Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non‐24‐hour sleep‐wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT₁ and MT₂, belonging to the G protein‐coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT₁ and MT₂. Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT₁ and MT₂ by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT₁ or MT₂. None of the mABs cross‐reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR‐KO mice as control. MT₂ expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta‐cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.     

Angiostatin overexpression in Morris hepatoma results in decreased tumor growth but increased perfusion and vascularization.

Growth of malignant tumors is dependent on sufficient blood supply. Thus, inhibition of tumor angiogenesis is emerging as a promising target in the treatment of malignancies. Human angiostatin (hANG) is one of the most potent inhibitors of endothelial cell proliferation, angiogenesis, and tumor growth in vivo. However, its mechanisms operating in vivo are not well understood. METHODS: To obtain more information about functional changes in the angiogenic process, we established Morris hepatoma (MH3924A) cell lines expressing hANG (hANG-MH3924A). The effects of hANG expression on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) were measured in coculture experiments in vitro. To evaluate changes in tumor perfusion and blood volume, H2 15O and 68Ga-DOTA-albumin (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid) were used for PET studies in vivo. Additionally, immunohistologic quantification of vascularization, apoptosis, and proliferation as well as gene array analyses were performed. RESULTS: Our in vitro experiments demonstrate reduced proliferation and increased apoptosis in HUVECs when being cocultured with hANG-MH3924A. In support, tumor growth of hANG-MH3924A is diminished by 95% in vivo. However, tumor perfusion and blood volume are increased in hANG-MH3924A corresponding to an increased microvessel density. Furthermore, hANG-transfected tumors show changes in expression of genes related to apoptosis, stress, signal transduction, and metabolism. CONCLUSION: hANG expression leads to inhibition of tumor growth, increased apoptosis, and changes in the expression of multiple genes involved in stress reactions, signal transduction, and apoptosis, which indicates a multifactorial reaction of tumors. An enhanced microvessel density is seen as part of these reactions and is associated with increased perfusion as measured by PET.     

Auricular Acupressure as a Treatment for Anxiety in Prehospital Transport Settings

Background: Auricular acupuncture at the relaxation point has been previously shown to be an effective treatment for anxiety in the preoperative setting. The purpose of this prospective, randomized, blinded study was to determine whether auricular acupressure can reduce stress and anxiety during ambulance transport.

Methods: Patients who required ambulance transport secondary to medical conditions were randomized to receive auricular acupressure at the relaxation point (n = 17) or at a sham point (n = 19). A visual analog scale was used to assess state anxiety as well as patient anticipation of hospital medical treatment (estimated waiting period for treatment, anticipated pain during treatment, attitude toward the physicians, and treatment outcomes). These variables were assessed at baseline and on arrival to the hospital.

Results: Patients in the relaxation group reported significantly less anxiety than patients in the sham group on arrival to the hospital (visual analog scale mean +/- SD: 37.6 +/- 20.6 to 12.4 +/- 7.8 mm vs. 42.5 +/- 29.9 to 46.7 +/- 25.9 mm, respectively;P = 0.002). Similarly, patient perception of pain during treatment (mean visual analog scale +/- SD: 32.7 +/- 27.7 to 14.5 +/- 8.1 mm vs. 17.2 +/- 26.1 to 28.8 +/- 21.9 mm, respectively;P = 0.006) and treatment outcomes of their illnesses (mean visual analog scale +/- SD: 46.7 +/- 29.4 to 19.1 +/- 10.4 mm vs. 35.0 +/- 25.7 to 31.5 +/- 20.5 mm, respectively;P = 0.014) were significantly more positive in the relaxation group than in the sham group. No differences were found in the other variables assessed.     

Transjugular intrahepatic portosystemic shunting (TIPS) with balloon-expandable and self-expanding stents: Technical and clinical aspects after 3 1/2 years’ experience

Purpose To evaluate prospectively our experience with transjugular intrahepatic portosystemic shunt (TIPS) using four different metallic stents. Methods Between November 1991 and April 1995, 57 patients (41 men and 16 women; age 35–72 years, mean 54 years) underwent the TIPS procedure. Techniques for portal vein localization before and during TIPS were fluoroscopy, computed tomography (CT) studies, wedged hepatic venography, arterial portography, and ultrasound. After predilation we deployed balloon-expandable (n=48) and self-expanding (n=45) metallic stents. Fifteen patients underwent variceal embolization. Initial follow-up angiograms (mean 6.9 months, range 3–24 months) were obtained in 39 of these patients. Results Fifty-three patients (93%) had successful TIPS placement. The mean decrease in portal pressure was 42.7%. Besides fluoroscopy, the most helpful techniques for portal vein localization were venography and CT. Residual stenosis (n=1) and late shortening (n=4) of Wallstents resulted in shunt dysfunction. The technical problems encountered with the Palmaz stent resulted from its lack of flexibility. We combined balloon-expandable and self-expanding stents in 12 patients. The 30-day and late follow-up (mean 11.9 months) percutaneous reintervention rates were 11.3% and 64.2%, respectively. There were no clinically significant complications related to the TIPS insertions. Conclusion An ideal stent does not exist for TIPS, and the authors recommend combining a Palmaz stent with a flexible self-expanding stent.