and used as reference category in estimating the age-adjusted OR of developing a tumor with absent or high AR expression versus one with the reference AR expression. (b) In the ER-negative group, there were no differences between low, moderate or high AR expression, so all three categories of positive AR expressions were pooled together and used as reference in a binomial logistic model estimating OR of having AR-absent instead of AR-positive tumors.Testosterone tertiles were categorized according to the distribution of women in natural menopause. Although the likelihood ratio test did not reveal any significant interaction between age and testosterone, data were separately analyzed in order to take into account the hormonal pattern modification during years after the menopause in women <65 and those ≥65 years of age.P values refer to two-sided statistical tests; differences with P≤0.05 were considered significant. Analyses were performed with the Stata statistical package, 9.2 (2007) release .Ninety-five percent confidence intervals (95% CI) were estimated. Linear trends in OR were tested using ordinal variables of testosterone tertiles. All P=0.072), more frequently had a BMI ≥30 kg/m2 , more frequently had an ER-negative tumor , and more frequently had lower mean testosterone levels . Furthermore, no association between AR expression and testosterone levels was found for women who had had surgical menopause [see Additional file Of 528 postmenopausal women, 451 (85.4%) were naturally menopausal and 74 (14.0%) had had a surgical menopause . Compared to naturally menopausal women, those who had had surgical menopause were more frequently in the ≥65 year age class , as did the frequency of women with ER-positive or PR-positive tumors: 53%, 82%, 89%, and 94% (P for trend <0.001), and 42%, 61%, 69%, and 77% (P for trend <0.001), respectively. In the HER2-positive group, the incidence of women with poor and moderate AR expression tended to be higher than that of women with AR-absent or AR-high expression (58% and 52% vs. 41%). In these naturally menopausal women, AR expression did not appear to be related to BMI or nodal status.Table P=0.002), although a trend across the AR expression categories was not shown. When women defined by ER status were separately analyzed, those in the ER-negative group showed significantly lower mean testosterone levels than those in the ER-positive group . In the ER-positive group, women with high AR expression had the highest testosterone level (linear contrast: Bonferroni adjusted P=0.022), yet women in the AR-absent category showed high testosterone levels, although not significantly different from those in low and moderate combined AR categories (linear contrast: Bonferroni adjusted P=0.216). In the ER-negative group, women with AR-positive expressions had lower, not significantly, testosterone levels than women in the AR-absent category.Mean testosterone levels by categories of AR expression are presented in Table Logistic regression analysis further illustrated that ER-negative and ER-positive groups showed different age-adjusted relationships between testosterone and AR expression Table . For womIn the present study, we explored the role of AR in breast cancer by evaluating relationships between AR expression, serum testosterone levels, and some patient  and tumor characteristics  in a cohort of postmenopausal patients. The association of testosterone and AR expression was more evident in patients who experienced natural menopause than in the whole cohort, which included patients with surgical menopause  was present in about 85% of tumors in our cohort, a percentage comparable to that of the other studies ,16, whicOur finding that AR are more frequently, although not significantly, expressed in older than in younger postmenopausal patients has also been reported in several other studies ,10,12. TOur finding that elevated serum testosterone levels are significantly related with both ER-positivity  and AR-pSumming up our reasoning on hormone-dependent breast cancer growth, we suggest that most of the findings reported in the literature, including high intratumor concentrations of androgens and estrogens, elevated expression of estrogen-producing and androgen-producing enzymes, increased expression of ER and AR, can be explained by an androgen excess. Furthermore, looking at breast cancer growth under the viewpoint of the androgen excess, the elevated intratumor levels of androgens and estrogens should be regarded as two different sides of the same endocrine abnormality of the woman with cancer, i.e., an androgen excess, thus bypassing the problem of whether androgens inhibit or stimulate breast cancer growth.P=0.043). It is noteworthy that in women with ER-negative tumors the association between testosterone levels and AR expression substantially weakened and virtually disappeared when we classified as AR-negative those tumors with ≤10% or with ≤30% of stained cells, respectively. In the AR-absent/ER-negative subset, elevated androgen levels cannot stimulate cancer growth either directly or after conversion into estrogens, but they probably stimulate increased production of some other substance which is responsible for cancer growth through binding to its specific receptor. We suggest that such a substance may be the epidermal growth factor (EGF), whose synthesis and function is under the control of androgens [In the present study, we regarded the AR-absent group as the negative group and classified the other tumors as poorly positive, moderately positive and highly positive according to the percentage of stained cells. The AR-absent group included about 13% of the patients in our cohort: it was characterized by high serum testosterone levels, comparable to those found in patients with highly positive-AR expression and substantially higher than those in poorly and in moderately AR-positive groups. About 47% of AR-absent tumors were also ER-negative, representing approximately 6% of the whole cohort. Our finding that elevated testosterone levels were associated with AR-absent expression in ER-negative tumors identified a particular subset of cancers whose growth may be stimulated by androgens. The positive association between testosterone and tumor size remained significant in this group: mean testosterone levels were 0.348±0.176 for tumor size <2 cm and 0.482±0.188 for tumor size ≥2 cm . In the ER-negative subset, HER2 overexpression was found significantly associated with AR positivity .HER2 expression was examined in 70% of the tumors of our cohort. We did not find a significant association between HER2 and AR expression in the whole cohort, but when we divided women by ER status, HER2 overexpression showed a significant inverse relationship with AR-high expression in the ER-positive subset  ,49-51. ASumming up findings in the present study, we have shown that (a) relationships between AR expression and tumor characteristics are in agreement with reports in the literature; (b) the association with AR expression confirms testosterone levels as a marker of hormone-dependent disease; (c) a subset of patients characterized by AR-absent expression and elevated levels of testosterone has been identified; and (d) the contemporary evaluation of ER status, AR expression, and circulating testosterone levels may identify different subsets of cancers whose growth may be influenced by androgens. These findings provide further support to the androgen excess theory of breast cancer, which points to androgen excess as a stimulatory hormonal alteration common to several breast cancer types, both ER-positive and ER-negative . Breast AR: Androgen receptors; BMI: Body mass index; DHEA: Dehydroepiandrosterone; EGF: Epidermal growth factor; EGFR: Human epidermal growth factor receptor; HER1: Human epidermal growth factor receptor 1; HER2: Human epidermal growth factor receptor 2; ER: Estrogen receptor; INT: Fondazione IRCSS Istituto Nazionale dei Tumori; 95% CI: Ninety-five percent confidence intervals; OR: Odds ratio; PR: Progesterone receptor; RIA: Radioimmunoassay; TPM: Testosterone, prognosis, mammary cancer.The authors declare that they have no competing interests.Giorgio Secreto and Andrea Micheli: conception and design of the study, interpretation of data and drafting of the manuscript. Elisabetta Meneghini: analysis and interpretation of data and drafting of the manuscript. Elisabetta Venturelli: acquisition and interpretation of data, and drafting of the manuscript. Maria Luisa Carcangiu and Biagio Paolini: evaluation of AR expression. Roberto Agresti, Patrizia Cogliati, Giuseppina Saragò, Cristina Pellitteri: acquisition of data. Franco Berrino and Massimo Gion: critical revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/599/prepubA word file containing tables and relating comments of supplementary analyses on all  and on surgical postmenopausal women. Additional Table S1: Androgen receptor (AR) expression and characteristics of all postmenopausal breast cancer women. Additional Table S2: Serum testosterone (mean ± SD) by androgen and estrogen receptor expression in breast cancer women who had had a surgical menopause. Additional Table S3: Odds ratios of AR expression by testosterone tertiles in all postmenopausal women with ER-positive breast cancer. Additional Table S4: Odds ratios of AR expression by testosterone tertiles in postmenopausal women with ER-negative breast cancer .Click here for file


Partial androgen insensitivity syndrome (PAIS) is associated with impaired male genital development and can be transmitted through mutations in the androgen receptor (AR). The aim of this study is to develop a cell model suitable for studying the impact AR mutations might have on AR interacting proteins. For this purpose, male genital development relevant mouse cell lines were genetically modified to express a tagged version of wild-type AR, allowing copurification of multiprotein complexes under native conditions followed by mass spectrometry. We report 57 known wild-type AR-interacting proteins identified in cells grown under proliferating and 65 under nonproliferating conditions. Of those, 47 were common to both samples suggesting different AR protein complex components in proliferating and proliferation-inhibited cells from the mouse proximal caput epididymus. These preliminary results now allow future studies to focus on replacing wild-type AR with mutant AR to uncover differences in protein interactions caused by AR mutations involved in PAIS. Often mutations in the androgen receptor (AR) are involved which interfere with ligand binding, DNA binding, or increase or decrease intramolecular interactions between AR domains . Where nin vivo , the sigin vivo  and MFVDin vivo  were genin vivo , 7 and tin vivo . By focuThe N-TAP was designed by modifying the C-terminal tandem affinity tag (C-TAP) from Fernández et al., 2009 [5 COS-1 or Hela cells/well were seeded into 12-well tissue culture plates in DMEM, containing 10% charcoal-stripped serum. Cells were transiently transfected using Fugene (Roche) or Lipofectamine 2000 (Invitrogen) with 25 ng AR or N-TAP-mAR, 500 ng of pGRE-luciferase and 25 ng pTK-RL according to manufacturer's instructions. 12–16 hours after transfection, the medium was replaced with DMEM, containing 10% charcoal-stripped serum + or −10 nM dihydrotestosterone . 24 h later, cells were harvested and lysed in 25 mM glycine (pH 7.8), 15 mM MgSO4, 4 mM EGTA, 1% triton × 100 and 1 mM dithiothreitol. Luciferase assays were performed with reagents from nanolight technology and the ratio of luciferin : renillaluciferase activity was measured using a Turner TD-20/20 luminometer. Standard error bars relate to three independent transfection experiments.In total 10d.p.f.) mouse vas deferens and show features of Wolffian duct mesenchymal cells and androgen responsiveness [Androgen responsive cell lines PC1 and MFVD from the mouse urogenital tract served as control cell lines in their nonmodified state. The PC 1 cell line was a gift from Araki et al.  and the siveness . Both cesiveness , 5 in th5–103 cells/14 cm diameter dish and G418 resistant clones were selected with 750 μg/mL G418 (PC1) and 250 ug/mL G418 (MFVD). Single colonies coming up were picked with cloning rings, grown up and tested for N-TAP-mAR expression by Western blot analysis and immunocytochemistry using the FLAG M2 antibody (Sigma) and the AR-N20 antibody (Santa Cruz).The control cell lines PC1 and MFVD were transfected with the ScaI  linearised N-TAP-mAR vector using Lipofectamine 2000 (Invitrogen). After 48 hours, transfected cells were replated in dilutions from 10PC1 cells, derived from the mouse proximal caput epididymus and immortalized by expression of a temperature sensitive SV 40 large T-antigen, were continuously cultured at 33°C, the permissive temperature of large T in the presence of 5 nM mibolerone. At physiological temperature (37°C), cell growth is partly inhibited and T-antigen-expressing cells can survive . Large Tβ-mercaptoethanol). Samples were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membrane, blocked and probed in phosphate-buffered saline  (pH 7.3), 0.05% (vol/vol) Tween 20) containing 10% nonfat dry milk. Primary antibodies were used at recommended dilutions and horseradish peroxidase-conjugated secondary antibodies (Dako) and ECL Plus Blotting Detection Reagents (Amersham) or SuperSignal West Femto (Thermo Scientific) were used as described in the manufacturer's instructions. SRC-1 (128E7) and CTNNB1 (9587) antibodies were purchased from NEB, FLAG M2 (F3165) from Sigma, and N20-AR (Sc-816), NR3C1 (Sc-8992), SMARCC1 (Sc-9748), ACTB (Sc-81178) from Santa Cruz.Cells from subconfluent cultures were washed, trypsinized, and pelleted by centrifugation and washed 3x in cold PBS. For whole cell lysates, cells were lysed in SDS loading buffer , washed 3x with prewarmed PBS, trypsinised with 1 mL trypsin-EDTA (Sigma)/plate 5 minutes 37°C, inactivated with medium, pooled in 50 mL Falcon, spun1200 rpm 5 mins RT, and washed 3x with cold (4°C) PBS. Cells were transferred to a 2 mL microfuge tube and pelleted by centrifugation at 6500 rpm 2-3 minutes at 4°C. Pellets (2 × 200 μL) were resuspended in 2 × 1.8 mL hypotonic buffer , 5 nM mibolerone, 1 mM Pefabloc (Roche), and Phosstop (Roche)) by pipetting up and down and vortexing vigorously on highest setting for 15 seconds followed by 45 minute incubation on ice vortexing every 10 minutes. Nuclei were spun down at 4°C 13.000 RPM for 5 minutes, the supernatant, called here cytosol fraction, transferred to a clean tube and NaCl was added to 15 mM. This cytosol fraction was added to FLAG-M2 coupled magnetic beads (ca. 5 mg) after keeping an aliquot as cytosol input fraction for PAGE. Coupling of the magnetic beads (Dynabeads M-270 Epoxy from Invitrogen) to the FLAG M2 antibody (Sigma F3165) was performed according to manufacturer's instructions. Cytosol extracts on beads rotated for 1 hour minimum at 4°C. Nuclei were taken up in sucrose buffer , 5 nM mibolerone, 1 mM Pefabloc (Roche), Phosstop (Roche)) 150 μL/100 μL cell pellet and resuspended. 0.3 units micrococcal nuclease from Sigma (0.1 U/μL in 10 mM Tris/0.1 mM CaCl2 pH8) for 100 μL nuclear extract were added and incubated for 30 min at 37°C. Extracts were then diluted with 1 volume of sucrose buffer and sonicated 6 × 10 seconds on ice with 10 sec bursts and 10 sec cool downs alternating. Extracts were finally loaded onto FLAG M2-coupled magnetic beads and rotated for at least 1 hour at 4°C in cold room. Supernatants were kept as flow through and aliquots before loading on to the beads as nuclear input. After the incubation period, beads loaded with cytosol preparations were washed 3x with hypotonic wash buffer , 5 nM mibolerone, 1 mM Pefabloc (Roche) and Phosstop (Roche), 0.5% NP40, 0.05% sodium deoxycholate, 0.005% SDS, 15 mM NaCl) or sucrose buffer (nuclear preps) followed by 3 washes with TEV buffer  supplemented with Protease Inhibitor Cocktail (Roche), mibolerone (5 nM), and phosphatase inhibitors (Phosstop and Pefabloc (Roche), and NaCl to15 mM NaCl in cytosolic and 150 mM NaCl in nuclear TEV buffer. The beads were finally taken up in 100 uL TEV buffer and bound protein complexes were eluted with 10 units of TEV protease (GST-tag)  at RT for 1 hour. Protein concentration in the eluted fraction was determined by Bradford assay. The beads were kept in PBS for recycling.The protein extraction protocol is a modified version of a chromatin extraction protocol for co-purification of histones by Saade et al., 2009 . Cells wμM i.d. × 150 mm, 3 μM particle size). Peptides were loaded onto a precolumn  from the autosampler with 0.1% formic acid for 5 minutes at a flow rate of 10 μL/min. After this period, the valve was switched to allow elution of peptides from the precolumn onto the analytical column. Solvent A was water + 0.1% formic acid and solvent B was acetonitrile + 0.1% formic acid. The gradient employed was 5–50% B in 45 minutes. The LC eluant was sprayed into the mass spectrometer by means of a new objective nanospray source. All m/z values of eluting ions were measured in an Orbitrap Velos mass analyzer, set at a resolution of 30000. Data dependent scans (Top 20) were employed to automatically isolate and generate fragment ions by collision-induced dissociation in the linear ion trap, resulting in the generation of MS/MS spectra. Ions with charge states of 2+ and above were selected for fragmentation.Colloidal coomassie stained protein bands  were excised out of a 5% PAGE after separation in 7 slices covering a size spectrum of 48 kDa to the top of the gel. Gel slices were briefly washed in MilliQ water and kept in water at −80°C until submitted to “Cambridge Proteomics Services” where all LC-MS/MS experiments were performed using an Eksigent NanoLC-1D Plus  HPLC system and an LTQ Orbitrap Velos mass spectrometer . Separation of peptides was performed by reverse-phase chromatography used at a flow rate of 300 nL/min and an LC-Packings  PepMap 100 column . Briefly, all MS/MS data were converted to mgf (text) files. These files were then submitted to the Mascot search algorithm  and searched against Uniprot Mouse database, using a fixed modification of carbamidomethyl and variable modifications of oxidation (M).Stable MFVD and PC1 clones expressing N-TAP-mAR were developed as described in Different protein purification protocols for producing nuclear and cytosol cell extracts were tested. Commercially available purification kits and a modified version of a chromatin purification protocol (FLAG-antibody capture) were compared and the latter was chosen. FLAG-antibody capture gave a good recovery of N-TAP-mAR when preparing extracts from M7  or P17 chttp://www.matrixscience.com/). Only those ions scores that exceed a significance threshold of 0.05  contributed to the score. This would translate into 1500 peptides falling within the mass tolerance window to have a score of ≥45. Known AR-interacting proteins (http://androgendb.mcgill.ca) were identified among the protein hits and are listed in The results from the LC-MS/MS search are portrayed as peptide matches and grouped as protein hits using a simple parsimony algorithm  involved. Another overrepresented pathway “chromatin remodeling by hSWI/SNF ATP-dependent complexes” involves genes SMARCA4, ACTB, NR3C1, NF1, ARID1A. A third overrepresented BIOCARTA pathway is “control of gene expression by vitamin D receptor” with EP300, SMARCA4, MED1, NCOA2, and ARID1A involved . All 3 pThe Gene list of known AR-interacting proteins identified in the nuclear FLAG purifications of the 33°C samples  and RB1 missing. “Chromatin remodeling by hSWI/SNF ATP-dependent complexes” has NR3C1 and NF1 missing and gained 3 new components with ARID1B, SMARCC1, and SMARCD1. SMARCA4, ACTB, and ARID1A are present. The pathway “control of gene expression by vitamin D receptor” has now 5 components present EP300, SMARCA4, but not MED1 and NCOA2 anymore. ARID1A is still present and the 2 new components SMARCC1 and SMARCD1.Identical gene lists submitted to DAVID were also submitted to the gene ontology online tool g : Profiler for functional characterization. “Regulation of transcription” was not a high scoring biological process” on this occasion, whereas “gene expression” scored high at both temperatures, with many genes overlapping in both categories . Also heKegg pathway: “pathways in cancer” is only detected in the 37°C samples, with 9 components represented . The gene ontology subgroups for “biological processes” (BPs), “anatomical structure morphogenesis”, and “embryo development” are overrepresented in the 37°C samples and overlapping components are involved . “EmbryoAmong cellular component profiling, the nBAF complex is solely detected in the 33°C samples with SMRCC1, SMARCD1, SMRCA4, and ARID1A (not listed in tables). These proteins are also components of the SWI/SNF chromatin remodelling complex.The observation that some AR-interacting proteins were only detectable in proliferating cells and others only in nonproliferating cells  could siNo major differences in expression were observed when extracts were probed with AR-N20 and ACTB control antibodies, but differences were found elsewhere. NR3C1 is expressed at much higher levels in nonproliferating versus proliferating P17 cells, and expression levels of SMARCC1 were slightly lower in nonproliferating (37°C) PC1/P17 when compared to proliferating cells (33°C). The difference in the amounts of AR copurified SMARCC1 in proliferating versus nonproliferating is not as striking as it is for NR3C1, and the amounts at “low expression” temperatures are probably too small to be detected by mass spectroscopy. The proliferation status of P17 cells seems to affect SMARCC1 and NR3C1 expression levels per se as SMARCC1 expression is up and NR3C1 expression is down in proliferating cells. However, the loss of NR3C1 expression in proliferating cells is unlikely to account for all the loss in AR binding observed here. Similar loss of SMARCC1 expression in nonproliferating cells does not account for all the loss in AR binding detected. Therefore, expression levels of coregulators and binding affinity to AR may contribute to the differences observed in the 33°C and 37°C coregulator profiles. The N-TAP-mAR itself appears to increase GR expression levels by at least 3 fold under nonproliferating conditions comparing GR expression in PC1 and P17 input samples .We have developed the epithelial and mesenchymal mouse cell lines P17 and M7 for copurification of AR-associated protein complexes under native conditions. The two cell lineages are derived from the proximal caput epididymus (P17) and the mesenchyme of the fetal vas deferens (M7) of the mouse. The decision to carry out preparative scale N-TAP-mAR purification on PC1 cells rather than MFVD cells was based on the observation that CTNNB1 and SRC-1 could not be copurified from MFVD cells with our extraction protocol, whereas in P17 copurifications both coactivators were easily detected. Another advantage of choosing the PC1 cell line was the more abundant AR expression and the rapid growth compared to the mesenchymal cells. One confluent dish of PC1 gave about 5–10 times as much cell pellet than 1 confluent dish of MFVD.To demonstrate proof of principle we used the newly developed purification protocol to detect differences in AR cofactor binding in cells grown under proliferating (33°C) and nonproliferating (37°C) conditions. As expected, many of the copurified proteins confirm the role of the AR in chromatin remodelling machinery, transcriptional complexes and associated with the cytoskeleton. The purification protocol we use is relatively crude and results in enrichment of 200–500 bp DNA fragments after a micrococcal nuclease digestion. The isolation protocol is nondenaturing and keeps the chromatin fraction as intact as possible. No size fractionation step is included, which could allow isolation of larger chromatin fractions, especially heterochromatic fractions which are not degraded by micrococcal nuclease. This fraction could contribute to a background of unspecific binding, which is difficult to control for as this might not occur in the PC1 control sample, where the “anchor” in form of the N-TAP-mAR is missing. On the other hand, the purification protocol is selecting for stable interactions, because no cross-linking step is included in our purification procedure.We have not tested whether novel interacting proteins identified with this approach are indeed associated with AR or whether they are just contaminants. These potential interacting proteins could be novel AR-interacting proteins, but could also bind unspecific to the FLAG-M2-coupled magnetic beads. Unspecific binding to the FLAG antibody-coupled magnetic beads is estimated at 5% based on the recovery of protein from the FLAG purification of the PC1 control sample. This would mean that 1 out of 20 identified proteins is not part of the AR-associated complex. Unfortunately we were not able to reduce this background with an additional His purification step, because the FLAG purified and protease (TEV) eluted fraction was not able to specifically bind Nickel resin, probably caused by complex components covering up the His tag. In this study we concentrate only on a small proportion of all the potential AR-binding partners identified, representing the already known AR-interacting proteins. We show that our approach has the potential to differentiate between proteins which preferably form part of the AR complexes in proliferating or nonproliferating conditions, and those proteins where interaction is independent of the proliferation status of the cells.in vitro for ligand-dependent transactivation by nuclear hormone receptors, such as vitamin D3 receptor, retinoid X receptor, and peroxisome proliferator-activated receptor PPAR-γ [in vivo for reconstitution of glucocorticoid-receptor-(NR3C1-) dependent transcription [Common pathways enriched for or overrepresented in proliferating and nonproliferating AR FLAG purifications were the biological processes “transcriptional regulation” and “gene expression”. Considering that the androgen receptor is classified as a transcription factor, high scores in those categories are expected. Scoring surprisingly high were BIOCARTA pathways not involving AR at all such as “chromatin remodelling by hSWI/SNF ATP-dependent complexes” and “control of gene expression by vitamin D receptor (VDR)” , which ir PPAR-γ . It has cription , chromatcription  and in ncription  but has cription , 21, whiFor male genital development and PAIS relevant biological processes are “embryo development”, which is here represented with 13 genes identified by g-profiler and 11 identified by DAVID. Of those 10 are overlapping . The embIn future we aim to replace the endogenous AR with N-TAP-mAR. We will hopefully be able to apply the FLAG purification protocol tested in this study to identify differences in the proteome caused by the respective AR mutation. The protein purification approach taken here and shown to identify differences in co-factor recruitment of AR under proliferating and nonproliferating conditions is encouraging to undertake further experiments aiming to identify specific interaction protein profiles for AR mutants associated with PAIS.