The Translational Role of Animal Models for Estrogen-Related Functional Bladder Outlet Obstruction and Prostatic Inflammation

The prevalence of LUTS and prostatic diseases increases with age both in humans and companion animals, suggesting that a common underlying cause of these conditions may be age-associated alterations in the balance of sex hormones. The symptoms are present with different and variable micturition dysfunctions and can be assigned to different clinical conditions including bladder outlet obstruction (BOO). LUTS may also be linked to chronic non-bacterial prostatitis/chronic pelvic pain syndrome (CP/CPPS), but the relationship between these conditions is unknown. This review summarizes the preclinical data that supports a role for excessive estrogen action in the development of obstructive voiding and nonbacterial prostatic inflammation. Preclinical studies that are emphasized in this review have unequivocally indicated that estrogens can induce functional and structural changes resembling those seen in human diseases. Recognizing excessive estrogen action as a possible hormonal basis for the effects observed at multiple sites in the LUT may inspire the development of innovative treatment options for human and animal patients with LUTS associated with functional BOO and CP/CPPS

A real-time health notification system aimed at enhancing the interaction between animal care staff and researchers promotes animal welfare

Regardless of the microbiological status of an animal facility, research animals may experience health problems, leading to pain, suffering and distress. Simple and efficient tools are needed to collect data systematically, allowing researchers to react and resolve animals' health issues. We have developed a real-time notification method for recording clinical observations, which caretakers can input into the ELLI record-keeping system, accompanied by a picture or video. A browser-based interface system sends alerts using a three-tier scale (+, 120 hours; ++, 72 hours; +++, 24 hours) by email and/or SMS. The percentage of animal health notifications for rodents was 1.31% in 2016, 1.33% in 2017 and 1.58% in 2018, with 34-44% for coat and skin conditions (wounds, bites and scratches). All other notifications, including environment and behaviour, procedure-specific indicators (weight loss, bleeding and abnormal secretions) and other abnormalities such as eye and teeth malformations, ranged from 5% to 10% during the three-year period. Researchers displayed good compliance by reacting to the notifications within the expected time frame. Most health notifications concerned genetically modified (GM) animals without a predetermined harmful phenotype, regardless of being on project licence or maintenance licence. Health notification records may be useful retrospectively not only to review the health and welfare issues of new GM lines but also to evaluate the actual severity of procedures. The health notification system described here provides valuable information to the veterinarian and the animal welfare body by helping to address specific health conditions and to improve animal welfare and implement the 3Rs

Infection with the enteric pathogen C. rodentium promotes islet-specific autoimmunity by activating a lymphatic route from the gut to pancreatic lymph node

In nonobese diabetic (NOD) mice, C. rodentium promotes priming of islet-specific T-cells in pancreatic lymph nodes (PaLN), which is a critical step in initiation and perpetuation of islet-autoimmunity. To investigate mechanisms by which C. rodentium promotes T-cell priming in PaLN, we used fluorescent imaging of lymphatic vasculature emanating from colon, followed dendritic cell (DC) migration from colon using photoconvertible-reporter mice, and evaluated the translocation of bacteria to lymph nodes with GFP-C. rodentium and in situ hybridization of bacterial DNA. Fluorescent dextran injected in the colon wall accumulated under subcapsular sinus of PaLN indicating the existence of a lymphatic route from colon to PaLN. Infection with C. rodentium induced DC migration from colon to PaLN and bacterial DNA was detected in medullary sinus and inner cortex of PaLN. Following infection with GFP-C. rodentium, fluorescence appeared in macrophages and gut-derived (CD103+) and resident (CD103-/XCR1+) DC, indicating transportation of bacteria from colon to PaLN both by DC and by lymph itself. This induced proinflammatory cytokine transcripts, activation of DC and islet-specific T-cells in PaLN of NOD mice. Our findings demonstrate the existence of a direct, enteric pathogen-activated route for lymph, cells, and bacteria from colon, which promotes activation of islet-specific T-cells in PaLN.</p

CC chemokine ligand 2 (CCL2) stimulates aromatase gene expression in mammary adipose tissue

Obesity is a risk factor for postmenopausal breast cancer. Obesity-related inflammation upregulates aromatase expression, the rate-limiting enzyme for estrogen synthesis, in breast adipose tissue (BAT), increasing estrogen production and cancer risk. The regulation of aromatase gene (CYP19A1) in BAT is complex, and the mechanisms linking obesity and aromatase dysregulation are not fully understood. An obesity-associated factor that could regulate aromatase is the CC chemokine ligand (CCL) 2, a pro-inflammatory factor that also activates signaling pathways implicated in CYP19A1 transcription. By using human primary breast adipose stromal cells (ASCs) and aromatase reporter (hARO-Luc) mouse mammary adipose explants, we demonstrated that CCL2 enhances the glucocorticoid-mediated CYP19A1 transcription. The potential mechanism involves the activation of PI.4 via ERK1/2 pathway. We also showed that CCL2 contributes to the pro-inflammatory milieu and aromatase expression in obesity, evidenced by increased expression of CCL2 and CYP19A1 in mammary tissues from obese hARO-Luc mice, and subcutaneous adipose tissue from obese women. In summary, our results indicate that postmenopausal obesity may promote CCL2 production in BAT, leading to exacerbation of the menopause-related inflammatory state and further stimulation of local aromatase and estrogens. These results provide new insights into the regulation of aromatase and may aid in finding approaches to prevent breast cancer

Evaluation of the estrogenic effects of dietary perinatal Trifolium pratense

This study was designed to investigate the potential estrogenic effects of perinatal dietary phytoestrogens on the rat uterus. Pregnant rats were divided to three groups provided the following diets: (1) rat chow, (2) rat chow with 7.5% Trifolium (T.) pratense, or (3) rat chow supplemented with 17β-estradiol (0.5 mg/kg). The dams in each group were kept on the same diet during pregnancy and lactation. Female offspring were euthanized on day 21 at which time body and organ weights were recorded and tissue samples were taken for histology. Immunohistochemistry was performed to detect estrogen receptor alpha (ERα) and progesterone receptor (PR) levels. Our results revealed estrogen-like biological effects of perinatal T. pratense exposure. Relative uterus and ovary weights in the experimental groups were increased compared to control. The number of uterine glands and luminal epithelium heights were also increased. However, there were no statistically significant changes detected in the immunostaining intensity of ERα and PR between the groups

In vivo Induction of Functional Inhibitory IgG Antibodies by a Hypoallergenic Bet v 1 Variant

Allergic sensitization to the major allergen Bet v 1 represents the dominating factor inducing a vast variety of allergic symptoms in birch pollen allergic patients worldwide, including the pollen food allergy syndrome. In order to overcome the huge socio-economic burden associated with allergic diseases, allergen-specific immunotherapy (AIT) as a curative strategy to manage the disease was introduced. Still, many hurdles related to this treatment exist making AIT not the patients' first choice. To improve the current situation, the development of hypoallergen-based drug products has raised attention in the last decade. Herein, we investigated the efficacy of the novel AIT candidate BM4, a hypoallergenic variant of Bet v 1, to induce treatment-relevant cross-reactive Bet v 1-specific IgG antibodies in two different mammals, Wistar rats and New Zealand White rabbits. We further analyzed the cross-reactivity of BM4-induced Wistar rat antibodies with the birch pollen-associated food allergens Mal d 1 and Cor a 1, and the functional capability of the induced antibodies to act as IgE-blocking IgG antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to determine the titers of rat IgG1, IgG2a, IgG2b, and IgE, as well as rabbit IgG and IgE antibodies. To address the functional relevance of the induced IgG antibodies, the capacity of rat sera to suppress binding of human IgE to Bet v 1 was investigated by using an inhibition ELISA and an IgE-facilitated allergen-binding inhibition assay. We found that the treatment with BM4 induced elevated Bet v 1-specific IgG antibody titers in both mammalian species. In Wistar rats, high BM4-specific IgG1, IgG2a, and IgG2b titers (10(4)to 10(6)) were induced, which cross-reacted with wild-type Bet v 1, and the homologous allergens Mal d 1 and Cor a 1. Rat allergen-specific IgG antibodies sustained upon treatment discontinuation. Sera of rats immunized with BM4 were able to significantly suppress binding of human IgE to the wild-type allergens and CD23-mediated human IgE-facilitated Bet v 1 binding on B cells. By contrast, treatment-induced IgE antibody levels were low or undetectable. In summary, BM4 induced a robust IgG immune response that efficiently blocked human IgE-binding to wild-type allergens, underscoring its potential therapeutic value in AIT

Novel Lignan and Stilbenoid Mixture Shows Anticarcinogenic Efficacy in Preclinical PC-3M-luc2 Prostate Cancer Model

Peer reviewe

Signature of circulating small non-coding RNAs during early fracture healing in mice

Fracture healing is a complex process with multiple overlapping metabolic and differentiation phases. Small non-coding RNAs are involved in the regulation of fracture healing and their presence in circulation is under current interest due to their obvious value as potential biomarkers. Circulating microRNAs (miRNAs) have been characterized to some extent but the current knowledge on tRNA-derived small RNA fragments (tsRNAs) is relatively scarce, especially in circulation.In this study, the spectrum of circulating miRNAs and tsRNAs was analysed by next generation sequencing to show their differential expression during fracture healing in vivo. Analysed tsRNA fragments included stress-induced translation interfering tRNA fragments (tiRNAs or tRNA halves) and internal tRNA fragments (i-tRF), within the size range of 28–36 bp. To unveil the expression of these non-coding RNAs, genome-wide analysis was performed on two months old C57BL/6 mice on days 1, 5, 7, 10, and 14 (D1, D5, D7, D10, and D14) after a closed tibial fracture.Valine isoacceptor tRNA-derived Val-AAC 5′end and Val-CAC 5′end fragments were the major types of 5′end tiRNAs in circulation, comprising about 65 % of the total counts. Their expression was not affected by fracture. After a fracture, the levels of two 5′end tiRNAs Lys-TTT 5′ and Lys-CTT 5′ were decreased and His-GTG 5′ was increased through D1-D14. The level of miR-451a was decreased on the first post-fracture day (D1), whereas miR-328-3p, miR-133a-3p, miR-375-3p, miR-423-5p, and miR-150-5p were increased post-fracture. These data provide evidence on how fracture healing could provoke systemic metabolic effects and further pinpoint the potential of small non-coding RNAs as biomarkers for tissue regeneration.</p

[C-11]carfentanil PET imaging for studying the peripheral opioid system in vivo : effect of photoperiod on mu-opioid receptor availability in brown adipose tissue

Purpose Photoperiod determines the metabolic activity of brown adipose tissue (BAT) and affects the food intake and body mass of mammals. Sympathetic innervation of the BAT controls thermogenesis and facilitates physiological adaption to seasonal changes, but the exact mechanism remains elusive. Previous studies have shown that central opioid signaling regulates BAT thermogenesis, and that the expression of the brain mu-opioid receptor (MOR) varies seasonally. Therefore, it is important to know whether MOR expression in BAT shows seasonal variation. Methods We determined the effect of photoperiod on BAT MOR availability using [C-11] carfentanil positron emission tomography (PET). Adult rats (n = 9) were repeatedly imaged under various photoperiods in order to simulate seasonal changes. Results Long photoperiod was associated with low MOR expression in BAT (beta = -0.04, 95% confidence interval: - 0.07, - 0.01), but not in muscles. We confirmed the expression of MOR in BAT and muscle using immunofluorescence staining. Conclusion Photoperiod affects MOR availability in BAT. Sympathetic innervation of BAT may influence thermogenesis via the peripheral MOR system. The present study supports the utility of [C-11]carfentanil PET to study the peripheral MOR system.Peer reviewe

Evaluation of [F-18]F-DPA PET for Detecting Microglial Activation in the Spinal Cord of a Rat Model of Neuropathic Pain

Purpose Recent studies have linked activated spinal glia to neuropathic pain. Here, using a positron emission tomography (PET) scanner with high spatial resolution and sensitivity, we evaluated the feasibility and sensitivity of N,N-diethyl-2-(2-(4-([18F]fluoro)phenyl)-5,7-dimethylpyrazolo[1,5-a] pyrimidin3-yl)acetamide -([F-18]F-DPA) imaging for detecting spinal cord microglial activation after partial sciatic nerve ligation (PSNL) in rats.Procedures Neuropathic pain was induced in rats (n = 20) by PSNL, and pain sensation tests were conducted before surgery and 3 and 7 days post- injury. On day 7, in vivo PET imaging and ex vivo autoradiography were performed using -[F-18]F-DPA or -[C-11]PK11195. Ex vivo biodistribution and PET imaging of the removed spinal cord were carried out with -[F-18]F-DPA. Sham-operated and PK11195pretreated animals were also examined.Results Mechanical allodynia was confirmed in the PSNL rats from day 3 through day 7. Ex vivo autoradiography showed a higher lesion-to-background uptake with -[F-18]F-DPA compared with -[C-11]PK11195. Ex vivo PET imaging of the removed spinal cord showed -[F-18]F-DPA accumulation in the inflammation site, which was immunohistochemically confirmed to coincide with microglia activation. Pretreatment with PK11195 eliminated the uptake. The SUV values of in vivo -[F-18]F-DPA and -[C-11]PK11195 PET were not significantly increased in the lesion compared with the reference region, and were fivefold higher than the values obtained from the ex vivo data. Ex vivo biodistribution revealed a twofold higher -[F-18] F-DPA uptake in the vertebral body compared to that seen in the bone from the skull.Conclusions[F-18]F-DPA aided visualization of the spinal cord inflammation site in PSNL rats on ex vivo autoradiography and was superior to -[C-11]PK11195. In vivo -[F-18]F-DPA PET did not allow for visualization of tracer accumulation even using a high-spatial-resolution PET scanner. The main reason for this result was due to insufficient SUVs in the spinal cord region as compared with the background noise, in addition to a spillover from the vertebral body.</p