To study relationship between androgen receptor (AR) CAG repeat polymorphism, insulin resistance (IR), β-cell function and other clinical/biochemical parameters in ethnic South Asian adults. A case , control  study, was conducted and 110 males were invited. Anthropometry, blood pressure and biochemical parameters  were measured. IR and β-cell function was calculated. A multiple-linear-regression analysis was performed, using number of AR CAG repeats as the continuous dependent variable.Sample size was 100 . Mean age was 49.6 ± 10.7 years. CAG repeat length did not show any significant correlation with IR or β-cell function. In all males there was a significant correlation between number of AR CAG repeats and systolic blood pressure , diastolic blood pressure , total cholesterol  and low-density lipoprotein cholesterol . Only total cholesterol  and estrogen  were significantly associated with number of AR CAG repeats in regression analysis. In conclusion, AR CAG repeat length did not show any significant correlation with IR or β-cell function. Positive association of AR CAG with systolic and diastolic blood pressure and negative association of AR CAG with total and low-density lipoprotein cholesterol deserves further attention. Males of South Asian origin, including Sri Lankans are at an increased risk of developing insulin resistance (IR) and type-2 diabetes (T2DM), in comparison to European males . CentralActivity of testosterone is predominantly related to the activation of the androgen receptor (AR). Exon 1 of the AR gene contains a polymorphic CAG repeat sequence which encodes a variable length poly-glutamine stretch (AR CAG) . The lenThe study was conducted as a case–control study. One hundred and ten males between 21 and 65 years of age were invited for the study from the National Hospital of Sri Lanka (NHSL), which included an equal proportion of males with diabetes mellitus (cases) and males without diabetes (controls). Sample size to determine a difference of AR CAG repeats of 1 (SD 1.75) between the cases and controls , with a non-response rate of 10% was 108. Hence, a total of 110 subjects (55 in each study group) were invited for the study. Those on anti-testosterone treatment, those who have undergone prostatic surgery or pelvic irradiation or those who are diagnosed with klinefelters syndrome were excluded. Ethical approval for the study was obtained from the Ethics Review Committee (ERC), Faculty of Medicine, University of Colombo. Informed written consent was obtained from all research participants.1C was also evaluated in all research participants. IR (HOMA-IR) and β-cell function (HOMA-β) was calculated based on the Homeostatic model assessment (HOMA) method.A structured interviewer-administered questionnaire was used for data collection. The questionnaire evaluated socio-demographic and clinical details such as age, diabetes status and presence of other chronic diseases. Height, weight, waist circumference, hip circumference and blood pressure were measured according to standard methods. Following hormonal studies were performed by a chemiluminescent immunoassay (CLIA) using commercially available kits; total testosterone, sex hormone binding globulin (SHBG), luteinising hormone (LH), follicular stimulating hormone (FSH), estrogen (E2) and fasting insulin. In addition total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triacylglyceride (TAG), serum albumin, fasting blood sugar (FBS) and HbADNA was extracted from peripheral lymphocytes in whole blood. A region in the exon 1 of the Androgen receptor gene present in the X chromosome was amplified by PCR. The PCR fragment was purified and sequenced at Macrogen Inc Korea. The sequence obtained was visualized in Bioedit sequence editor and the alignment was processed using the Mega 4 software with a known AR gene sequence obtained from the NCBI database as a reference. The final sequence was again blasted against the sequences that were reported in the Genebank in order to clarify the amplified nucleotide sequence and count the CAG repeats.Data were analyzed using SPSS version 17.0 . A multiple linear-regression analysis was performed in all patients with number of AR CAG repeats as the continuous dependent variable and total cholesterol, LDL cholesterol, systolic blood pressure, diastolic blood pressure, IR, free testosterone, estrogen and diabetes status as the continuous or dichotomous independent variables and confounding factors. In all analyses a p value ≤ 0.05 was considered statistically significant.Sample size was 100 (response rate—90.9%). There were 53 patients with diabetes  and 47 patients without diabetes . Mean age (± SD) was 49.6 ± 10.7 years (cases—52.2 ± 10.8 years and controls—47.3 ± 10.2 years). The clinical and biochemical characteristics of the cohort are summarized in Table r = 0.05; p = 0.68), cases  or controls . Similarly no significant correlation was observed between the AR CAG repeat length and β-cell function (HOMA-β %) in all males , patients with  and without diabetes . The mean number of CAG repeats in the AR gene did not show any significant difference with the presence of hypertension, ischaemic heart disease, stroke, cirrhosis and chronic kidney disease. Similarly the mean number of CAG repeats in the AR gene did not show any significant difference with the presence of metabolic syndrome defined according to the NCEP-ATPIII criteria.The mean CAG repeat length (± SD) of the AR in the study cohort was 22.4 ± 3.1 ranging from 14 to 31 as shown in Fig. r = 0.09; p = 0.41), waist circumference  and hip circumference . A similar observation was noted independently in both males with and without diabetes (data not shown). In all males there was a significant positive correlation between the number of AR CAG repeats with the systolic  and diastolic blood pressure . However, this association was not observed independently in males with and without diabetes.In the study cohort there was no significant correlation between the number of AR CAG repeats and BMI , HbA1c , serum insulin , triglycerides , HDL cholesterol , total testosterone , free testosterone , bio-available testosterone , albumin , SHBG , FSH  LH  and Estrogen . A similar finding was seen in males with and without diabetes independently (data not shown). In all males a significant negative correlation was observed between the number of AR CAG repeats and total cholesterol  and LDL cholesterol . A higher negative correlation between the number of AR CAG repeats and total cholesterol  and LDL cholesterol  was observed in males with diabetes. However, no significant correlation was observed in males without diabetes. The backward regression model demonstrated statistical significance, the Cox & Snell R-Square and Nagelkerke R Square values were 0.379 and 0.339 respectively. The final results indicated that only total cholesterol  and estrogen  were significantly associated with the number of AR CAG repeats.In the study cohort the number of AR CAG repeats did not shown any significant correlation with FBS  of the AR in the present study cohort was 22.4 ± 3.1 (range 14–31), which is comparable to results observed previously in white Caucasian males (22.0 ± 3.2 [range 17–32]) . A largeWe were unable to demonstrate any significant relationship between the number of AR CAG repeats and the presence of diabetes, hypertension, ischaemic heart disease, stroke, cirrhosis and chronic kidney disease. Similarly the AR CAG repeat length did not show any significant correlation with IR, β-cell function, FBS and HbA1c. In patients with diabetes IR and β-cell function is likely to be modified by treatment and progression of the disease. Furthermore, it is not unexpected that AR CAG had no relationship with FBS and HbA1C in our population, because patients with diabetes are on treatment with diet, oral hypoglycaemics and insulin with the aim of meeting diabetes treatment targets and in patients without diabetes the parameters are remaining within the normal range. However, previous studies have demonstrated a significant relationship between AR CAG repeat polymorphism and body fat mass, serum leptin, serum insulin and IR among healthy males , 14. HenThe positive association of AR CAG with systolic and diastolic blood pressure deserves further attention. The fact that these findings were not independently observed in those with and without diabetes may suggest a false positive result. However, since this has previously been observed in adolescents, hypo-gonadal males and in males with diabetes, this association deserves further study, to establish or refute any relationship and to identify the aetio-pathological mechanisms , 19, 20.There are several limitations which needs to be appreciated. It is a single-centre study on a limited number of patients. However, the study findings can serve as cornerstones for further studies involving a larger number of patients. Furthermore, most of the participants in the diabetes cohort were treated with medications that could affect BMI, waist, HbA1C and blood pressure levels, which could distort associations between AR CAG repeat length and other variables. The AR CAG distribution in our population is similar in comparison to other populations both Asian and Caucasian, implying representativeness of the sample. It is also important to note that this cross-sectional study can only describe relationships between AR CAG and other variables and does not provide evidence of causal effects.


Mahuang Fuzi Xixin decoction (MFXD) has been widely used to treat AR in clinics in Asian countries. This study investigated the effect and possible therapeutic mechanisms of MFXD in the treatment of AR. A Wistar rat model of ovalbumin- (OVA-) induced AR was established and then treated with three doses of MFXD; AR symptoms, serum total immunoglobulin E, histamine, histopathological features, and release and expression of factors related to type 1 helper T (Th1) and type 2 helper T (Th2) responses were analyzed. Our study demonstrated that MFXD has a good therapeutic effect on OVA-induced allergic inflammation in an AR rat model as manifested in reduced frequencies of sneezing and nasal scratching and in reduced serum levels of total IgE and HIS. In addition, MFXD regulates imbalance in Th1/Th2 cells caused by AR by simultaneously attenuating Th1 and Th2 responses, such as by reducing the serum levels of IFN-γ and IL-4 and mRNA expression levels of IFN-γ, IL-4, GATA-3, and STAT-6. This study provided valuable information on the immunoregulatory effect of MFXD for the treatment of AR in future clinical studies.Allergic rhinitis (AR) is one of the most common allergic diseases, which adversely affect patients' quality of life.  Allergic rhinitis (AR) is a type I allergic disease induced by an immunoglobulin E- (IgE-) mediated inflammation and characterized by paroxysmal nasal obstruction, rhinorrhea, nasal itching, and sneezing , 2. As a+ T lymphocytes, which in turn release cytokines that stimulate B lymphocytes to differentiate into plasma cells; as a result, production of immunoglobulin E (IgE) is promoted. Individuals become sensitized when IgE antibodies bind to receptors on mast cells and eosinophils; when they are exposed to allergens once again, IgE-mediated inflammation is stimulated, resulting in AR symptoms [The allergic sensitization procedure for AR was well established in the 1970s. When persistently exposed to certain concentrations of allergens, an antigen-presenting cell presents the allergens to CD4symptoms –7.γ (IFN-γ), which is called the imbalance of Th1/Th2 [The imbalance in type 1 helper T (Th1) cells and type 2 helper T (Th2) cells has been considered the main induction factor in IgE-mediated allergic inflammation , 8–11. W Th1/Th2 , 12–14.  Th1/Th2 .The most popular medications currently used for AR are oral H1 antihistamines and intranasal corticosteroids, which are constantly combined with immunotherapy; these medications can control this allergic disease within either short or long term , 16. HowMahuang Fuzi Xixin decoction (MFXD) is an extract of a classical Chinese traditional formula consisting of Ephedrae , Radix Aconiti Lateralis , and Asarum  at a dry weight ratio of 2 : 3 : 1; MFXD is used to treat common cold, migraine, asthma, rheumatoid arthritis, and AR [Ephedrae has been widely used in China to treat asthma and common cold, and alkaloids such as ephedrine and pseudoephedrine are its main effective constituents [Aconitum alkaloids, especially lowly toxic monoester alkaloids, such as benzoylaconine, benzoylhypaconine, and benzoylmesaconine, have been identified as the main pharmacologic components of Radix Aconiti Lateralis, making it as an effective treatment against rheumatoid arthritis and asthma [Asarum has been generally used to treat common cold, migraine, and bronchitis, and its main effective components are the essential oils methyleugenol, α-pinene, and safrole [, and AR , 21. Ephtituents , 23. Simd asthma , 25. Asa safrole , 27. MFX safrole . HoweverIn this study, a rat model of OVA-induced AR was used to investigate the effect of MFXD on allergic inflammation and Th1 and Th2 immune responses associated with AR were further examined to elucidate the possible therapeutic mechanisms of MFXD.Specific pathogen-free (SPF) adult male Wistar rats (180 ± 20 g) were obtained from the Experimental Animal Center of Southern Medical University (number 44002100009161), and this study was approved by the Institutional Animal Care and Use Committee of Southern Medical University, Guangzhou, China . Rats were housed in the SPF Experimental Animal Center of Southern Medical University with a relative humidity of 40–70% and at a temperature of 20–24°C under lighting controls (12 h light/dark cycle). All rats had free access to standard food and water and were allowed to be acclimated for seven days before the experiment.Ephedrae  was immersed in water (2700 mL) for 30 min and boiled for 20 min. Radix Aconiti Lateralis  and Asarum  were subsequently added and then simmered for another 90 min. Filtered water extract was concentrated to 1.52 g·mL−1 under reduced pressure and then reconstituted in distilled water to achieve the required dose for all subsequent experiments.The preparation of MFXD was conducted as described in Treatise on Febrile Diseases, an ancient Chinese medical book. μm; Agilent Technologies, USA) with a mobile phase consisting of acetonitrile (A) and 0.1% aqueous solution of formic acid (B); the following gradient program was used: 0% A at 0–2 min, 0% A–5% A at 2–5 min, 5% A at 5–8 min, 5% A–20% A at 8–15 min, 20% A–35% A at 15–28 min, 35% A–50% A at 28–31 min, 50% A–55% A at 31–36 min, 55% A–95% A at 36–45 min, and 95% A–100% A at 45–55 min. The injection volume was 1 μL, the flow rate is 0.4 mL/min, and the column temperature was 25°C.UPLC-MS/MS was used to analyze the chemical composition of MFXD. Chromatographic analysis was performed on an Agilent 1290 Infinity LC system  and on a 6410B Triple Quadrupole Mass Spectrometer . In brief, the analytes of MFXD were separated on a Zorbax SB-Aq column  dissolved in 1 mL of physiological saline once every other day for 2 weeks. The rats were subsequently challenged through nasal instillation with 50 μL of OVA solution  into each nasal cavity once daily from day 15 to day 21. Forty AR rats were randomly divided into five groups (eight rats per group), namely, AR model, MFXD (1.9 g/kg), MFXD (3.8 g/kg), MFXD (7.6 g/kg), and loratadine  groups. Rats in three MFXD groups were orally administered daily with three doses MFXD 1 h before nasal challenge for 10 days (day 22 to day 31) according to our preliminary study; rats in the loratadine and AR model groups were simultaneously treated intragastrically with loratadine solution (1 mg/kg) and filtered water, respectively. The rats in the control group were sensitized with Al(OH)3, challenged with saline, and orally administered with filtered water synchronously. During the period of oral administration, all of the AR rats were challenged through nasal instillation with 50 μL of 5% OVA solution once every other day to maintain the nasal stimulation.AR rat models were established as described previously  with minμL of 5% OVA solution. After the evaluation of nasal symptoms, blood samples were collected from rats by using the abdominal aortic method under anesthesia; sera were obtained through centrifugation (3000 rpm) for 10 min and then stored at −80°C. Nasal mucosa samples were removed for further histopathological and qRT-PCR analyses.On the last day (day 31), the rats were placed in observation cages for approximately 10 min for acclimatization after oral administration. The frequencies of sneezing and nasal scratching in the rats were counted for 30 min immediately after the last challenge involving nasal instillation with 50 μm thick sections and stained with hematoxylin and eosin (HE) and toluidine blue . Histopathological changes were evaluated and photographed using an orthorhombic optical photomicroscope .Nasal mucosa samples were fixed in 4% paraformaldehyde  for 24 h and then embedded in paraffin. Paraffin-embedded tissue samples were cut into 4 γ and IL-4 were measured via enzyme-linked immunosorbent assay (ELISA) according to the manuals of rat HIS and IgE Elisa Assay kits  and rat IFN-γ and IL-4 ELISA kits , respectively. The minimum detection limits of IgE, HIS, IFN-γ, and IL-4 were 0.05 U/mL, 0.5 ng/mL, 0.625 pg/mL, and 1.56 pg/mL, respectively.Serum levels of total IgE and HIS and those of IFN-μg/mL ionomycin , and 3.0 μg/mL Brefeldin A (BFA)  for 6 h in 5% CO2 humidified incubator . Cells were subsequently surface-stained with fluorescein isothiocyanate- (FITC-) labeled anti-rat CD3 and allophycocyanin- (APC-) labeled anti-rat CD4 antibodies  at room temperature for 30 min in the dark and then fixed and permeabilized with intracellular fixation and permeabilization buffer , respectively, according to the manufacturer's instruction. After being washed in phosphate-buffered saline (PBS) and after centrifugation, the cells were divided equally and then incubated with phycoerythrin- (PE-) labeled anti-rat IFN-γ, PE-labeled anti-rat IL-4, and PE-labeled anti-rat lgG1 antibodies  at room temperature for 15 min in the dark. The stained cells were washed once and detected by a FACSCalibur flow cytometer , and the results were analyzed with CellQuest software .Peripheral blood mononuclear cells (PBMCs) of rats were separated from the blood sample according to the instruction provided in Rat Peripheral Blood Lymphocyte Separation Kit  and placed in a tube containing RPMI 1640 . The PBMCs were stimulated with 81 ng/mL phorbol 12-myristate 13-acetate , 1.34 ® Bioscience, Shanghai, China) according to the manufacturer's instruction under the following temperature conditions: 37°C for 15 min and 98°C for 5 min. qPCR was performed on an Applied Biosystems 7500 Real-Time PCR System  by using 10 μL of qPCR MasterMix, 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), and 1 μL of cDNA using Bestar® SYBR Green qPCR MasterMix Reagent . The forward primer and reverse primer sequences of IFN-γ, IL-4, T-bet, GATA-3, STAT-1, STAT-6, and β-actin  are listed in β-actin by using cycle threshold (Ct) values.Total RNA was isolated from the nasal mucosa by using a Total RNA Extraction Kit . Complementary DNA (cDNA) was synthesized through reverse transcription reaction of the extracted RNA by using a Bestar™ qPCR RT Kit  or Tamhane's T2 test , and p < 0.05 indicated statistical significance.The results of the four experiments were analyzed using SPSS for Windows version 16.0  and expressed as mean ± standard deviation (SD). Between-groups comparisons were performed using one-way ANOVA and analyzed by LSD Mahuang, Fuzi, and Xixin were analyzed by UPLC-MS/MS. Mahuang, Fuzi, and Xixin, respectively, and eight peaks were found in the three herbs. Nine compounds were detected based on the reference standards shown in Mahuang were identified as norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, and methylephedrine, respectively. Peaks 10–12 derived from Fuzi were identified as benzoylmesaconine, benzoylaconine, and benzoylhypaconine, respectively. Peak 13 derived from Xixin was identified as 3,4,5-trimethoxytoluene.The components of 10 groups of MFXD consisting of different batches of p < 0.01), wherein the rats did not show obvious sneezing and nasal scratching. After oral administration of MFXD, the nasal symptoms in rats of AR were relieved evidently. The frequencies of sneezing and nasal scratching in MFXD (3.8 g/kg) and MFXD (7.6 g/kg) groups significantly decreased compared with those in the AR model group . The frequencies of sneezing and nasal scratching in the MFXD (7.6 g/kg) group were 5.38 and 16.63, respectively, demonstrating the effective therapeutic effect of MFXD on the nasal symptoms in an AR rat model and that such effect is as good as that in the positive group.The frequencies of sneezing and nasal scratching in rats were counted for 30 min to evaluate the therapeutic effect of MFXD after the last nasal challenge with OVA solution. As shown in p < 0.01; p < 0.01). In addition, the concentrations of serum HIS in the high-dose MFXD group and positive group were 20.58 and 19.07 ng/mL, respectively, which were also significantly reduced compared with that in the AR model group .AR is a type I allergic disease induced by IgE-mediated inflammation and characterized by the release of HIS . As summHE and TB staining examinations were conducted to evaluate the effect of MFXD on the histopathological changes in the nasal mucosa. The HE  and TB  stainingγ and IL-4 are the immune cytokines mainly released by Th1 and Th2 cells, respectively [γ and IL-4 measurements were performed to indirectly indicate the status of Th1 and Th2 responses. As depicted in γ and IL-4 in AR model rats significantly increased compared with those in the control group. After oral administration of MFXD and loratadine, the serum levels of IFN-γ and IL-4 significantly decreased (except in the low-dose MFXD group), especially in MFXD (7.6 g/kg) rats (p < 0.01), compared with those in the model group. The serum IFN-γ levels in control, AR model, and MFXD (7.6 g/kg) groups were 0.94, 1.34, and 1.15 pg/mL, respectively, and the corresponding serum IL-4 levels of these groups were 5.52, 15.21, and 8.19 pg/mL, respectively. To evaluate the status of Th1/Th2 balance, we calculated the IFN-γ/IL-4 values in all groups . In addition, the IFN-γ/IL-4 values in MFXD (3.9 g/kg), MFXD (7.6 g/kg), and positive groups significantly increased  compared to that in the model group.IFN-ectively . IFN-γ al groups . Compare+CD4+ lymphocytes and the percentages of CD3+CD4+IFN-γ+ Th1 and CD3+CD4+IL-4+ Th2 cells were analyzed and measured in PBMCs via flow cytometry. As shown in Figures +CD4+IFN-γ+ Th1 cells was significantly higher in the AR model group than in the normal control group (p < 0.01), whereas no obvious changes were observed among the model, MFXD, and positive groups. As shown in Figures +CD4+IL-4+ Th2 cells in the AR model group was 5.16%, significantly higher than the 1.55% in the normal control group (p < 0.01). However, the percentage of CD3+CD4+IL-4+ Th2 cells significantly decreased after administration with MFXD and positive reagent (p < 0.01), and the percentages in MFXD (7.6 g/kg) and positive groups were 2.47% and 2.53%, respectively. Moreover, the ratio of CD3+CD4+IFN-γ+ Th1 and CD3+CD4+IL-4+ Th2 cells was calculated (p < 0.01) but did not significantly differ from that in the control group (p > 0.05).Th1 and Th2 cells differentiated from CD3lculated , and theγ activates the T-bet gene and induces the expression of T-bet through the Janus kinase/signal transducer and activator of transcription 1 signal transduction pathway [γ, IL-4, T-bet, GATA-3, STAT-1, and STAT-6 in the nasal mucosa were significantly higher in the AR model group than in the control group. After intragastric administration of MFXD and positive drug, the expression levels of IFN-γ and STAT-6 in all groups significantly decreased and the expression levels of IL-4 and GATA-3 in MFXD (7.6 g/kg) and positive groups significantly decreased compared with those in the AR model group. γ/IL-4, T-bet/GATA-3, and STAT-1/STAT-6 values, which were calculated to evaluate Th1 and Th2 responses. IFN-γ/IL-4, T-bet/GATA-3, and STAT-1/STAT-6 values in the AR model group significantly decreased compared with those in the control group, and T-bet/GATA-3 ratios were significantly higher in MFXD and positive groups than that in the model group. Moreover, compared with that in the AR model group, the STAT-1/STAT-6 values in MFXD (7.6 g/kg) and positive groups significantly increased and the IFN-γ/IL-4 values in administered groups also tended to increase.T-bet and GATA-3 are important transcription factors that directly or indirectly regulate the differentiation and development of Th1 and Th2 cells . IFN-γ a pathway , 33. In  pathway . As show3 as an adjuvant [3 suspension and nasal instillation with 5% OVA solution. In the AR model group, the rats displayed extremely frequent sneezing and nasal scratching and significantly increased serum levels of total IgE and HIS; moreover, obvious epithelial disruption, leucocyte and mast cell infiltration, and cilia cell reduction were observed in nasal mucosa sections; these results indicated that the rat model of AR was successfully established and presented evident AR responses.AR is one of the most common allergic diseases that do not only adversely affect patients' quality of life but also induce multiple relevant complications , 5. Reseadjuvant , 36. In MFXD is a classical Chinese traditional formula that has been widely used in clinics to treat AR in Asian countries , 21. In γ, T-bet, and SATA-1, leading to the imbalance in Th1/Th2 cells [γ content, the percentage of CD3+CD4+IFN-γ+ Th1 cells in PBMCs, and the mRNA expression levels of IFN-γ, T-bet, and SATA-1 were all significantly increased compared with those in the normal control group; the same trend was observed for the serum IL-4 content, the percentage of CD3+CD4+IL-4+ Th2 cells in PBMCs, and the mRNA expression levels of IL-4, GATA-3, and SATA-6. In this study, Th1 and Th2 responses were simultaneously enhanced in a rat model of AR, consistent with the results of Kim et al. who also used a mouse model of AR [γ promotes allergen penetration through the respiratory epithelium and aggravates allergic inflammation [γ in a rat model of OVA-induced AR obviously increased within 24 h after induction of AR and then decreased [AR is a Th2-polarized allergic disease, and imbalance of Th1/Th2 cells is speculated to be an important factor that results in AR –11. Diffh2 cells . Studiesh2 cells , 38 or ih2 cells , 40, wheel of AR . Two reael of AR , especiaammation , 44. Sececreased .γ/IL-4; CD3+CD4+IFN-γ+ Th1 (%)/CD3+CD4+IFN-γ+ Th1 (%) in PBMCs; and mRNA expression of IFN-γ/IL-4, T-bet/GATA-3, and STAT-1/STAT-6. After oral administration of MFXD, all of the values tended to increase and high dose of MFXD significantly enhanced the values, consistent with the result for the positive reagent, indicating that MFXD can regulate the AR-induced imbalance in Th1/Th2 cells.Although Th1 and Th2 responses were enhanced simultaneously in a rat model of AR, the imbalance in Th1/Th2 cells significantly changed. Compared with those in the control group, the following ratios significantly decreased in the AR model group: ratios of serum IFN-γ and IL-4 and mRNA expression levels of IFN-γ, IL-4, GATA-3, and STAT-6. This study provides some valuable information on the immunoregulatory effect of MFXD on the treatment of AR in future clinical studies.In conclusion, MFXD exerts a good therapeutic effect against OVA-induced allergic inflammation in a rat model of AR as seen in the reduced frequencies of sneezing and nasal scratching in rats and in the reduced serum levels of total IgE and HIS. MFXD regulates the AR-induced imbalance in Th1/Th2 cells by attenuating Th1 and Th2 responses simultaneously, such as reducing the serum levels of IFN-


Klk4 transcription is known to be regulated in other cell types by androgen receptor (AR) and progesterone receptors (PR). In this study, we determined the possible role of fluoride in down-regulation of KLK4 expression through changes in AR and PR. Immunohistochemical localization showed that both AR and PR nuclear translocation was suppressed in fluoride exposed mice. However, when AR signaling was silenced in mouse ameloblast-lineage cells (ALCs), expression of both Pgr and Klk4 were increased. Similar to the effect from AR silencing, fluoride also upregulated Pgr in ALCs, but downregulated Klk4. This finding suggests that though suppression of AR transactivation by fluoride increases Prg expression, inhibition of PR transactivation by fluoride has a much greater effect, ultimately resulting in downregulation of Klk4 expression. These findings indicate that in ameloblasts, PR has a dominant role in regulating Klk4 expression. We found that when AR was retained in the cytoplasm in the presence of fluoride, that co-localized with heat shock protein 90 (HSP90), a well-known chaperone for steroid hormone receptors. HSP90 also known to regulate TGF-β signaling. Consistent with the effect of fluoride on AR and HSP90, we found evidence of reduced TGF-β signaling activity in fluorosed ameloblasts as reduced immunolocalization of TGFB1 and TGFBR-2 and a significant increase in Cyclin D1 mRNA expression, which also possibly contributes to the reduced AR signaling activity. In vitro, when serum was removed from the media, aluminum was required for fluoride to inhibit the dissociation of HSP90 from AR. In conclusion, fluoride related downregulation of Klk4 is associated with reduced nuclear translocation of AR and PR, and also reduced TGF-β signaling activity, all of which are regulated by HSP90. We suggest that a common mechanism by which fluoride affects AR, PR, and TGF-β signaling is through inhibiting ATP-dependent conformational cycling of HSP90.Fluorosed maturation stage enamel is hypomineralized in part due to a delay in the removal of matrix proteins to inhibit final crystal growth. The delay in protein removal is likely related to reduced expression of kallikrein-related peptidase 4 (KLK4), resulting in a reduced matrix proteinase activity that found in fluorosed enamel.  Enamel formed in the presence of high levels of fluoride has a delayed removal of matrix proteins  and progesterone receptor (PR) . In vitro, we found that fluoride together with aluminum induced a cytoplasmic retention of AR, suggesting the possibility that fluoride requires aluminum to inhibit the chaperone function of HSP90 to regulate AR nuclear translocation.To explore the intracellular mechanisms that mediate effects of fluoride on Guide for the Care and Use of Laboratory Animals.All animal procedures were carried out with approval by the University of California-San Francisco Institutional Animal Care and Use Committees. The experiments reported herein were conducted in compliance with the Animal Welfare Act and in accordance with the principles set forth in the National Research Council's Klk4 expression in the presence of fluoride resulted in decreased KLK4 activity in the enamel matrix, 3-week-old female Wistar rats  were divided into two groups, with the groups given either deionized drinking water or deionized drinking water supplemented with 100 ppm (5.3 mM) fluoride as sodium fluoride  ad libitium for 4 weeks. After 4 weeks, the rats were euthanized, and the mandibular incisors were dissected to allow access to separately dissected the enamel matrix and enamel organ for KLK4 activity assays, and quantitative real-time polymerase chain reaction (qPCR) of the relative expression of Klk4 mRNA.To determine whether the reduced ad libitium for 4 weeks. After 4 weeks, the mice were euthanized, and mandibles were obtained for morphology, immunohistochemical, and qPCR analyses.Three-week-old C57BL/6J female mice (Jackson Laboratory) were divided into two groups, with the groups given either deionized drinking water or deionized drinking water supplemented with 50 ppm (2.6 mM) fluoride as sodium fluoride (Sigma-Aldrich) drc package  mandibular maturation-stage incisor enamel organs that were dissected according to landmarks described previously . Mrpl19 was used as a reference gene for enamel organ samples and Eef1a1 for ALCs. Primer sequences are listed in Table t-test with Benjamini & Hochberg correction following ANOVA  using primer sets for Mouse mandibles were fixed in 4% paraformaldehyde in 0.06 M sodium cacodylate buffer (pH 7.3) at 4°C for 24 h. After decalcification in 8% EDTA (pH 7.3), samples were processed for routine paraffin embedding and sagittally sectioned. The sections were incubated with 10% swine and 5% goat sera followed by incubation with rabbit anti-human AR , rabbit anti-mouse TGFBR2 , rabbit anti-human TGFB1 , and rabbit anti-mouse PR  antibodies respectively overnight at room temperature. A biotinylated swine anti-rabbit IgG F(ab')2 fraction  was used as the secondary antibody for 1 h at room temperature incubation. Following incubation with alkaline phosphatase conjugated streptavidin  for 30 min, immunoreactivity was visualized using a Vector® Red kit (Vector Laboratories) resulting in pink/red color for positive staining. Counter-staining was performed with methyl green (Dako). Negative control was done with normal rabbit sera.5 cells/cm2) or 8-well chamber slides (1.5 × 105 cells/cm2). After 24 h, the cells were treated with fluoride (0 or 1 mM) as sodium fluoride (Sigma-Aldrich), followed by total RNA extraction. Other cells, initially cultured in the medium with FBS for 24 h, were exposed to 2% TCM® serum replacement  for 24 h. After 24 h, cells were transfected with antisense oligonucleotides targeting AR, LNA™ GapmeRs  to silence Ar mRNA, using Lipofectamine 2000 Transfection Reagent . Cells were then harvested for total RNA and extracts were analyzed for Klk4, Ar, Pgr, Tgfb1, Tgfbr2, and Ccnd1 expression. For immunofluorescent staining, some cells were treated with fluoride as described above. Other cells were initially cultured in FBS containing medium for 24 h. After culturing in the medium with 2% TCM® serum replacement for another 24 h, cells were treated with 0 or 1 μM dihydrotestosterone/DHT . Some of the DHT treated cells were further exposed to aluminum  as aluminum chloride (Sigma-Aldrich) and/or fluoride (0 or 1 mM) as sodium fluoride (Sigma-Aldrich). Aluminum concentration was equivalent to the range of those in calf serum   for 10 min at room temperature and washed with PBS. They were then permeablized with 0.25% Triton-X 100 and incubated with 10% swine and 5% goat sera to block non-specific binding. Cells were then simultaneously incubated with AR  and HSP90 (Santa Cruz Biotech) antibodies that were fluorescently tagged with Zenon Rabbit IgG Labeling Kit  per manufacturer's instructions. AR antibody was labeled with Alexa Fluor 594 and HSP90 antibody was labeled with Alex Fluor 488. After antibody incubation, cells were washed with PBS, then counterstained with DAPI and mounted with mounting medium.Klk4 mRNA expression was significantly reduced . To further investigate the effect of fluoride on molecular profiles in maturation ameloblasts, we therefore used the mouse model.To determine the ultimate effect of fluoride on KLK4 synthesis, we assessed synthesis of KLK4 in fluorosed rat enamel, by comparing KLK4 proteinase activity in the enamel matrix of rats given either 0 or 100 ppm fluoride in drinking water. Enamel matrix protein extracts of rats given 100 ppm F in drinking water had reduced matrix KLK4 proteolytic activity when compared with control rats Figure , and sigd Figure . Similarin vitro, we found that ALCs grown in serum containing medium with 1 mM fluoride had significantly less Klk4 expression as compared to ALCs grown in medium without fluoride  . In fluorosed ameloblasts, immunostaining for both TGFB1 and TGFR-2 was reduced . Further immunostaining showed that the increased cytoplasmic AR in the presence of both fluoride and aluminum co-localized with HSP90  and in vitro (with ALCs) experimental system. Indeed, in our ALC culture, DHT induced nuclear translocation of AR, but did not upregulate Klk4 expression (data not shown). Jedeon et al. showed that in rat HAT-7 ameloblasts, activation of AR by testosterone (a primary androgen) increased expression of both Klk4 and Ar  is such a candidate molecule. HSP90 is an abundant molecular chaperone that functions by facilitating protein folding and stabilization  fluoride supplemented water have a serum fluoride level around 3–5 μM (Guy et al., in vitro, fluoride levels are required to be much higher to show effects similar to those found in vivo (Sharma et al., in vivo, and at these fluoride levels, cell proliferation and morphology of the ALCs were not affected, indicating that the effects of fluoride on the ALCs used for these in vitro studies are specific and not due to a generalized effect of fluoride on cell toxicity. The need for higher levels of fluoride in ALC culture may be related to the mechanisms by which fluoride may enter the cell, as compared to in vivo.Finally, it is important to mention that studies of the cellular effects of fluoride require attention to biologically relevant levels of fluoride. We do not yet know how fluoride enters ameloblasts, and the effect of the unique process of enamel mineralization, such as changes in the extracellular pH. Klk4 transcription in maturation ameloblasts. Our data support the possibility that this effect occurs through the action of fluoride in combination with aluminum, possibly through an effect on HSP90. These studies raise many questions related to the regulation of ameloblast maturation, and how fluoride can alter this process at a cellular level, to result in fluorosis. The cellular effect of fluoride may be related to fluoride entry into the cell, which may be unique to ameloblasts, and additional studies will be needed to address this possibility. It should also be noted that the in vitro studies were all done using female rats and mice. Though there is no convincing evidence that fluorosis is sex-linked, it is reasonable that future studies of fluorosis mechanisms include both male and female mice.In conclusion, fluoride targets activation of AR and PR and thus inhibits AR- and PR- driven All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A new approach to modelling pedestrians' avoidance dynamics based on a Fokker–Planck (FP) Nash game framework is presented. In this framework, two interacting pedestrians are considered, whose motion variability is modelled through the corresponding probability density functions (PDFs) governed by FP equations. Based on these equations, a Nash differential game is formulated where the game strategies represent controls aiming at avoidance by minimizing appropriate collision cost functionals. The existence of Nash equilibria solutions is proved and characterized as a solution to an optimal control problem that is solved numerically. Results of numerical experiments are presented that successfully compare the computed Nash equilibria to the output of real experiments (conducted with humans) for four test cases. Thus, we may consider that the two players are aimed at minimizing the probability of such an event and we set Qp is the following characteristic function:X(1)(t) and X(2)(t), and assuming that r is small enough, we haveB denotes the open ball |y−x|<r in f(1) and f(2) are smooth enough as being solutions to the FP equations .Wp( f(1),f(2)) as follows:ρ is defined as ρ=CrD, and C≥0 denotes the relative strength of the interaction, tuned to balance the weights of the other terms in the cost functionals Jp in  to minimizeu(2) as follows:fp)(∈Up)–(3.2).u(1),u(2)∈U(1)×U(2), we have For all This theorem states that the existence of a minimum of Note that theorem 4.p=1,2, are in the following admissible set:We recall that our NE Next, we discuss the existence of solutions to the optimal control problems –4.6). F. F4.6). The objective functional (4.1) is sequentially weakly lower semicontinuous (w.l.s.c.), bounded from below, coercive onU(1)×U(2)and it is Fréchet differentiable.The proof of this lemma is straightforward, once one recalls that the PDFs are non-negative functions.The next result states the existence of an optimal control Assume thatthatsatisfies (2.6) and that the objective is given by (4.1). Then, there exist PDFsp=1,2, andsuch thatsolves the FP constraints (4.2)–(4.5) andminimizesinU(1)×U(2).um)∈U(1)×U(2). As U(1)×U(2) is reflexive and uml) such that H1⊂⊂C is compact, we have strong convergence Boundedness from below of uml), the solution of the FP equations results in the bounded sequence ( fml)∈[L2)∩C)]2 while the sequence of the time derivatives, (∂tfml), is bounded in [L2)]2. Therefore, both sequences converge weakly to fml), which we denote by the same index, that converges strongly in [L2)]2.In correspondence of the sequence f), recall the structure of b given in fml converges strongly. Finally, considering these limiting sequences in the weak formulation of the FP problems, it follows that Concerning the terms ∇⋅(given in  and the ution of –4.5) wi wib and p(2) denote the Lagrange multipliers associated with the FP equations  and the control is zero at the initial and final times (controls switch on at t=0 and switch off at t=T), the solution of the gradient equation with homogeneous Dirichlet boundary conditions results in Note that in the optimality system, the following reduced H−1. Therefore, it is necessary to determine the corresponding reduced H1 gradient. This is done based on the following fact:We wish to apply a gradient-based optimization scheme where the residual of  is used.H1 inner product and integrating by parts, we have that the H1 gradient can be obtained by solving the following boundary-value problem:k=1,…,D, and Δ denotes the Laplace operator in time.Using the definition of the The solution to this problem provides the appropriate gradient to be used in a gradient update of the control that includes projection to satisfy the given control constraints.5.We are now ready to consider four different test cases to discuss the validity of our FP-Nash approach to pedestrian avoidance and test the efficiency and robustness of our optimization set-up for determining the NE pair ial game –3.4). T. T(u¯(1)A1,A2)=xt(0) is the starting point of the pedestrians X, and x=. The terminal potentials for both the pedestrians are defined aspth pedestrian. The parameter α is set to be 100 and ν=1.The motion of the pedestrians/players are represented by the motion of their PDFs. In the plots, the trajectories of the players depict the trajectories of the mean of the position computed using their respective PDFs. The initial density for the players are defined as follows:ρ.The differential game consists in determining NE strategies We solve the optimal control problem –4.6), w, w4.6), Dp)( and Ap)eparture and (A)rrival locations of pedestrian A for p=1, and B for p=2.In the following, we use the notations 5.1.Ω=× for a time interval  for T=3. Player A starts its motion from the departure point D(1)= and player B starts its motion from the point D(2)=. The drift v(1) for player A is , thus it moves along the x-axis. The drift v(2) for player B is  and, thus, it moves down diagonally. The arrival position of player A is A(1)= and for player B is A(2)=. The spatial and the temporal domains are divided into 50 uniformly distributed subintervals. The settings for the game are shown in The test case Huber-135 corresponds to the experiment in  where tra,b shows the plots of the pedestrian mean positions for ρ=0.01, 200, respectively. We can see that, for ρ=0.01, the two players meet at time t=1.0 as denoted by the black dot. In the case ρ=200, pedestrian A goes around pedestrian B to avoid intersection as is shown from the position of the players at time t=1.0 with the black dot. Figure 3c,d show the results of the corresponding Monte Carlo SDEs simulation for ρ=0.01, 200, respectively. In both the cases, we see that the two players reach their final target. This is comparable with the results of the corresponding experiment with humans presented in × for a time interval  for T=5. Pedestrian A starts its motion from the departure point D(1)= and player B starts its motion from the point D(2)=. The arrival position of player A is A(1)= and that for player B is A(2)=. The spatial and the temporal domains are divided into 50 uniformly distributed subintervals. The settings for the game are shown in In the next test case, we consider an experiment from . Two para,b shows the zoomed-in plots of the mean of the PDFs for ρ=0.01,200, respectively. We note that, for ρ=0.01, the two pedestrians meet at time t=2.5 as denoted by the black dot. In the case ρ=200, players A and B move around each other to avoid intersection as is shown by their positions at time t=2.5 with a black dot. Figure 5c,d shows the Monte Carlo simulations for ρ=0.01,200, respectively. For ρ=200, we see that, around the region where trajectories cross, the layout of the Monte Carlo simulation is comparable with the one from × for a time interval  for T=5. Player A starts moving from the departure point D(1)= and player B starts its motion from the point D(2)=. The terminal position of player A is A(1)= and that for player B is A(2)=.In the next test case, we consider another experiment from . In thisa,b shows the zoomed-in plots of the mean pedestrian positions for ρ=0.01,150, respectively. We note that, for ρ=0.01, the two players meet at time t=2.5 as denoted by the black dot. In the case ρ=150, players A and B move around each other to avoid intersection as is shown by their positions at time t=2.6 with a black dot. Figure 8c,d shows the results of Monte Carlo simulation for ρ=0.01,150, respectively. Figure 8ρ=150, the results of Monte Carlo simulation show even more geometric similarities with the ones resulting from the human experiments in × for a time interval  for T=1. Player A starts its motion from the departure point D(1)= and player B starts its motion from the point D(2)=. The terminal position of player A is A(1)= and that of player B is A(2)=.In the final test case, we consider an experiment from , which wfigure 9a,b shows the zoomed-in plots of the mean of the PDFs for ρ=0.01,200, respectively. We note that, for ρ=0.01, the two pedestrian meet at time t=0.5 as denoted by the black dot. In the case ρ=200, player A moves faster near the time of intersection t=0.5 to avoid intersection with player B. After avoiding intersection, player A goes slower to reach its terminal target. Figure 10b,e is in very good accordance with respect to the trajectories followed by the real pedestrian as well as with respect to the velocity profiles. By observing the ‘temporal correspondence between trajectories’ as in figure 10e, we note that one pedestrian moves faster near the time of intersection ‘tcross’ to avoid intersection with the other pedestrian. After avoiding intersection, the first player goes slower to reach its terminal target. In comparison to the previous Turnwald experiments, which involved early visual information weakening the static game assumption, in this case the real experiment is set up as a static (or blind or simultaneous) Nash game, and the results of our computational model are strikingly similar to the results of the real experiment.We remark that figure 106.A new approach to modelling pedestrian's avoidance dynamics based on a FP-Nash game framework was presented and investigated theoretically and numerically. This approach attempts to explain why the observed dynamics arise, while classical phenomenological approaches prescribe how the dynamics should be.Based on FP equations, a Nash differential game was formulated where the game strategies represent controls aiming at avoidance by minimizing appropriate collision cost functionals. The existence of a Nash equilibria solution was proved and characterized as a solution to an optimal control problem. The resulting FP-Nash computational strategy for pedestrian motion was successfully benchmarked with results of real experiments from cognitive psychology studies. The proposed FP-Nash approach represents a powerful new paradigm for the theoretical investigation and computational simulation of differential games with non-convex cost functionals, and it provides a very rich framework to model a large class of processes involved in pedestrian motion.


Since Huggins defined the androgen-sensitive nature of prostate cancer (PCa), suppression of systemic testosterone (T) has remained the most effective initial therapy for advanced disease although progression inevitably occurs. From the inception of clinical efforts to suppress androgen receptor (AR) signaling by reducing AR ligands, it was also recognized that administration of T in men with castration-resistant prostate cancer (CRPC) could result in substantial clinical responses. Data from preclinical models have reproducibly shown biphasic responses to T administration, with proliferation at low androgen concentrations and growth inhibition at supraphysiological T concentrations. Many questions regarding the biphasic response of PCa to androgen treatment remain, primarily regarding the mechanisms driving these responses and how best to exploit the biphasic phenomenon clinically. Here we review the preclinical and clinical data on high dose androgen growth repression and discuss cellular pathways and mechanisms likely to be involved in mediating this response. Although meaningful clinical responses have now been observed in men with PCa treated with high dose T, not all men respond, leading to questions regarding which tumor characteristics promote response or resistance, and highlighting the need for studies designed to determine the molecular mechanism(s) driving these responses and identify predictive biomarkers. TMPRSS2-ERG) rearrangements).Since the landmark studies of Huggins and colleagues showed the androgen-sensitive nature of prostate cancer (PCa) , suppresFrom the inception of clinical efforts to suppress AR signaling by reducing AR ligands, it was also recognized that the administration of T to men with CRPC can result in substantial clinical responses, though the reports were largely anecdotal and remissions were highly variable, potentially due to variability and inadequate increases in the levels of circulating T that were achieved ,3,4. In Notably, biphasic responses to hormone concentrations are not unique to PCa and the AR: When exposed to estradiol (E2), estrogen receptor (ER)-responsive MCF-7 breast cancer cell line adapted to proliferate in the absence of estradiol undergo an apoptotic response ,13,14. EMany questions regarding the biphasic responses of prostate tumors to supraphysiologic androgen concentrations remain, primarily regarding the mechanisms driving this response and how best to exploit this phenomenon clinically. To date, efforts to enhance efficacy of SPT have focused on concomitant manipulation of the androgen receptor (AR), i.e., androgen cycling to induce AR upregulation and increased sensitivity, but preclinical studies suggest other approaches and drug combinations may be reasonable to pursue. Here we review the preclinical and clinical literature on androgen-mediated growth repression and discuss cellular pathways and mechanisms likely to be involved in mediating this response. Although meaningful clinical responses have now been observed in men with CRPC treated with SPT , not allThe AR plays a critical role in the normal development of the prostate gland, although initial morphogenic activity occurs via mesenchymal AR, not epithelial AR . In the In contrast to its role in promoting differentiation in normal prostate epithelial cells, AR signaling in PCa acquires a critical oncogenic role and promotes growth and survival. The mechanism for this conversion is not fully understood but appears to involve in part a gain of function in AR induced regulation of myelocytomatosis oncogene cellular homolog (MYC) expression ,17. DuriThe clinical relevance of continued AR signaling in promoting CRPC tumor growth is confirmed by the clinical responses to agents targeting residual androgen pathway activity including the striking clinical response observed with novel ligand synthesis inhibitors such as abiraterone (ABI), and potent AR inhibitors such as enzalutamide (ENZ) ,48,49,50Interestingly, despite his clear demonstration that androgens were a critical driver of PCa progression, Huggins himself proposed that both hormonal deprivation and hormonal excess  might be used for therapeutic benefit . A numbeImportantly, simply discontinuing ADT and allowing androgen recovery to a eugonadal state does not appear to enhance survival; studies of intermittent androgen suppression in metastatic disease, which also allows gradual T recovery to physiological levels, demonstrated a trend toward inferior survival compared to continuous ADT . In thisAlthough historical support for the clinical benefit of androgen therapy for PCa has been mixed, the paradoxical inhibitory response of PCa to supraphysiological androgens has been demonstrated in multiple in vitro and in vivo studies. These studies are summarized below, and a more detailed review of findings in each pre-clinical model is provided in less androgen. Progression to the CR state is often accompanied by an increase in AR levels and transcriptional activity, suggesting the elevated AR levels in the more refractory tumor cells sensitizes these models to repression at lower androgen doses. In the transition from the androgen sensitive (AS) LNCaP 104S variant to the androgen independent (AI) 104R line  media), the repressive dose of R1881 is left-shifted two orders of magnitude, from 1 nM (10−9) to 0.01 nM (10−11), accompanied by increased AR expression and activity suggesting an increased sensitivity to androgen signaling [Within a tumor model, the response of cells to androgen-repressed growth is often more pronounced in castration resistant (CR) variants that have been derived after serial passage in androgen-depleted media, whereby the growth repressing effects can occur at 10–100 fold ignaling . An AI signaling ,60. Simiignaling .Exemplifying the known heterogeneity of advanced disease, PCa cell lines demonstrate a diversity of androgen-mediated growth responses. Importantly, an androgen-repressed growth phenotype is not exclusively associated with elevated AR levels, nor do low AR levels preclude androgen-repressed growth. Some PC cells with relatively low AR such as ARCaP still show a strong androgen-repressed phenotype . In contNotably, despite compelling preclinical evidence that the androgen-repressive effect is often magnified in CR tumor variants, clinical observations do not necessarily support this. In the early studies of Fowler and Whitmore 45 of 52 men had unfavorable responses to exogenous T, and the proportion of men who had an unfavorable response was higher in those who had been on prolonged hormone suppression (94%) compared to castration-naïve men (25%), or men in early stages of hormone suppression (36%) . It is pSeveral contemporary trials of T treatment have been conducted  .Szmulewitz et al. conducted a randomized trial evaluating transdermal T at 25, 5.0 or 75 mg/day in 15 men with CRPC but minimal metastatic disease . NotablyIn contrast to these studies in which T was administered in continuous manner, the group of Denmeade and Isaacs has pioneered an approach termed Bipolar Androgen Therapy (BAT) . OverexpSchweizer et al. reported the first clinical experience with this approach: 16 men with asymptomatic metastatic CRPC were treated with 400 mg of T intramuscularly (IM) monthly . Notablyn = 30) [In a follow up study recently reported by Teply et al, men with CRPC who had progressed on ENZ went on to receive BAT (n = 30) . This stIn another study in hormone naive patients, 29 men with low metastatic burden or biochemically recurrent disease who achieved PSA < 4 ng/dL after 6 months of ADT were treated with alternating 3 month cycles of BAT , followed by 3 months of ADT alone . The prin = 180) randomized trial  in asymptomatic mCRPC patients who have failed on abiraterone. In this study, BAT is being compared with ENZ with a primary end point of progression-free survival (PFS). While the more substantive clinical benefit observed in the studies of BAT vs. other contemporary and historical studies of androgen treatment may reflect the bipolar dosing strategy, it is likely that it also is related to the fact that these are the only studies in which supraphysiologic androgens have been achieved. Importantly, the relative dearth of patients experiencing clinical deterioration on T treatment compared to historical studies highlights the importance of patient selection, which, in modern studies, was limited to asymptomatic patients with limited metastatic disease.BAT is currently being tested in a large , the fraction of androgen-sensitive LNCaP cells in G0–G1 phase significantly decreased (at 36 and 48 h), reflected by an increase in cells in the S and G2/M phases, whereas at growth inhibitory doses of DHT greater than 0.1 nM, an increase in the number of cells in G0–G1 phase was observed, with a significant decrease in cells in the S and G2/M phases ,79. G1 aSPT in PC-3 cells with exogenous expression of AR has also shown a G0/G1 growth arrest. Litvinov et al. reported that overexpression of AR in PC-3 cells caused growth inhibition via G1 arrest, associated with increased expression of the cyclin dependent kinase (CDK) inhibitor p21 (WAF1/CIP1) , as welCDKN1B gene [Exposure of LNCaP and LNCaP-derived sublines to SPT has been shown to repress the expression of SKP2 . SKP2 isN1B gene . The encN1B gene ,79. LNCaN1B gene ,79,80,86One proposed mechanism involves AR-mediated repression of MYC (transcription), with concomitant repression of MYC target genes, which include the ubiquitin ligase SKP2, and thereby upregulation of the G1 cyclin dependent kinase (CDK) inhibitor p27Kip1 which is regulated by SKP2-mediated degradation ,80,83. AIn the androgen-sensitive LNCaP 104-S cells, R1881 increased levels of MYC and SKP2 expression and decreased levels of p27kip1 . In contSKP2 is also a transcriptional target of E2F , and chaTo date, these findings have not been validated across models that respond or resist SPT, and the mechanism(s) by which MYC or SKP2 are regulated by SPT have not been identified.While the role of androgen has been shown to be anti-apoptotic in androgen-dependent PCa cells , androgeNormally, licensing factors are degraded in M phase or early G1 phase to allow for relicensing and re-initiation of DNA replication in the next cell cycle . LitvinoUsing LNCaP, LAPC4 and CWR22Rv1 PCa cells, a cyclic proteasome-dependent degradation of AR during G1 was observed, along with co-immunoprecipitation (co-IP) of AR with replication complexes (RC), and co-IP of AR with origin of replication complex 2 (ORC2) in four of seven human CRPC metastases, suggesting AR may function as a licensing factor for DNA replication in cells that are androgen-sensitive for growth. In contrast, they do not observe binding of AR to RCs in cells in which liganded AR does not drive growth  ,71. The –6 M T in the 22Rv1 model [However, while cyclic degradation of AR in 22Rv1 cells, and association of AR with RC’s in 22Rv1 and VCaP cells was similarly observed, a repressive effect of androgen on 22RV1 does not occur; androgen remains stimulatory at 10v1 model . These oTo the extent that AR and AR splice variants are drivers of CRPC progression, mechanisms that repress their expression may lead to decreased cell growth. Studies by the Balk group have shown that AR gene expression is directly repressed by the AR through recruitment of lysine-specific histone demethylase 1 (LSD1) to an AR Binding Site (ARBS) region termed ARBS2 of which a segment of ~400 bp is highly conserved across species . AgonistWhether SPT results in dynamic changes of AR expression associated with growth inhibition in the clinical setting has not been assessed, but has been suggested in several pre-clinical models in vivo. Thelen et al. compared the growth of castration sensitive VCaP cells maintained in 10% fetal bovine serum (FBS) to a subline adapted to growth in 1 nM T after implantation in intact (non-castrate) nude mice . NotablyNakata examined growth inhibition by androgen in JDCaP-hr, an AR and ARV7 positive cell line derived from the castration recurrent outgrowth of a JDCaP xenograft (generated from the skin metastasis of a Japanese CRPC patient) ,99. ExprA recent trial of BAT in patients who progressed on ENZ did not show a clear correlation between response and modulation of AR-FL or AR-V7 transcript levels in circulating tumor cells . HoweverAs decreased AR signaling following ligand depletion leads to oncogenic changes in the AR-cistrome, transitioning back from low- to high-T conditions may reprogram the AR-cistrome toward a more differentiated state ,100. In Alternatively, Ruan et al. have recently demonstrated that SKP2 can bind and stabilize expression of Twist protein, leading to acquisition of epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) characteristics . GeneticThe repression or induction of specific genes involved in transcriptional programming and differentiation has also been linked to androgen induced proliferative arrest in PCa cells. SOX2 is an androgen repressed gene that is upregulated in CRPC and has been shown to stimulate epithelial-to-mesenchymal transition (EMT) as well as mediate lineage plasticity from (AR)-dependent luminal epithelial cells to AR-independent basal-like cells ,104,105.PDS5B  was initially discovered as a gene induced in LNCaP PCa cell lines and rat prostate cells undergoing androgen induced proliferative arrest . It was Promyelocytic leukemia zinc finger protein  is another androgen induced transcription factor gene widely involved in regulation of proliferation, differentiation, and stem cell maintenance that could potentially play a role in androgen-mediated growth repression . PLZF exSenescence is an irreversible cell cycle arrest associated with changes in cell morphology and gene expression that occurs during the normal embryogenesis . ExogenoSeveral mechanisms of induction of senescence by high dose androgens in PCa cells have been described. Roediger et al. demonstrated a dose-dependent induction of G1/G0 cell cycle arrest and senescence associated beta galactosidase activity  in LNCaP, C4-2 and AR-expressing PC-3 PCa cell lines treated with R1881 at 1 nM or higher concentrations, and in malignant prostate tissues treated ex vivo with R1881 at 10 nM or higher concentrations . FormatiN-acetylcysteine, or by inhibition of TGF beta/BMP mediated SMAD phosphorylation. The authors propose that utilization of high dose androgen therapy as early as after radical prostatectomy, or possibly biochemical relapse, when cancer cells are still dispersed and solitary may have most efficacy as stable induction of the self-sustained quiescent state in their studies only occurred at low cell density.In contrast to senescence, quiescence is a reversible growth arrest in G0/G1 that generally requires persistence of an external growth condition for its maintenance. However, Bui et al. recently reported the androgen-mediated induction of a self-sustained quiescent state in LNCaP and VCaP cells that was dependent on culturing cells at low density, and associated with induction of oxidative stress, a sustained redox imbalance, and transforming growth factor-beta (TGF beta)/bone morphogenic protein (BMP) signaling . TreatmeAlthough it is well established that ADT causes DNA damage, recent data have also shown that high androgen concentrations induce dsDNA breaks (DSB) in PCa cells that can lead to chromosomal rearrangements such as the TMPRSS2-ERG fusion , and maySimilar to the dual growth-promoting and growth-repressing effects of androgens in PCa, estrogens occupy a similarly paradoxical role in the biology and treatment of breast cancer, although with a more substantial history of clinical use. The efficacy of synthetic estrogens for advanced breast cancer was first described by Haddow in 1944, followed by a number of clinical trials that made estrogens the standard of care in postmenopausal patients with advanced breast cancer from the early 1960s onwards . A When the non-steroidal anti-estrogen tamoxifen was introduced in the 1970s for the treatment of advanced breast cancer, it was compared in clinical trials to DES or EE as the current standard of care. Notably, the response rate was generally comparable to that seen with estrogen treatment, but the consistently superior side effect profile of tamoxifen resulted in its uniform adoption for the first line treatment of advanced breast cancer. Estrogen therapy was essentially abandoned until studies in the 1990’s demonstrating the efficacy of this approach in patients who were resistant to anti-estrogens began to emerge. A number of studies evaluating DES or EE in heavily pre-treated post-menopausal women who were resistant to prior hormonal therapies including tamoxifen and/or aromatase inhibitors demonstrated objective response rates in approximately 30% of patients ,123. Theor in vivo to estradiol deprivation  induces sensitivity to estradiol-mediated growth inhibition [Similar to the data for PCa, work by several investigators demonstrated that long term adaptation of breast cancer cells in vitro hibition ,125. In hibition . Notablyhibition .A number of mechanisms for estrogen-induced apoptosis have been described. Work by the group of C. Jordan has delineated a SRC-dependent estradiol-mediated induction of endoplasmic reticulum stress and inflammatory responses that initiates an unfolded protein response, followed by apoptosis through the intrinsic  pathway with subsequent recruitment of the extrinsic (death receptor) pathway to complete the process . An estrHigh AR levels appear to sensitize a number of PC preclinical models to the inhibitory effect of SPT, raising the possibility that tumor AR expression might be useful in predicting response to SPT. Consistent with this hypothesis, high baseline PSA levels were associated with response in the BAT CRPC study, suggesting that SPT responders may have more active AR-signaling . HoweverThese data indicate that AR expression by itself is not necessarily correlated with androgen-driven growth suppression, and that a cell must possess the appropriate molecular mechanisms to engage AR as a growth suppressor or as an oncogene . As discAs discussed above, preclinical data suggest the induction of dsDNA breaks may be a mechanism mediating the anti-tumor effects of BAT, and an extreme response to BAT in a patient with inactivating mutations in the DNA damage response (DDR) genes ATM and BRCA2 has been reported . HoweverEarlier trials using physiologic dose T showed limited clinical activity compared to the supraphysiologic levels achieved in the BAT studies, suggesting the anti-tumor efficacy of SPT may reflect the level of intratumoral androgens achieved on therapy, an effect that may be influenced by steroid transport and metabolizing enzymes such as SLCO1B3 and UGT2B17. Expression and/or genetic variation in SLCO1B3 can modulate cellular T uptake in PCa cells in vitro, and genetic variants of SLCO1B3 linked to more efficient T uptake were associated with a shorter time to progression in men with CRPC on ADT ,131. In UGT2B17, responsible for the irreversible glucuronidation and ensuing elimination of T and DHT, is highly polymorphic and deletion variants of this enzyme (primarily expressed in the liver) are known to influence circulating steroid levels . As suchAlthough a significant body of preclinical evidence, and emerging clinical data, support the concept of high dose androgen therapy for the treatment of CRPC, a number of important questions remain unanswered. What is the optimal dosing schedule of T therapy? Given the diversity of mechanisms observed in preclinical studies, what are the specific mechanisms mediating clinical anti-tumor efficacy in a particular individual? Can rational drug combinations be designed to improve efficacy or sensitize tumors that are not inhibited by high dose androgen alone? Can biomarkers be identified to predict response or resistance to SPT? What, if any, is the impact of stromal AR signaling on the response to high dose androgen therapy?The relative success of the modern studies employing the BAT approach are encouraging, but the extent to which the clinical benefit observed in these studies reflects the bipolar dosing strategy or the documented achievement of truly supra-physiological androgen levels remains unclear. Preclinical studies have consistently found that SPT delivered on a continuous basis represses the growth of PCa cells, and no studies  have directly compared continuously administered SPT with BAT. Whether rapid cycling of SPT (vs. continuous SPT) is necessary to achieve the clinical benefit or delays resistance by preventing adaptive AR-downregulation remains to be determined. Ultimately, the predominant mechanism of SPT action may dictate the ideal dosing strategy, with continuous treatment likely providing improved efficacy if the main mechanism of SPT action relates to changing from a “low-T” oncogenic AR transcriptome to that of a more differentiating SPT transcriptome , and BATUnderstanding the mechanisms driving the anti-tumor efficacy of high dose androgen is particularly relevant for designing rational drug combinations. For example, the observation that androgens can induce DSB in conjunction with a recent report of an extreme response to BAT in a patient with inactivating ATM and BRCA2 mutations, provides support for the concept that DNA damage can sensitize to SPT . These dA further consideration which merits discussion is the potential for aromatase-mediated conversion of exogenous T to E2. PCa cells can variably express one or both of ER-alpha (which can promote PCa proliferation) and ER-beta (which can inhibit PCa proliferation); thus the net effect of a possible increase in estrogen signaling may be adverse or beneficial depending on the relative level of each . WhetherThe potential ability of T therapy to re-sensitize CRPC tumors to AR-axis inhibition is intriguing, and if borne out, may represent an important clinical approach for delaying disease progression. Illustrating the potential ability of androgen-repletion to ‘ug’ mechanisms of androgen sensitivity, culture of the androgen-repressed LNCaP 104-R1 cells in androgen rich media gave rise to a subline that was again androgen-sensitive for growth .  driving these responses and identify predictive biomarkers are needed in order to optimize this approach and identify rational treatment combinations.The clinical utility of potential treatment combinations will clearly depend on the relative importance of the various proposed mechanism, and the extent to which the diversity of mechanisms observed in the laboratory is recapitulated in human tumor specimens. Clinical studies with built-in collection of biospecimens will be critical to assessing the molecular changes associated with response or resistance to SPT, and for identifying potential predictors of response. There may be subsets of men whose tumors will be variably responsive to SPT or particular combinations based on the status of steroid transport and metabolizing genes, DNA damage repair genes, AR expression, MYC dependence, and/or RB loss or alterations in other cell cycle regulators. Ultimately, high dose T therapy is likely to represent yet another avenue for applying the principles of precision medicine for optimizing the care of men with CRPC.


The androgen receptor (AR) is the main driver of prostate cancer (PC) development and progression, and the primary therapeutic target in PC. To date, two functional ubiquitination sites have been identified on AR, both located in its C-terminal ligand binding domain (LBD). Recent reports highlight the emergence of AR splice variants lacking the LBD that can arise during disease progression and contribute to castrate resistance. Here, we report a novel N-terminal ubiquitination site at lysine 311. Ubiquitination of this site plays a role in AR stability and is critical for its transcriptional activity. Inactivation of this site causes AR to accumulate on chromatin and inactivates its transcriptional function as a consequence of inability to bind to p300. Additionally, mutation at lysine 311 affects cellular transcriptome altering the expression of genes involved in chromatin organization, signaling, adhesion, motility, development and metabolism. Even though this site is present in clinically relevant AR-variants it can only be ubiquitinated in cells when AR retains LBD suggesting a role for AR C-terminus in E2/E3 substrate recognition. We report that as a consequence AR variants lacking the LBD cannot be ubiquitinated in the cellular environment and their protein turnover must be regulated via an alternate pathway. Prostate cancer (PC) is the most common male malignancy in developed countries accounting for 15% of newly diagnosed male cancer cases in 2012 . Globally, in 2012 an estimated 307 000 men died from PC with deaths resulting from advanced, metastatic disease. PC is driven by the androgen receptor (AR) and tumours will initially respond to androgen deprivation therapy (ADT). However, the main clinical challenge for PC results from the inevitable recurrence of resistant tumours, termed castrate resistant prostate cancer (CRPC) for which there are no curative treatments and prognosis is extremely poor. Recently developed second generation anti-androgens are characterized by a response rate of only 50% highlighting the need for new targeted therapies . The intUbiquitination is a reversible covalent process that results in an attachment of a ubiquitin molecule to specific substrates that regulates multiple cellular processes, including protein degradation . UbiquitCurrently, two AR ubiquitination sites have been identified at K845 and K847, both in the C-terminal ligand binding domain (LBD) of AR . SeveralIn this manuscript, we report the discovery of a novel N-terminal domain (NTD) AR ubiquitination site at K311 which regulates both AR protein stability and is also critical for AR transcriptional activity and binding to p300 in the presence of androgen. Mutation of this site causes retention of AR on chromatin and abrogates its activity toward target genes. When this ubiquitination site is lost transcription of over a thousand genes is altered with global changes to gene expression in chromatin organization, cellular adhesion and motility, signal transduction, development and cellular metabolism pathways. Additionally, we report that AR-Vs are not ubiquitinated and hence their activity is neither regulated through this site nor by the MDM2 E3 ligase.in vitro mutagenesis . Lentiviral AR careers were prepared as previously described , anti-AR-v7 (Precision Antibodies), anti-H2A.Z (escribed .l-glutamine (Invitrogen) supplemented with 10% (v/v) foetal calf serum (FCS) at 37°C in 5% CO2. Transfections were performed using TransIT-LT1 reagent (MirusBio) following the manufacturer's instructions.LNCaP, HEK293T, CWR22Rv1 and COS-7 cells  were cultured in RPMI 1640 medium with 2 mM For luciferase assays, cells were transfected with 50 ng of pARE3-luc, 50 ng of pCMV-β-gal and 10 ng of pFLAG-His-AR and pMYC-MDM2 as required. All reactions were balanced with pCMV empty vector. Cells were cultured in steroid-depleted media (SDM) for 48 h followed by supplementation with 10 nM dihydrotestosterone (DHT) for an additional 24 h. Cells were lysed and incubated in 1× Promega luciferase assay reagents according to the manufacturer's instruction, and luciferase counts were established and normalized to β-galactosidase activity. Results were normalized to AR expression alone in steroid depleted conditions.In vitro ubiquitination assays were performed on purified N-terminal AR protein (aa 1–518) using a Ubiquitin Conjugation Reaction Buffer Kit (SK10) and incorporating the following enzymes: UBE1 (E-305), UBCH5 (E2–616) and MDM2 (E3–204)  according to manufacturer's instructions. Monoubiquitination assays were performed using a modified ubiquitin lacking the seven lysine ubiquitin acceptor sites, Ub-K0 (UM-NOK) and polyubiquitination assays performed using wild type ubiquitin (U-100H). The E3 Ligases CHIP, E6AP and hPIRH2 were generated via GST purification and in vitro ubiquitination reactions repeated, reactions utilizing purified E3 ligases were conducted in the presence of Ubiquitin aldehyde (Cat U-201) to inhibit any potential deubiquitinase contamination from the E3 purification stage. All reactions were activated by ATP and quenched with E1 stop buffer.Ubiquitination of N-terminal AR was confirmed via western blot using anti-AR and anti-ubiquitin antibodies (as outlined). Samples submitted for proteomic analysis were resolved via PAGE and stained using a silver stain II kit (Sigma) according to manufacturer's instructions or with coomassie stain. Bands representing monoubiquitinated AR were excised and sent for analysis at Newcastle University Protein and Proteome Analysis (NUPPA) facility.4HCO3 pH 7.8 and mashed to small pieces. The gel pieces were washed with 60% acetonitrile and washed with NH4HCO3 pH 7.8 prior to reduction in 50 μl of 10 mM DTT in 100 mM NH4HCO3 for 1 h at 56°C. Subsequently, cysteines were alkylated by adding 50 mM iodoacetamide in 100 mM NH4HCO3 and incubating in the dark at room temperature for 30 min. Gel pieces were repeatedly washed with 100 mM NH4HCO3 and 50% acetonitrile, and dehydrated using 100% acetonitrile. Proteins were in gel digested with 8 ng of trypsin (Promega) in 50 mM NH4HCO3, 1 mM CaCl2 and incubated overnight, shaking in a thermomixer at 37°C. The digest was stopped through addition of 5% of TFA. The gel pieces were washed twice with 60% acetonitrile containing 2% TFA and the washes were pooled with the aqueous supernatant after the digest. The volume of the digests was reduced to 10 μl in a speedvac and 10% was analysed by LCMSMS.The excised gel piece was put into a small volume of NHPeptides were concentrated on a Pepmap C-18 trap column (300 μm ID × 5 mm) and separated on a Pepmap C18 reversed phase column   using a linear gradient over 42 min from 96% A (0.05% formic acid), 4% B  to 35% A, 65% B and a flow rate of 300 nl/min.m/z 400 after accumulation of 5 × 105 ions. The 10 most intense ions of the survey scan were sequenced concurrently to full scan acquisition in the orbitrap by collision-induced dissociation  in the LTQ. Maximum filling times were 50 ms for the full scans and 100 ms for the MSMS scans. Precursor ion charge state screening was enabled and unassigned charge states and singly charged ions were rejected. Dynamic exclusion was enabled for 180 s with a maximum dynamic exclusion list of 50 entries and a relative mass window from −0.5 Da to + 1 Da.The spray voltage was 1.6 kV and the temperature of the heated capillary was set to 200°C. Survey scans were acquired with enabled lockmass from 400–1600 Da at a resolution of 30 000 at 6 cells/90-mm dish, transfected 24 h later with 1 μg of each plasmid as indicated, incubated for 48 h, and lysed directly into lysis buffer ). Lysates were incubated with 1 μg of antibodies as indicated for 16 h at 4°C, and antibodies were pulled down using protein G-Sepharose beads. For denaturing IPs, cells were subjected to 20 μM MG132 proteasomal inhibitor treatment for the final 16 h followed by collection into lysis buffer supplemented with 2% SDS and subsequently denatured at 100°C for 10 min  and incubated on ice for 10 min. Nuclei were collected by centrifugation at 1200 g for 5 min at 4°C. Supernatant was used as a cytosolic fraction and cleaned by centrifugation at 200 g for 5 min at 4°C. Nuclei were washed in 1 ml of buffer A without glycerol and resuspended in 1ml of buffer B  and incubated on ice for 30 min. Finally, sample was centrifuged at 1500 g for 5 min at 4°C and the chromatin pellet was washed in buffer B seven times followed by denaturing immunoprecipitation.For chromatin isolation, cells were trypsinized and resuspended in 1ml of Buffer A  according to the manufacturer's instructions and incubated in culture medium for 96 h prior to cell lysis and analysis by western blotting as described previously  or qPCR.®Green on 384-well optical reaction plates with the ABI 7900HT real-time PCR system. Results were normalized to HPRT1 expression. Primers were KLK2 F: 5΄-AGCATCGAACCAGAGGAGTTCT-3΄, R: 5΄-TGGAGGCTCACACACTGAAGA-3΄; HPRT1 F: 5΄-GAACGTCTTGCTCGAGAGATGTG-3΄, R: 5΄-CCAGCAGGTCAGCAAAGAATTT-3΄; PSA F: 5΄-ACTGCATCAGGAACAAAAGCGT-3΄, R: 5΄-TGTGGGAAGCTGTGGCTGAC-3΄; NDRG1 F: 5΄-ACAACCCCCTCTTCAACTACG-3΄, R: 5΄-GCCAATAATGCTTTTCAGCCCA-3΄.For qPCR RNA was extracted using Trizol (Invitrogen) according to manufacturer's instruction, and quantified using Nanodrop cDNA synthesis using the oligo dT primers and data analysis was performed as described previously . BrieflyPSA gene enhancer forward (F) 5΄-GGGGTTTGTGCCACTGGTGAG-3΄; reverse (R) 5΄-GGGAGGCAATTCTCCATGGTT-3΄; enhancer-promoter F 5΄-TAGAAGACGTGGAAGTAGCTG-3΄; R 5΄-AACCTCATGGATCCGGTGTCC-3΄; promoter F 5΄-TCTAGTTTCTGGTCTCAGAG-3΄; R 5΄-TTGCTGTTCTGCAATTACTAG-3΄; intron 1 F 5΄-CCAAGGACCTCTCTCAATGC-3΄; R 5΄-AGGGAATGAGGAGTTCTCAG-3΄; exon 3 F 5΄-CACACCCGCTCTACGATATGAG-3΄; R 5΄-GAGCTCGGCAGGCTCTGACAG-3΄; 3΄ UTR (untranslated region) F 5΄-TACTGGCCATGCCTGGAGAC-3΄; R 5΄-TGGCTCACAGCCTTCTCTAG-3 as previously described by Lee et al.  for 72 h followed by 10 nM DHT treatment for 2 h. Cell were fixed for 20 min in 4% formaldehyde, permeabilized for 20 min in 0.1% Triton X-100 in PBS followed by blocking in 1% BSA, staining and imaging using Zeiss LSM 700 confocal microscope.in vitro ubiquitination assays, incorporating purified N-terminal AR fragment (aa1-538), E1 (UBE1), E2 (UBCH5α) and various E3 ligase enzymes potentially linked to AR function to examine specificity of AR ubiquitination. By incorporating a modified ubiquitin lacking all seven lysine ubiquitin acceptor sites (UbK7R), we demonstrated AR N-terminal mono-ubiquitination by MDM2  were recently identified in the C-terminus, both targeted by RNF6 . Previou5/K847 we2 Figure –B, PIRH22 Figure . Mass sp2 Figure , highlig2 Figure .in vitro ubiquitination experiments with MDM2 and a carboxy-terminal AR construct comprising both DNA binding domain (DBD) and ligand binding domain (LBD) which demonstrated a strong ubiquitination of this C-terminal construct by MDM2 , and is absent in all the other regions of the gene. However, K311R AR levels at the promoter region are not changed as much in response to androgen stimulation and most importantly this mutant is retained throughout the whole PSA gene in response to androgen stimulation suggesting failure to effectively serve its function as a transcription factor  by at least 2-fold In the presence of androgen and 771 in steroid depleted conditions when compared to cells expressing WT AR. Most significantly altered genes included KLK3 (PSA) and KLK2  of AR, however we failed to detect any ubiquitination of this domain by MDM2, in contrast to efficient ubiquitination of full length AR . However, in the PC346 model derived from a primary human prostate tumour subcutaneously implanted into athymic mice that were subsequently castrated, an androgen-independent subline, PC346DCC emerged that contained the K311R AR mutation . InteresAR phosphorylation of serine 308 (S308), adjacent to K311, is associated with a longer time to disease specific death  and may Potentially, the role of the LBD in K311 AR ubiquitination could be important if ubiquitination occurred mainly outside of the nucleus as the LBD contains a nuclear export signal (NES) which has been previously reported to signal for poly-ubiquitination and degradation but it is blocked by binding to androgens which inhibits NES . ConsequSupplementary DataClick here for additional data file.


This population-based cohort study investigated the risk of developing peripheral arterial occlusive disease (PAOD) in patients with Bell’s palsy.We used longitudinal claims data of health insurance of Taiwan to identify 5,152 patients with Bell’s palsy newly diagnosed in 2000–2010 and a control cohort of 20,608 patients without Bell’s palsy matched by propensity score. Incidence and hazard ratio (HR) of PAOD were assessed by the end of 2013.The incidence of PAOD was approximately 1.5 times greater in the Bell’s palsy group than in the non-Bell’s palsy controls (7.75 vs. 4.99 per 1000 person-years). The Cox proportional hazards regression analysis measured adjusted HR was 1.54  = 1.35–1.76) for the Bell’s palsy group compared to the non-Bell’s palsy group, after adjusting for sex, age, occupation, income and comorbidities. Men were at higher risk of PAOD than women in the Bell’s palsy group, but not in the controls. The incidence of PAOD increased with age in both groups, but the Bell’s palsy group to control group HR of PAOD decreased as age increased. The systemic steroid treatment reduced 13% of PAOD hazard for Bell’s palsy patients, compared to those without the treatment, but not significant.Bell’s palsy appears to be associated with an increased risk of developing PAOD. Further pathophysiologic, histopathology and immunologic research is required to explore the underlying biologic mechanism. Peripheral arterial occlusive disease (PAOD) is a circulation disease caused by atherosclerosis of peripheral vessels. It features with pain, pale, paresthesia and pulseless of limbs. The prevalence of PAOD ranges from 3.9%-26.2% among worldwide population., 2 The iBell’s palsy features with a sudden onset of unilateral facial paresis or paralysis. The symptom is caused by the inflammatory facial nerve, resulting from compression of the narrowest portion of the fallopian canal , 7. The A recent study reported that patients with Bell’s palsy had a 2-fold increased risk of stroke. To exploThe insurance program of Taiwan is a universal insurance program, reformed from all 13 insurance systems in 1995, providing comprehensive coverage for 99% of all residents. This study used the longitudinal health insurance database (LHID 2000), consisting of claims data of 1,000,000 insured individuals randomly selected from all 23 million insured population. Information on demographic data, inpatient and outpatient cares, date of clinic visit or hospitalization and prescriptions were available in the database for the period from 1996 to 2013. Diagnoses were coded in the format of the International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM). The authority had replaced the original identification numbers with surrogate numbers before the data were released to protect the people privacy. Our research was approved by the Research Ethics Committee of China Medical University and Hospital (CMUH104-REC2-115).From the LHID 2000 medical claims, we identified patients with Bell’s palsy (ICD-9-CM 351.0x), aged 20 years and older without the history of PAOD, newly diagnosed in 2000–2010, and included them in the Bell’s palsy group. The date with Bell’s palsy diagnosed was defined as the index date. Only patients who received systemic steroid treatment within 10 days after the date with Bell’s palsy diagnosed using anatomical therapeutic chemical (ATC) code (H02AB) were included in the present study. A non-Bell’s palsy controls were also selected from population without Bell’s palsy and PAOD, matched by propensity score to balance the two groups to augment their comparability. We estimOur primary outcome was PAOD . Both Bell’s palsy and control groups were followed from the index date until they had the diagnosis of PAOD, or were censored for deaths or withdrawal from the insurance system, or the end of 2013.The distribution of categorical variables  were compared between Bell’s palsy and non-Bell’s palsy groups by the Chi-square test. The Student’s t-test was used to compare mean ages between the 2 cohorts. The incidence density rates of PAOD  were calculated for the Bell’s palsy and non-Bell’s palsy groups by potential risk factors, such as sex and age , and comorbidity (no/yes). The Kaplan-Meier method was used to draw cumulative incidence curves of PAOD for both groups and the curves were examined using log-rank test. Univariate and multivariate Cox proportional hazards regression models were used to assess hazard ratios (HRs) of PAOD for Bell’s palsy patients compared to the non-Bell’s palsy group by PAOD-associated factors. The multivariate model included variable of sex, age, occupation, income, and comorbidities . HRs of PAOD were evaluated by sex, age group, and comorbidity. We further evaluated whether the systemic steroid treatment could reduce the development of PAOD for Bell’s palsy patients.We deemed a two-tailed p value less than 0·05 significant. SAS 9.4 software  was performed data management and statistical analysis.After matching participants in a 1:4 ratio by the propensity score, 5152 patients with Bell’s palsy and 20608 individuals without Bell’s palsy were included. During the mean follow-up period of 7.66 years, there were 303 patients diagnosed with PAOD in the Bell’s palsy group, with an incidence density rate of 7.75 per 1000 person-years, and 790 patients diagnosed with PAOD in the non-Bell’s palsy group, with an incidence density rate of 4.99 per 1000 person-years. Cumulative incidence curves of PAOD in patients with and without Bell’s palsy were shown in Further data analysis evaluating treatment effectiveness of systemic steroid showed that the non-users were at 1.4-fold greater PAOD risk than users (9.45 vs. 6.81 per per 1000 person-years) . Bell’s To the best of our knowledge, this is the first retrospective cohort study using population data to investigate the risk of developing PAOD following the Bell’s palsy occurrence. We demonstrated a 54% increased hazard of subsequent PAOD in Bell’s palsy cohort compared with controls. Besides, the effect is more prominent in young patients and patients without comorbidity.Several studies have investigated mechanisms to explain the association between PAOD and Bell’s palsy. Bell’s palsy is acknowledged widely as reactivation of latency virus of geniculate ganglion, with HSV-1 and VZV are known more common pathogens , 17. VirHowever, the diagnosis accuracy is a common concern of using medical registry database, especially for Bell’s palsy and stroke because the sudden onset of unilateral facial palsy of both disorders may receive more attention from physicians. In this aspect, our study outcome of increased risk of PAOD in Bell’s palsy cohort supported that Bell’s palsy played the pathologic role in cardiovascular disease risk and contained vascular risk components in this idiopathic neurologic inflammation disease.Our study showed that the PAOD risk in patients with Bell’s palsy increased for those with older ages, diabetes, hypertension and dyslipidemia. Our findings are compatible with previous study., 4, 27 IIt is interesting to note that the incidence of PAOD increased with age in both cohorts with and without Bell’s palsy. But, the Bell’s palsy group to non-Bell’s palsy group HR was greater in younger than in the elderly, indicating the impact of Bell’s palsy is relatively greater for younger Bell’s palsy patients. On the other hand, the corresponding relative adjusted HR for the elderly was 1.21 (95% CI = 0.98–1.49). Our data analysis by comorbidity showed that patients with cardiovascular diseases and diabetes have increased PAOD risk in both Bell’s palsy and control cohorts. Cardiovascular diseases and diabetes were more prevalent in the elderly in both cohorts. This is why the relative adjusted HR for the elderly was lower than that for the younger. The effect of Bell’s palsy is particularly prominent in the youngest subgroup and the subgroup without comorbidity. These findings support that Bell’s palsy is an independent risk factor for PAOD.15 Our further data analysis compared the PAOD risk between Bell’s palsy patients with and without systemic steroid treatment. Results showed that Bell’s palsy patients with and without systemic steroid treatment consistently had a significantly higher risk of PAOD than in subjects without Bell’s palsy. Our result also revealed that the systemic steroid treatment had a moderate effect in reducing the risk of the long term PAOD risk for patient with Bell’s palsy, but not significant. In an earlier study, Gilden reported that Bell’s palsy treatment only supported a short course of prednisone medication within 2 to 14 days after the onset of symptoms.[It is important to note the treatment effectiveness of systemic steroid treatment for Bell’s palsy patients. A recent study found a beneficial effect for Bell’s palsy patients in reducing stroke risk.symptoms. A short Our study had some potential limitations. First, diagnosis accuracy was always concerned in using medical registry database. However, in addition to verification coding by licensed medical record technician after physician competing discharge chart, routine sampling of charts to cross-over exam by specialists at the NHI Bureau to ensure the validity and accuracy of disease coding. Second, there’s some meaningful information not available in NHIRD, such as smoking, daily activity and obesity. However, we minimized the effect of these confounding factors using COPD, CAD and stroke as proxy in the data analysis. Third, information on histopathology of diseases is unavailable in the claims data, the histopathology relationship could not confirmed between Bell's palsy and PAOD. Finally, the patient with asymptomatic PAOD may not seek medical assistance and may not identified in our study. Therefore, it led to underestimate incidence of PAOD in both Bell’s palsy and control cohorts. Despite these limitations, our nationwide population-based study using information from the compulsory health insurance database over a 14-year period provided a sufficient sample size to investigate the association between Bell’s palsy and PAOD risk.The findings from this large-scale follow-up study could conclude that Bell’s palsy was associated with 49% increased risk of developing PAOD. Low-dose maintenance systemic steroid therapy for a longer period would be an option to reduce the risk and deserve study. Further research focused on pathophysiologic, histopathology and immunologic issues is also warranted to clarify the underlying biologic mechanism.