Maine Medical Center Research Institute | Date: 2016-06-01
The present invention provides antagonists and methods of use thereof in the treatment of cancer and abnormal immune suppression diseases.
News Article | May 29, 2017
Menopause is associated with health problems for women, such as weight gain and loss of bone density. During menopause, levels of follicle-stimulating hormone (FSH) also rise dramatically. An antibody targeting FSH that was previously shown to increase bone mass in mice also reduces body fat and increases metabolism in mice, according to a new study (Nature 2017, DOI: 10.1038/nature22342). The discovery, by two international teams, that blocking FSH in mice directly counteracts many of the symptoms that arise during menopause holds promise for treatments of myriad conditions, such as obesity, osteoporosis, cardiovascular disease, and cancer. FSH is produced by the pituitary gland in both male and female mammals, and, in addition to stimulating the growth of ovarian follicles in females, regulates numerous other reproductive processes. The teams, supervised by Mone Zaidi and Li Sun at the Icahn School of Medicine at Mount Sinai and Clifford J. Rosen at Maine Medical Center Research Institute, used a synthetic mouse antibody that targets a 13-amino-acid sequence of one subunit of FSH. They tested the antibody on populations of female mice that had their ovaries removed—and thus had high FSH levels—and on both male and female mice that were fed high-fat diets. In both cases, treatment with the antibody resulted in fat loss and increased metabolism. Kathleen Gavin, at the University of Colorado School of Medicine, says the results “are very intruiging and definitely worth more extensive study,” although she cautions that their relevance in humans will require substantial additional investigation.
News Article | May 31, 2017
Recombinant mouse Fsh was obtained from R&D (8576-FS). For the cyclic AMP assay, we used fully glycosylated Fsh24 (provided by T.R.K.)41. It is important for bone and fat assays to use the fully glycosylated form, owing to sub-optimal actions of the hypoglycosylated glycoform41. Recombinant mouse Fsh (Fshα–Fshβ chimaera, 2 μg) was passed through resin (Pierce Co-Immunoprecipitation Kit, 26149, Thermo Scientific) with immobilized Fsh antibody or goat IgG. Elution, flow-through, and consecutive wash fractions were collected and immunoblotted with a monoclonal Fsh antibody (Hf2) (generated by GenScript). The immunoprecipitated eluate (from above) was reduced (DTT, Sigma), alkylated (iodoaceteamide, Sigma), and trypsinized (trypsin, Promega). Peptides were analysed by reversed phase (12 cm/75 μm, 3 μm C beads, Nikkyo Technologies) LC-MS/MS (Ultimate 3000 nano-HPLC system coupled to Q-Exactive Plus mass spectrometer, Thermo Scientific). Peptides were separated using a gradient increasing from 6% buffer B/94% buffer A to 50% buffer B/50% buffer A in 34 min (buffer A: 0.1% formic acid; buffer B: 0.1% formic acid in 80% acetonitrile) and analysed in a combined data dependent (DDA)/parallel reaction monitoring (PRM) experiment42. Six Fsh peptides were targeted. Tandem MS spectra were recorded at a resolution of 17,500 with m/z of 100 as the lowest mass. Normalized collision energy was set at 27, with automatic gain control (AGC) target and maximum injection time being 2 × 105 and 60 ms, respectively. Tandem MS data were extracted and queried against a protein database containing the Fshα–Fshβ chimaera sequence concatenated with an Escherichia coli background database and known common contaminants43using Proteome Discoverer 1.4 (Thermo Scientific) and MASCOT 2.5.1 (Matrix Science). Acetyl (Protein N-term) and Oxidation (M) were chosen as variable modifications while all cysteines were considered carbamidomethylated. 10 ppm and 20 mDa were used as mass accuracy for precursors and fragment ions, respectively. Matched peptides were filtered using 1% false discovery rate calculated by Percolator44and, in addition, required that a peptide was matched as rank 1 and that precursor mass accuracy was better than 5 ppm. The area of the three most abundant peptides per protein45was used to estimate approximately the abundance of matched proteins. The crystal structure of the human FSH in complex with the entire ectodomain of the human FSHR was used as the template (PDB code 4AY9) for comparative modelling46. The sequence of the epitope on mouse Fshβ differs by only two amino acids (DLVYKDPARP QK→DLVYKDPARP QK). Several models of the modified Fshβ epitope were constructed using ICM software47. Restrained minimization was carried out to remove any steric clashes. The final model was selected on the basis of the lowest Cα r.m.s.d. value after superimposition on the template structure (0.2 Å). The structure of the Fshr resembles a right-hand palm, with the main body as the palm and the protruding hairpin loop as the thumb46. The Fshβ binds in the small groove generated between the palm and the thumb. The electrostatic surfaces generated reveal a complementary surface charge between the Fshr and Fshβ. Colonies of male and/or female wild-type C57BL/6J mice, nu/nu mice, male Fshr+/− mice, male ThermoMice and male PhAMexcised mice, originally obtained from The Jackson Laboratory, were maintained in-house at Icahn School of Medicine at Mount Sinai and/or Maine Medical Center Research Institute. Mice were subjected to standard 12-h light–dark cycles (6 am to 6 pm) and fed as below. For thermoneutrality experiments, mice were housed in temperature-controlled cages (30 °C). All protocols were approved by the Institutional Animal Care and Use Committees of the respective institutions. Three-month-old wild type C57BL/6J mice (n = 15) were injected intraperitoneally with a single dose of antibody (100 μg per mouse), with groups of three mice being killed at 0, 2, 6, 12 or 24 h. Collected plasma was subject to in-house ELISA, in which two rabbit anti-goat IgGs, one of which was labelled with HRP (HRP–IgG from Jackson ImmunoResearch, Cat. # 305-035-046 and unlabelled IgG from Thermo Scientific, # 31133), were used to sandwich-capture goat IgG. Depending on the experiment, 3-, 6- or 8-month-old C57BL/6J mice were pair-fed or allowed ad libitum access to a high-fat diet (DIO Formula D12492, 60% fat; Research Diets, Inc.) or regular chow (Laboratory Rodent Diet 5001; LabDiet) for up to 8 weeks, during which cumulative food intake was measured near-daily, in addition to measurements of body weight around twice per week. For pair-feeding, the amount of chow consumed ad libitum by the IgG group was given to the antibody-treated group. For the ad libitum protocol used at Maine Medical Center, both groups were allowed free access to food, with the leftover chow being counted to determine food intake. For specific experiments (Extended Data Fig. 6d–f) performed at Mount Sinai, the antibody-treated group was allowed ad libitum access to food and the same amount of chow was given to the IgG group, with the leftover chow being counted to determine the food intake of the IgG group. Antibody was injected at doses between 100 and 400 μg per mouse as noted in the individual figure legends. Numbers of mice per group are indicated in the figure legends. Several complementary approaches, namely quantitative nuclear magnetic resonance (qNMR), microcomputed tomography (micro-CT), dual energy X-ray absorptiometry (DXA), osmium micro-CT for bone marrow fat quantification, and tissue weight measurements, were used to examine total body fat, as well as fat volume or weight in different adipose tissue compartments. For qNMR, live mice were placed into a thin-walled plastic cylinder, with freedom to turn around. An Echo3-in-1 NMR analyser (Echo Medical) was used to measure fat, lean and total mass, according to the manufacturer’s instructions. For micro-CT, we followed the protocol described in ref. 48. VivaCT-40 (Scanco AG) with a detector size of 1,024 × 256 pixels was used for imaging fat and measuring fat volume in thoracolumbar compartments. Mice were anaesthetized by purging the chamber with 5% isoflurane and O for 5–10 min (X.E.G.) or with Avertin (C.J.R.) and positioned with both legs extended. The torso of each mouse was scanned at an isotropic voxel size of 76 μm (45 kV, 133 μA) and a 200-ms integration time. Two-dimensional grey scale image slices were reconstructed into a 3D tomogram, with a Gaussian filter (σ = 0.8, support = 1) applied to reduce noise. Scans were reconstructed between the proximal end of L1 and the distal end of L5. The head and feet were not scanned or evaluated because of the relatively low adiposity in these regions, and to allow a decrease in scan time and radiation exposure for the animals. Regions of fat were manually traced and thresholded at 5% maximum grayscale value. The high resolution of this method allows the imaging of both sWAT and vWAT. An automated algorithm was used to quantify the volume of sWAT and vWAT using previously described methods49, 50. BMD and body fat measurements were performed using a Lunar Piximus DXA, with a precision of <1.5%51. Anaesthetized mice were subject to measurements, with the cranium excluded. The instrument was calibrated each time before use by employing a phantom per the manufacturer’s recommendation. Osmium staining for marrow fat was performed in collaboration with the Small Animal Imaging Core and the Physiology Core at Maine Medical Center Research Institute, using previously published methods52. Briefly, tibias were isolated, fixed with 10% formalin for 24 h, washed, and then decalcified for 14 days in EDTA. Upon further washing, bones were stained for 48 h in 1% osmium tetraoxide. Following subsequent washes, bones were scanned in PBS with an energy level of 55 kVp, and intensity of 145 μA using the VivaCT-40 (Scanco AG). The integration time was set to 500 ms at a maximum isotropic voxel size of 10.5 μm at a high-resolution setting. Two voxels of interest (VOIs) were selected as shown in Fig. 2c. Indirect calorimetry was performed, as described previously49, using the Promethion Metabolic Cage System (Sable Systems) located in the Physiology Core of Maine Medical Center Research Institute. Data acquisition and instrument control were performed using MetaScreen software (v.2.2.18) and raw data processed using ExpeData (v.1.8.2) (Sable Systems). An analysis script detailing all aspects of data transformation was used. The study consisted of a 12-h acclimation period followed by a 72-h sampling duration. Each metabolic cage in the 16-cage system consisted of a cage with standard bedding, a food hopper, water bottle, and ‘house-like enrichment tube’ for body mass measurements, connected to load cells for continuous monitoring, as well as 11.5-cm running wheel connected to a magnetic reed switch to record revolutions. Ambulatory activity and position were monitored using XYZ beam arrays with a beam spacing of 0.25 cm. From these data, mouse pedestrial locomotion and speed within the cage were calculated. Respiratory gases were measured using the GA-3 gas analyser (Sable Systems) equipped with a pull-mode, negative-pressure system. Air flow was measured and controlled by FR-8 (Sable Systems), with a set flow rate of 2,000 ml/min. Oxygen consumption (VO ) and carbon dioxide production (VCO ) (not shown) were reported in ml per minute. Water vapour was measured continuously and its dilution effect on O and CO were compensated mathematically in the analysis stream. Energy expenditure (EE) was calculated using: kcal/h = 60*(0.003941*VO +0.001106*VCO ) (Weir Equation) and respiratory quotient (RQ) was calculated as VCO /VO . Ambulatory activity and wheel running were determined simultaneously with the collection of the calorimetry data. We used two independent methods to derive resting energy expenditure (REE) and active energy expenditure from the time-dependent calorimetry and activity data. First, we determined REE as the average EE of 30-min intervals of no activity, and active EE as the average EE of 15 min of the most active states. Second, we used penalized spline regression to estimate the continuous REE (or resting metabolic rate (RMR)) and active EE related to physical activity (AEE (PA)), using four equidistant knots per day in the spline function and optimizing the activity-related preprocessing parameters with respect to the regression residuals53, 54. Sleep hours were determined as any inactivity lasting greater than 40 s or more. This latter analysis provided us with an independent verification for a lack of relationship between physical activity and EE53, 54 (Extended Data Fig. 2m). Mice were tested after 4 weeks of treatment with antibody or IgG for glucose and insulin tolerance. For GTT, mice were placed in a clean cage with water and starved overnight (16 h), following which glucose (1 g/kg) was administered intraperitoneally, and blood glucose levels measured at 0, 15, 30, 60, 90 and 120 min post-injection using the OneTouch Ultra Glucometer (LifeScan, Inc.) per manufacturer’s instructions. For ITT, antibody- or IgG-treated mice were fed ad libitum and injected intraperitoneally with insulin (1 U/kg). Glucose levels were measured at 0, 15, 30, 45, 60 and 120 min after injection. In the ThermoMouse, a luciferase reporter construct, Luc2-T2A-tdTomato, is inserted into the Ucp1 locus on the Y chromosome10 (Extended Data Fig. 1e). Activation of Ucp1 expression leads to upregulation of Luc2, which can be quantified in vivo by radiance (luminescence) measurements using IVIS Spectrum In vivo Imaging System (Perkin Elmer) following the injection of d-luciferin (10 μl g–1). Three-month-old male ThermoMice were treated with Fsh antibody or goat IgG (200 μg per day per mouse) for 2.5 (under thermoneutral conditions at 30 °C) or 8 weeks (at room temperature) while being pair-fed on high-fat diet, followed by d-luciferin injection and radiance capture from the ventral and/or dorsal surfaces of the entire body and lower and upper body regions of interest (ROI). In separate experiments, Thermo cells (1.5 × 106) were implanted into both flanks of nu/nu mice, which were fed on normal chow and injected with antibody (200 μg per mouse per day) for 8 weeks, following which Luc2 radiance was quantified after d-luciferin. As basal levels of Ucp1 expression can be variable in transgenic mice, and could therefore confound data interpretation, we routinely perform an early time point for radiance capture at 5 min post-d-luciferin. This allows us to evaluate ‘basal’ Ucp1 expression, and mice whose measured total flux and/or average radiance at the 5-min time point is more than 1 s.d. from the mean of group are excluded. For independent confirmation, frozen sections of resected areas where cells had been implanted were examined for tdTomato fluorescence. We used immortalized dedifferentiated brown adipocytes derived from the ThermoMouse (Thermo cells), in which the Ucp1 promoter drives a Luc2-T2A-tdTomato reporter10, provided by S. Kajimura (UCSF). 3T3.L1 cells were purchased from ATCC. Cell lines were not authenticated, nor were they tested for mycoplasma. Tissues were subject to haematoxylin & eosin staining or immunocytochemistry by protocols described earlier55. Images were captured using the Keyence or Zeiss microscope. Immunocytochemistry for Fshr used standard protocols and an anti-Fshr antibody (Lifespan, Cat. #LS-A4004). tdTomato and mito-Dendra2 fluorescence was examined in frozen, 15-μm sections. Quantitative PCR was performed using appropriate primer sets using Prism 7900-HT (Applied Biosystems Inc.)56. For cAMP measurements, cells were treated for 20 min with Fsh and/or CL-316,243, with or without a 16-h pre-incubation with pertussis toxin (100 ng ml–1; European Pharmacopoeia) (in the presence of 0.1 mM IBMX). Cyclic AMP was measured in cell extracts using an ELISA kit (Cayman, 581001). For irisin and metrnl measurements, we used ELISAs (Phoenix, EK-067-29 and R&D, DY7867, respectively). Plasma Fsh and E levels were measured by ELISAs (Biotang, M7581 and M7619, respectively). Thirty-two mice fed on a high-fat diet were treated with antibody or IgG (200 μg per mouse per day) for 7 weeks. Half of each group was killed at the outset following blood draw, and the other half was injected with α-methyl-p-tyrosine (AMPT; 250 mg kg–1), with a supplementary dose (125 mg kg–1) 2 h later. After a further 2 h, both groups were killed following blood draw. Extraction and HPLC were conducted at the Core CTSI Laboratory at Yale Medical School (courtesy: R. Jacobs). From preliminary micro-CT data, we found a marked, up to threefold, difference in fat volumes with 4 or 5 mice per group. Using a pre-specified effect size (x −x )/s of 3, a normalized Z-score at α = 0.05 (Z ) of 1.96, and assuming that standard deviation (S) is half the width of the confidence interval (W) [N = 4Z 2S2/W2], 4 mice per group was calculated to be sufficient for 95% statistical significance at 0.8 power (α = 0.05, β = 0.20). Statistically significant differences between any two groups were examined using a two-tailed Student’s t-test, given equal variance. P values were considered significant at or below 0.05. Mice were randomly picked for injection with IgG or antibody to ensure equal distribution of body weight across the groups. Technicians who generated and analysed micro-CT and qNMR data at Dr. Guo’s laboratory at Columbia University and Dr. Buettner’s laboratory at Mount Sinai, respectively, were blind to the mouse groups (Figs 1 and 2, Extended Data Figs 3, 4, 6 and 9). Additionally, the technician at the Mount Sinai Imaging Core Facility who generated data with Thermo mice (Fig. 5) and Thermo cell implants into nu/nu mice (Fig. 3d) was also blind to the mouse groups. Note that the fundamental data were confirmed at Dr. Rosen’s laboratory at Maine Medical Center (Extended Data Table 1). For experiments with Thermo mice, basal Luc2 measurements following d-luciferin injections were made before sampling. Luc2 radiance at 5 min is expected to be low. We made a pre-specified determination that if Luc2 radiance in a given mouse exceeded 1 s.d. of the mean, that mouse was excluded. One mouse was excluded on the basis of this criterion. The authors declare that all data supporting the findings of this study are available within the paper and as Source Data files. Specifically, all Source Data for Figs 1, 2, 3, 4, 5 and Extended Data Figs 1, 2, 3, 4, 5, 6, 7, 8, 9 is available with the online version of the paper as Excel spreadsheets (for bar graphs) and as a PDF file (for the raw immunoblot scan in Extended Data Fig. 1a). Extended Data Table 1 attributes specific experimental sets to individual principal investigators.
Prudovsky I.,Maine Medical Center Research Institute
Nucleus (Austin, Tex.) | Year: 2012
Cycling eukaryotic cells rapidly re-establish the nuclear envelope and internal architecture following mitosis. Studies with a specific anti-nucleosome antibody recently demonstrated that the surface ("epichromatin") of interphase and mitotic chromatin possesses a unique and conserved conformation, suggesting a role in postmitotic nuclear reformation. Here we present evidence showing that the anionic glycerophospholipid phosphatidylserine is specifically located in epichromatin throughout the cell cycle and is associated with nucleosome core histones. This suggests that chromatin bound phosphatidylserine may function as a nucleation site for the binding of ER and re-establishment of the nuclear envelope.
Brown A.C.,Maine Medical Center Research Institute |
Muthukrishnan S.D.,Maine Medical Center Research Institute |
Oxburgh L.,Maine Medical Center Research Institute
Developmental Cell | Year: 2015
FGF, BMP, and WNT balance embryonic nephron progenitor cell (NPC) renewal and differentiation. By modulating these pathways, we have created an in vitro niche in which NPCs from embryonic kidneys or derived from human embryonic stem cells can be propagated. NPC cultures expanded up to one billion-fold in this environment can be induced to form tubules expressing nephron differentiation markers. Single-cell culture reveals phenotypic variability within the early CITED1-expressing NPC compartment, indicating that it is a mixture of cells with varying progenitor potential. Furthermore, we find that the developmental age of NPCs does not correlate with propagation capacity, indicating that cessation of nephrogenesis is related to factors other than an intrinsic clock. This in vitro nephron progenitor niche will have important applications for expansion of cells for engraftment and will facilitate investigation of mechanisms that determine the balance between renewal and differentiation in these cells. The embryonic mammalian kidney maintains nephron progenitor cells (NPCs) within a specific niche. Niche signals have been recapitulated in culture, allowing many million-fold expansion of NPCs. NPC propagation facilitates investigation of mechanisms governing their proliferation and differentiation and provides sufficient cell numbers to generate kidney tissue in vitro. © 2015 Elsevier Inc..
Yoon J.K.,Maine Medical Center Research Institute |
Lee J.-S.,Maine Medical Center Research Institute
Cellular Signalling | Year: 2012
R-spondins (RSPOs) are a family of cysteine-rich secreted proteins containing a single thrombospondin type I repeat (TSR) domain. A vast amount of information regarding cellular signaling and biological functions of RSPOs has emerged over the last several years, especially with respect to their roles in the activation of the WNT signaling pathway. The identification of several classes of RSPO receptors may indicate that this family of proteins can affect several signaling cascades. Herein, we summarize the current understanding of RSPO signaling and its biological functions, and discuss its potential therapeutic implications to human diseases. © 2011 Elsevier Inc..
Motyl K.J.,Maine Medical Center Research Institute
Discovery medicine | Year: 2011
Caloric restriction is associated with a reduction in body weight and temperature, as well as a reduction in trabecular bone volume and paradoxically an increase in adipocytes within the bone marrow. The nature of these adipocytes is uncertain, although there is emerging evidence of a direct relationship between bone remodeling and brown adipocytes. For example, in heterotrophic ossification, brown adipocytes set up a hypoxic gradient that leads to vascular invasion, chondrocyte differentiation, and subsequent bone formation. Additionally, deletion of retinoblastoma protein in an osteosarcoma model leads to increased hibernoma (brown fat tumor). Brown adipose tissue (BAT) becomes senescent with age at a time when thermoregulation is altered, bone loss becomes apparent, and sympathetic activity increases. Interestingly, heart rate is an unexpected but good predictor of fracture risk in elderly individuals, pointing to a key role for the sympathetic nervous system in senile osteoporosis. Hence the possibility exists that BAT could play an indirect role in age-related bone loss. However, evidence of an indirect effect from thermogenic dysfunction on bone loss is currently limited. Here, we present current evidence for a relationship between brown adipose tissue and bone as well as provide novel insights into the effects of thermoregulation on bone mineral density.
Rosen C.J.,Maine Medical Center Research Institute
New England Journal of Medicine | Year: 2011
A healthy 61-year-old white woman is concerned about a low vitamin D level detected during an assessment of her skeletal health. Her menopause began at 54 years of age. She has no history of falls, and there is no family history of hip fracture. She takes no medications or supplements. Her height is 157.5 cm (5 ft 2 in.), and her weight 59.1 kg (130 lb). The results of a physical examination are unremarkable, and the findings on laboratory studies are normal. The T score for bone mineral density at the hip is -1.5, and the serum level of 25-hydroxyvitamin D is 21 ng per milliliter (53 nmol per liter). What do you advise? Copyright © 2011 Massachusetts Medical Society.
Scheller E.L.,University of Michigan |
Rosen C.J.,Maine Medical Center Research Institute
Annals of the New York Academy of Sciences | Year: 2014
Marrow adipose tissue (MAT) is functionally distinct from both white and brown adipose tissue and can contribute to systemic and skeletal metabolism. MAT formation is a spatially and temporally defined developmental event, suggesting that MAT is an organ that serves important functions and, like other organs, can undergo pathologic change. The well-documented inverse relationship between MAT and bone mineral density has been interpreted to mean that MAT removal is a possible therapeutic target for osteoporosis. However, the bone and metabolic phenotypes of patients with lipodystrophy argues that retention of MAT may actually be beneficial in some circumstances. Furthermore, MAT may exist in two forms, regulated and constitutive, with divergent responses to hematopoietic and nutritional demands. In this review, we discuss the role of MAT in lipodystrophy, bone loss, and metabolism, and highlight our current understanding of this unique adipose tissue depot. © 2014 New York Academy of Sciences.
Maine Medical Center Research Institute | Date: 2015-02-26
The present invention relates to compositions and methods associated with cocktails of growth factors and small molecules that target specific cell signaling pathways, the cocktails having been formulated to allow/promote the expansion of progenitor cells (e.g., nephron progenitor cells) within a defined culture system.