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.     

A Novel Nomogram to Identify Candidates for Extended Pelvic Lymph Node Dissection Among Patients with Clinically Localized Prostate Cancer Diagnosed with Magnetic Resonance Imaging-targeted and Systematic Biopsies

Background

Available models for predicting lymph node invasion (LNI) in prostate cancer (PCa) patients undergoing radical prostatectomy (RP) might not be applicable to men diagnosed via magnetic resonance imaging (MRI)-targeted biopsies.

Results of physical therapy for idiopathic clubfoot: a long-term follow-up study

Physical therapy without anesthesia or plaster casts was used to treat 338 cases of clubfoot (CF). Our technique is based on progressive sequential manipulations at birth. We first reduce the varus and later the equinus component of the CF. The gentle stretches used in this technique are complemented by active physiotherapy stimulating the muscles, and then a simple splint is suited to the foot to fix its degree of realignment. When used alone, this technique achieves 77% good and fair results. In resistant cases, complementary surgery was used. We obtained 96% good and fair results.     

Computational modeling to predict effect of treatment schedule on drug delivery to prostate in humans.

PURPOSE: To evaluate a computational approach that incorporates experimental data in preclinical models to depict doxorubicin human tissue pharmacokinetics. EXPERIMENTAL DESIGN: Beagle dogs were given 2 mg/kg doxorubicin as i.v. bolus, 4-h infusion, or 96-h infusion. Concentrations in plasma, prostate (target tissue), heart (toxicity), and major tissues for disposition were determined and modeled. Model parameters were obtained after the bolus injection with model validation based on the 4-h and 96-h infusion data. Clinical pharmacokinetic data and scale-up gave doxorubicin profiles in human prostate and heart. RESULTS: In agreement with in vitro results, tissues were best modeled with two compartments, one rapidly and one slowly equilibrating. The developed tissue distribution model predicted concentrations for all three administration regimens well, with an average deviation of 34% (median, 29%). Interspecies scale-up to humans showed that the change from a bolus injection to a slow, 96-h infusion (a) had different effects on the drug partition and accumulation in heart and prostate, and (b) lowered the peak concentration in the plasma by approximately 100-fold but had relatively little effect on maximal heart concentration ( approximately 33% lower). The simulated drug exposure in a human prostate was above the exposure required to inhibit tumor proliferation but was 30 to 50 times below that needed for cell death. CONCLUSION: The present study shows a computation-based paradigm for translating in vitro and in vivo preclinical data and to estimate and compare the drug delivery and pharmacokinetics in target tissues after different treatment schedules.     

Intraprostatic chemotherapy: distribution and transport mechanisms.

PURPOSE: The present study evaluated the tissue distribution and targeting advantage of intraprostatic chemotherapy. EXPERIMENTAL DESIGN: We studied the delivery and spatial distribution of a fluorescent drug, doxorubicin, in the prostate of beagle dogs, after intraprostatic or i.v. administration. Drug concentrations were measured using high-performance liquid chromatography and confocal fluorescence microscopy. RESULTS: I.v. and intraprostatic injections yielded qualitatively and quantitatively different doxorubicin distribution in the prostate. A relatively homogeneous distribution was found after i.v. administration, whereas intraprostatic injection yielded a highly heterogeneous distribution with >10-fold higher concentrations localized in a cone-shaped glandular lobule bound by fibromuscular stroma, compared with other parts of the prostate. Compared with i.v. injection, intraprostatic injection yielded, on average, approximately 100-fold higher tissue-to-plasma concentration ratio, ranging from 963-fold near the injection site to 19-fold in the contralateral half of the prostate. The drug distribution within the prostate further suggests an important role for acinar flow in intraprostatic drug transport. CONCLUSIONS: Intraprostatic administration represents a viable option to deliver high drug concentrations within the prostate. The results further suggest the fibromuscular stroma separating the prostatic lobules as a major barrier to drug transport and convective flow as an important drug transport mechanism in the prostate.     

Biliopancreatic diversion with duodenal switch or gastric bypass for failed gastric banding: retrospective study from two institutions with preliminary results.

BACKGROUND: Of patients who have undergone gastric banding, 11-25% will require a major reoperation with band removal and conversion to another bariatric procedure after they have failed to lose sufficient weight or have developed dysphagia or reflux. The aim of this study was to evaluate the respective benefits of Roux-en-Y gastric band (RYGB) or biliopancreatic diversion with duodenal switch (BPD-DS) after failed gastric banding and whether 1 of the 2 procedures might be a better procedure for such cases. METHODS: RYGB or BPD-DS was performed according to the institutional protocols with synchronous band removal, irrespective of the reason for failure. RESULTS: Of the 53 patients, 32 underwent laparoscopic RYGB for a body mass index (BMI) of 43.1 +/- 6.4 kg/m(2) (BMI 45.8 +/- 6.4 kg/m(2) before laparoscopic adjustable gastric banding) and 21 underwent BPD-DS for a BMI of 46.0 +/- 5.5 kg/m(2) (BMI 49.6 +/- 5.2 kg/m(2) before laparoscopic adjustable gastric banding). BPD-DS required significantly longer operative times (239.7 +/- 55.8 versus 135 +/- 26.7 minutes) and resulted in more complications (62% versus 12.5%; P <.002). No patients died postoperatively. The 2 groups of patients had a similar BMI at 12 and 18 months after revision (BMI 33.4 +/- 5.6 kg/m(2) and 31.4 +/- 3.5 kg/m(2)). The weight loss was greater after BPD-DS than after RYGB compared with the prerevision weight loss (66.2% versus 58.8% excess weight loss) or initial weight (73% versus 61.8%), although this was not significant. CONCLUSION: Despite an excessive rate of complications that were, in part, related to the learning curve in this series, BPD-DS resulted in greater weight loss compared with RYGB. However, both procedures were successful after failed gastric banding. A more accurate definition of failure could help to determine the respective indications for revisional surgery.     

Mediation by B1 and B2 receptors of vasodepressor responses to intravenously administered kinins in anaesthetized dogs.

1. Vasodepressor responses to intravenous (i.v.) injection of bradykinin (BK) and des-Arg9-BK, a selective B1 kinin receptor agonist, were characterized following i.v. pretreatment with selective B1 ([Leu8]-des-Arg9-BK) and B2 (Hoe 140) kinin receptor antagonists in anaesthetized dogs. 2. Des-Arg9-BK (0.05-3.3 nmol kg-1) produced dose-dependent decreases in mean arterial blood pressure with a ED50 0.4 nmol kg-1. The vasodepressor effects evoked by des-Arg9-BK (0.6 nmol kg-1) and BK (0.2 nmol kg-1) were greater after i.v. and i.a. injections, respectively. 3. The vasodepressor response to BK (0.6 nmol kg-1) but not to des-Arg9-BK (0.6 nmol kg-1) was significantly (P < 0.001) blocked by pretreatment with the B2 receptor antagonist, Hoe 140. 4. The vasodepressor response to des-Arg9-BK (0.6 nmol kg-1) but not to BK (0.6 nmol kg-1) was significantly (P < 0.001) reduced by pretreatment with the selective B1 receptor antagonist, [Leu8]-des-Arg9-BK. Although both B1 and B2 receptor antagonists caused a transient fall in blood pressure, their inhibitory action was unlikely to be related to a desensitization mechanism. 5. Inhibition of prostaglandin synthesis with indomethacin prevented the vasodepressor response induced by arachidonic acid (1 mg kg-1, i.v.) but not that to BK or des-Arg9-BK (0.6 nmol kg-1). 6. These results suggest, firstly, that the vasodepressor responses to i.v. BK and des-Arg9-BK are mediated by the activation of B2 and B1 receptors, respectively; secondly, that prostaglandins are not involved in the vasodepressor responses to kinins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ainhum (dactylolysis spontanea): A Radiological survey of 6000 patients

Dactylolysis spontanea is an idiopathic condition affecting the fifth toe, and sometimes other toes, that is frequently bilateral, with lesions in different stages. Between 1977 and 1999, a total 6000 radiographic studies of the feet were reviewed in a mainly African American population in Chicago, Illinois. After an initial screening based on the Cole criteria, 581 patients were selected and re-examined, and amplification techniques were performed. After reviewing the complementary exams, 102 patients were diagnosed with dactylolysis spontanea or ainhum. Soft-tissue constriction was the most frequently presented radiological sign on the initial screening. Kurtosis at the digit plantar fold and marked rotation of the fifth toe were normal findings in asymptomatic patients. Demographics, comorbidities, and radiological findings were analyzed in the selected population. Associated diseases occurring in these patients appeared to have no specific etiologic correlation with ainhum. African Americans and the dark-skinned population are affected exclusively by this condition, presumably due to the fibrogenic tendency of these individuals. Early diagnosis and accurate staging of ainhum are facilitated by radiological examination of the feet. The findings suggest that this condition is underdiagnosed and overlooked because its low prevalence and variable clinical presentations that might mimic more common etiologies, including localized trauma.