Neuronal M3 muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth

The molecular pathways that promote the proliferation and maintenance of pituitary somatotrophs and other cell types of the anterior pituitary gland are not well understood at present. However, such knowledge is likely to lead to the development of novel drugs useful for the treatment of various human growth disorders. Although muscarinic cholinergic pathways have been implicated in regulating somatotroph function, the physiological relevance of this effect and the localization and nature of the receptor subtypes involved in this activity remain unclear. We report the surprising observation that mutant mice that selectively lack the M₃ muscarinic acetylcholine receptor subtype in the brain (neurons and glial cells; Br-M3-KO mice) showed a dwarf phenotype associated with a pronounced hypoplasia of the anterior pituitary gland and a marked decrease in pituitary and serum growth hormone (GH) and prolactin. Remarkably, treatment of Br-M3-KO mice with CJC-1295, a synthetic GH-releasing hormone (GHRH) analog, rescued the growth deficit displayed by Br-M3-KO mice by restoring normal pituitary size and normal serum GH and IGF-1 levels. These findings, together with results from M₃ receptor/GHRH colocalization studies and hypothalamic hormone measurements, support a model in which central (hypothalamic) M₃ receptors are required for the proper function of hypothalamic GHRH neurons. Our data reveal an unexpected and critical role for central M₃ receptors in regulating longitudinal growth by promoting the proliferation of pituitary somatotroph cells.     

Dose of aspirin in the treatment and prevention of cardiovascular disease: current and future directions

In meta-analyses of randomized trials of aspirin among patients with prior occlusive vascular disease events (secondary prevention), doses from 75 mg to more than 1500 mg daily provide similar benefits on myocardial infarction, stroke, and cardiovascular death. In acute myocardial infarction and during acute occlusive stroke, a loading dose of 162.5 to 325 mg is necessary to achieve a rapid clinical antithrombotic effect. In primary prevention trials, predominantly among men, aspirin (75 mg daily to 325 mg on alternate days) reduced the risk of a first myocardial infarction. In a large-scale trial in women, aspirin (100 mg on alternate days) reduced risk of a first stroke. In subgroup analyses of women older than age 65, aspirin significantly reduced first myocardial infarction and ischemic stroke. Direct comparisons of higher doses may yield additional cardiovascular benefits. At present, daily doses of 75 to 325 mg aspirin are sufficient for long-term treatment and prevention of cardiovascular disease.     

Pilot study of p62 DNA vaccine in dogs with mammary tumors

Our previous data demonstrated profound anti-tumor and anti-metastatic effects of p62 (sqstm1) DNA vaccine in rodents with various types of transplantable tumors. Testing anti-cancer medicine in dogs as an intermediary step of translational research program provides two major benefits. First, clinical data collected in target animals is required for FDA/USDA approval as a veterinary anti-cancer drug or vaccine. It is noteworthy that the veterinary community is in need of novel medicine for the prevention and treatment of canine and feline cancers. The second more important benefit of testing anti-cancer vaccines in dogs is that spontaneous tumors in dogs may provide invaluable information for human trials. Here, we evaluated the effect(s) of p62 DNA vaccine on mammary tumors of dogs. We found that p62 DNA vaccine administered i.m. decreased or stabilized growth of locally advanced lesions in absence of its overall toxic effects. The observed antitumor activity was associated with lymphocyte infiltration and tumor encapsulation via fibrotic reaction. This data justifies both human clinical trials and veterinary application of p62 DNA vaccine.     

Obesity and Mental Illness: Implications for Cognitive Functioning

A priority research and clinical agenda is to identify determinants of cognitive impairment in individuals with neuropsychiatric disorders (NPD). The bidirectional association between NPD and cognitive performance has been reported to be mediated and/or moderated by obesity in a subset of individuals. Obesity can be conceptualized as a neurotoxic phenotype among individuals with NPD as evidenced by alterations in the structure and function of neural circuits and disseminated networks, diminished cognitive performance, and adverse effects on illness trajectory. The neurotoxic effect of obesity provides a rationale for screening, treating, and preventing obesity in neuropsychiatric populations. Research endeavors that aim to refine mediators and moderators of this association as well as novel strategies to reverse the injurious process of obesity on cognition are warranted.     

Reflection of the effectiveness of heterosensory integration in measures of event-linked potentials

The phenomenon of bimodal event-linked potential mismatch and summation of unimodal responses were used to study the relationship between mismatch amplitude and the effectiveness of visual-auditory integration. The effectiveness of integration was assessed in terms of the extent to which experimental measures approached the parameters of the "ideal observer." The greater the effectiveness of integration, the greater the focus of mismatch shifted from the occipital to the frontal areas of the cortex. The greatest correlation between mismatch amplitude and the effectiveness of visual-auditory integration was seen in the left frontal cortex.     

Stable long-term blood formation by stem cells in murine steady-state hematopoiesis

Hematopoietic stem cells (HSCs) generate all mature blood cells during the whole lifespan of an individual. However, the clonal contribution of individual HSC and progenitor cells in steady-state hematopoiesis is poorly understood. To investigate the activity of HSCs under steady-state conditions, murine HSC and progenitor cells were genetically marked in vivo by integrating lentiviral vectors (LVs) encoding green fluorescent protein (GFP). Hematopoietic contribution of individual marked clones was monitored by determination of lentiviral integration sites using highly sensitive linear amplification-mediated-polymerase chain reaction. A remarkably stable small proportion of hematopoietic cells expressed GFP in LV-injected animals for up to 24 months, indicating stable marking of murine steady-state hematopoiesis. Analysis of the lentiviral integration sites revealed that multiple hematopoietic clones with both myeloid and lymphoid differentiation potential contributed to long-term hematopoiesis. In contrast to intrafemoral vector injection, intravenous administration of LV preferentially targeted short-lived progenitor cells. Myelosuppressive treatment of mice prior to LV-injection did not affect the marking efficiency. Our study represents the first continuous analysis of clonal behavior of genetically marked hematopoietic cells in an unmanipulated system, providing evidence that multiple clones are simultaneously active in murine steady-state hematopoiesis. Stem Cells2012;30:1961-1970.     

High-level recombinant protein expression in transgenic plants by using a double-inducible viral vector

We describe here a unique ethanol-inducible process for expression of recombinant proteins in transgenic plants. The process is based on inducible release of viral RNA replicons from stably integrated DNA proreplicons. A simple treatment with ethanol releases the replicon leading to RNA amplification and high-level protein production. To achieve tight control of replicon activation and spread in the uninduced state, the viral vector has been deconstructed, and its two components, the replicon and the cell-to-cell movement protein, have each been placed separately under the control of an inducible promoter. Transgenic Nicotiana benthamiana plants incorporating this double-inducible system demonstrate negligible background expression, high (over 0.5 × 10⁴-fold) induction multiples, and high absolute levels of protein expression upon induction (up to 4.3 mg/g fresh biomass). The process can be easily scaled up, supports expression of practically important recombinant proteins, and thus can be directly used for industrial manufacturing.     

A history of early stent thrombosis is associated with prolonged clot lysis time

It has been demonstrated that formation of compact plasma fibrin clots resistant to plasmin-mediated lysis characterises patients following in-stent thrombosis (IST). The relationship between defective fibrinolysis, reflected as prolonged clot lysis time (CLT) and IST is unclear. We sought to investigate whether patients with acute and subacute IST have impaired fibrinolytic capacity. We studied 41 definite IST patients, including 15 with acute and 26 with subacute IST experienced 2-73 months prior to enrollment, versus 41 controls matched for demographics, cardiovascular risk factors, concomitant treatment and angiographic/stent parameters. CLT, reflecting lysis of a tissue factor-induced plasma clot by exogenous tissue plasminogen activator, together with plasminogen activator inhibitor-1 (PAI-1) antigen and activity, thrombin-activatable fibrinolysis inhibitor (TAFI) antigen and activity, thrombomodulin (TM), plasminogen and α2-antiplasmin (α2AP) were measured. There were no inter-group differences in angiographic parameters, indication to the first PCI, culprit vessel or a type of stent. Patients with IST had 11% longer CLT (p=0.005) and 13% higher PAI-1 antigen (p=0.04) compared to controls. There were positive correlations in both groups between CLT and PAI-1 antigen and TAFI activity (all p<0.001). Multiple regression analysis showed that CLT (odds ratio [OR]=1.04 per 1 minute, 95% CI 1.01-1.08, p=0.02) and platelet count (OR=1.01 per 1,000/μl, 95% CI 1.00-1.02, p=0.034) were independent predictors of IST (R(2)=0.28, p<0.05). Concluding, impaired fibrinolytic potential, that is in part determined by plasma PAI-1 antigen and TAFI activity, characterises patients with a history of acute and subacute IST, which might help identify patients at higher risk of IST.