Adolescent suicide assessment and management in primary care.

BACKGROUND: To understand how suicide management occurs within the primary care setting in terms of follow-up assessments and referral practices. METHODS: At an initial primary care visit, adolescents (aged 12-20 years old) completed electronic screening. Data were focused on youth who endorsed a suicidal risk item while completing screening at two Midwestern primary care clinics. Data were collected through retrospective chart reviews to analyze actions taken by the primary care physician at the youth's initial visit and follow-up visit within the next 12 months. RESULTS: At initial visits 200 adolescents endorsed a suicidal risk item and 39 (19.5%) were considered to be concerning by their primary care physician. The average age was 14.7 years old (SD ± 2.0). Seventy-two percent (n = 144) were female, and 65% (n = 129) identified as Black. At initial visits, significant differences between suicidal concern groups were found in reporting active suicidal ideation, past suicide attempts, those who were referred to behavioral health counseling, and those who had a diagnosis of depression. Interestingly, only 13% (n = 25) of all patients who endorsed the suicide item were asked whether or not there were weapons in their home and primary care providers asked only 7% (n = 13) of all patients whether they had a safety plan. CONCLUSIONS: There was inconsistent follow-up for adolescents with a history of suicide concerns. At this time, national guidelines do not exist regarding primary care follow-up of youth with suicide concerns. Guidelines are a necessary precursor for practice improvement. TRIAL REGISTRATION: Clinical Trials Registry: NCT02244138 . Registration date, September 1, 2014.

Changes in PIK3CA mutation status are not associated with recurrence, metastatic disease or progression in endocrine-treated breast cancer.

The phosphatidylinositol-3-kinase pathway plays an important role in proliferation, migration and survival in breast cancer and may play a role in resistance to endocrine therapy. Pathway activation occurs as a result of mutations in PIK3CA or loss of functional PTEN. Matched primary and recurrent samples from 120 breast cancer patients treated with endocrine therapy were profiled with a qPCR-based mutation assay covering eight mutational hotspots in PIK3CA. PTEN was assayed by immunohistochemistry. Samples were well characterized with respect to anatomic location of recurrence (metastatic nodal or local recurrence as opposed to contralateral or ipsilateral new primary cancers). In total, 43 % of patients had at least one PIK3CA mutation at diagnosis, and 41 % had a mutation at the time of recurrence. Only 8 % of patients with local recurrence, metastatic disease or progression on primary endocrine treatment changed their PIK3CA mutation status (four gains, two losses, total 76). The most common changes in PIK3CA mutation status were seen in patients who developed a new cancer either in the treated or contralateral breast (64 %, three gains, four losses, total 11). PIK3CA mutation status does not change in the majority of patients with breast cancer and the acquisition of mutations in PIK3CA is not responsible for the development of endocrine resistance. PTEN loss at diagnosis is associated with a significantly shorter time to progression compared with tumours in which PTEN was retained. These are the most comprehensive data currently available correlating PIK3CA status, site of recurrence and endocrine resistance.

The challenge of establishing, growing and sustaining a large biobank: a personal perspective.

Laboratory medicine professionals have a unique understanding of the wealth that biological samples bring to clinical research, and of the need for quality standards for the collection, transportation, storage and analytical phases. The expertise of laboratory physicians and scientists also adds value to the interpretation and publication of the results of clinical research studies. This is an account of the evolution of over thirty five years of the Biobank/Clinical Research Clinical Trials Laboratory at one Canadian health sciences centre. The logistical, financial, and quality management challenges are presented in growing from a small-scale facility to one that now stores three million well-characterized samples from more than seventy countries, representing five continents and five major ethnic groups. This is an account of a journey, it is not intended as a guide as to how to create an 'ideal' biobank. Collaboration, collegiality, consistency, creativity and clinical collaborators, are the keys to progress, but there must first be a vision, one that can expand to embrace new opportunities.

A novel antidote-controlled anticoagulant reduces thrombin generation and inflammation and improves cardiac function in cardiopulmonary bypass surgery.

Heparin and protamine are the standard anticoagulant-antidote regimen used in almost every cardiopulmonary bypass (CPB) procedure even though both are associated with an array of complications and toxicities. Here we demonstrate that an anticoagulant aptamer-antidote pair targeting factor IXa can replace heparin and protamine in a porcine CPB model and also limit the adverse effects on thrombin generation, inflammation, and cardiac physiology associated with heparin and protamine use. These results demonstrate that targeting clotting factors upstream of thrombin in the coagulation cascade can potentially reduce the perioperative pathologies associated with CPB and suggest that the aptamer-antidote pair to FIXa may improve the outcome of patients undergoing CPB. In particular, this novel anticoagulant-antidote pair may prove to be useful in patients diagnosed with heparin-induced thrombocytopenia or those who have been sensitized to protamine, particularly patients who have insulin-dependent diabetes.

Survival following ventricular free wall rupture: a case series.

Ventricular rupture occurs in 10% of acute myocardial infarctions and is associated with significant mortality. We describe two cases of ventricular rupture post myocardial infarction who survived. We highlight salient points in their diagnosis and management and review the existing literature to determine what constitutes optimal management in these difficult cases.

The adrenergic pathway and heart failure.

Heart failure represents the endpoint to many triggering cardiovascular pathologies. However, there are molecular and biochemical features that remain common to the failing heart, despite the varying etiologies. Principal among these is heightened activation of the sympathetic nervous system and associated enhancement of adrenergic signaling pathways via the catecholamines, norepinephrine and epinephrine. During heart failure, several hallmark alterations in the adrenergic system contribute to loss of cardiac function. To specifically study these changes in a physiologically relevant setting, we and others have utilized advances in genetically engineered mouse technology. This chapter will discuss the many transgenic and knockout mouse models that have been developed to study the adrenergic system in the normal and failing heart. These models include genetically manipulated alterations of adrenergic receptors, linked heterotrimeric G proteins, and the regulatory G protein-coupled receptor kinases (GRKs). Among the more-interesting information gained from these models is the finding that inhibition of a particular GRK - GRK2 or beta adrenergic receptor kinase 1 (betaARK1) - is a potential novel therapeutic strategy to improve function in the setting of heart failure. Furthermore, we will discuss recent transgenic research that proposes an important role for hypertension in the development of heart failure. Overall, genetically engineered mouse models pertaining to this critical myocardial signaling system have provided novel insight into heart function under normal conditions and during states of dysfunction and failure.

Developing a spatial framework of common ecological regions for the conterminous United States.

In 1996, nine federal agencies with mandates to inventory and manage the nation's land, water, and biological resources signed a memorandum of understanding entitled "Developing a Spatial Framework of Ecological Units of The United States." This spatial framework is the basis for interagency coordination and collaboration in the development of ecosystem management strategies. One of the objectives in this memorandum is the development of a map of common ecological regions for the conterminous United States. The regions defined in the spatial framework will be areas within which biotic, abiotic, terrestrial, and aquatic capacities and potentials are similar. The agencies agreed to begin by exploring areas of agreement and disagreement in three federal natural-resource spatial frameworks--Major Land Resource Areas of the US Department of Agriculture (USDA) Natural Resources Conservation Service, National Hierarchy of Ecological Units of the USDA Forest Service, and Level III Ecoregions of the US Environmental Protection Agency. The explicit intention is that the framework will foster an ecological understanding of the landscape, rather than an understanding based on a single resource, single discipline, or single agency perspective. This paper describes the origin, capabilities, and limitations of three major federal agency frameworks and suggests why a common ecological framework is desirable. The scientific and programmatic benefits of common ecological regions are described, and a proposed process for development of the common framework is presented.

Regulation of myocardial betaARK1 expression in catecholamine-induced cardiac hypertrophy in transgenic mice overexpressing alpha1B-adrenergic receptors.

OBJECTIVES: Using a transgenic mouse model of myocardial-targeted overexpression of the wild-type alpha1B adrenergic receptor (AR) (Tg alpha43), we studied the role of the betaAR kinase (betaARK1) in the evolution of myocardial hypertrophy and its transition to heart failure (HF). BACKGROUND: Increased myocardial expression of betaARK1 has been shown to be associated with HF and certain models of hypertrophy. METHODS: Tg alpha43 mice and their nontransgenic littermate controls were treated with the alpha1AR agonist phenylephrine (PE) for 3, 7 or 14 days to characterize the cardiac consequences. RESULTS: Nontransgenic littermate control mice treated for 14 days with PE display cardiac hypertrophy with no increase in betaARK1 expression. However, Tg alpha43 animals show a reduced tolerance to 14-day PE treatment, demonstrated by reduced survival and severe cardiac hypertrophy. Moreover, PE treatment for three and seven days in Tg alpha43 mice resulted in an exaggerated hypertrophic response accompanied by significant cardiac biochemical abnormalities that are normally associated with HF, including fetal gene expression, reduced betaAR density and enhanced betaARK1 expression. We also found reduced myocardial stores of the sympathetic neurotransmitter neuropeptide Y. CONCLUSIONS: These data suggest that PE-treated Tg alpha43 mice have chronic activation of the cardiac sympathetic nervous system, which may be responsible for the appearance of apparent maladaptive hypertrophy with an evolution towards HF and sudden death. Thus, the cardiac phenotypes found in these mice are not the direct result of enhanced alpha1B AR signaling and suggest that betaARK1 is a key molecule in the transition of myocardial hypertrophy to HF.

Myocardial collagen changes and edema in rats with hyperdynamic sepsis.

OBJECTIVE: To determine if sepsis, which is accompanied by both systolic and diastolic myocardial dysfunction, involves changes in myocardial collagen, as myocardial collagen changes can affect both myocardial compliance and contractility. DESIGN: Prospective, randomized, controlled study. SETTING: Animal laboratory at a university-affiliated hospital. SUBJECTS: Male Sprague-Dawley rats, weighing 310 to 396 g. INTERVENTIONS: Cecal ligation and perforation (to induce sepsis) for 24 (n = 9) or 48 hrs (n = 9); sham laparotomy for 24 (n = 10) or 48 hrs (n = 9) with saline fluid resuscitation or normal control (n = 5) groups. MEASUREMENTS AND MAIN RESULTS: Collagen content and interstitial space were determined, using polarized light microscopy and a computer video densitometry system. At 24 and 48 hrs post surgery, heart rate and cardiac index were increased, and systemic vascular resistance index was decreased significantly in the sepsis vs. the sham rats. Collagen content was decreased significantly in the sepsis vs. the sham groups both at 24 and 48 hrs following surgery (1.83 +/- 0.79 [SD] % [24 hrs], 1.76 +/- 0.31% [48 hrs] vs. 2.83 +/- 0.73% [24 hrs], 2.25 +/- 0.72% [48 hrs]; p < .01). Interstitial space was increased significantly in the sepsis vs. the sham groups (13.9 +/- 3.5% [24 hrs], 15.6 +/- 5.2% [48 hrs] vs. 8.6 +/- 4.2% [24 hrs], 9.9 +/- 4.8% [48 hrs]; p < .01). CONCLUSIONS: Sepsis is accompanied by changes in myocardial collagen content and myocardial edema. These changes may contribute to the systolic and diastolic myocardial dysfunction, and particularly to the ventricular dilation, observed in sepsis.

The use of flow cytometry and fluorescein-labeled antibodies to measure specific milk proteins in bovine mammary epithelial cells.

A flow cytometric technique was developed to measure the relative concentration of whey protein and beta-casein in individual fixed and permeabilized bovine mammary epithelial cells. Primary bovine mammary epithelial cells were compared to mammary cells isolated from explants after a 24-h incubation and a bovine mammary epithelial transfected cell line (MAC-T). Cells were incubated with rabbit anti-bovine whey protein (alpha-lactalbumin + beta-lactoglobulin) or beta-casein primary antibodies followed by a fluorescein-labeled goat anti-rabbit IgG second antibody. The number and intensity of fluorescing cells were measured using an EPICS Profile Flow Cytometer. Primary and explant cells contained 3.3 and 2.8 times more whey protein than MAC-T cells. Explant epithelial cells contained 2.9 and 5.1 times more beta-casein than primary or MAC-T cells. The higher concentrations of specific proteins within the cells was attributed to either greater synthesis or reduced secretion. These data show that flow cytometry is capable of detecting differences in milk protein concentration in different mammary epithelial cell types.

Bovine mammary explant versus primary cell cultures: effect of bovine somatotropin and insulin-like growth factor-I on DNA content and protein synthesis.

Cellular DNA, milk protein content, and protein secretion by bovine mammary explants were compared to cultures of confluent and growing primary bovine mammary secretory cells over 4 d. Explants were obtained at slaughter from eight Holstein cows (120 +/- 35 d lactation). Primary cells were grown to confluence, cryopreserved, thawed, and cultured through five passages. Explants and cells were cocultured with liver and adipose tissue in the presence of somatotropin, insulin-like growth factor-I, and somatotropin + insulin-like growth factor-I. Cellular DNA and milk proteins were assayed using fluorescent probes and flow cytometry. Media proteins were assayed by densitometer scanning of electrophoresis gel bands. DNA content of explant, confluent, and growing primary cells increased similarly through the 96 h incubation. DNA content in G0G1 phase was increased by: (a) insulin-like growth factor-I in explant cells; (b) somatotropin, insulin-like growth factor-I, and their combination in confluent primary cells; and (c) the combination of somatotropin and insulin-like growth factor in growing primary cells. Approximately 65% of explant and confluent primary cells were in the G0G1 or differentiated phase compared to 47% for the growing primary cells. Whey protein content and secretion were similar among cell types. Explant cells contained and secreted more beta-casein than primary cells but secretion trends for beta-casein and k-casein were similar after 48 h for both cell types. Results suggest that primary cell cultures are comparable to explant cultures when used to study mechanisms of DNA and milk protein synthesis and secretion.

Effect of somatotropin and insulin-like growth factor-I on milk lipid and protein synthesis in vitro.

The objective of this research was to test the hypothesis that bST stimulates milk secretion through the action of IGF-I. Cocultures of bovine mammary, adipose, and liver tissues were incubated with increasing concentrations (0 to 1000 ng/ml) of bST, IGF-I, prolactin, or bST plus prolactin. In addition, cocultures of mammary and adipose tissues without liver tissue were incubated with IGF-I. The synthesis of milk lipids and proteins and the concentration of cellular DNA were measured. The addition of liver tissue depressed DNA concentration and the synthesis of lipids and proteins in mammary tissue. In mammary tissue, increasing bST concentration resulted in greater synthesis of FFA, total lipids, and proteins than that produced by increasing concentrations of IGF-I, and the effect on protein synthesis was linear. Conversely, in adipose tissue, increasing the concentration of IGF-I in the presence of liver resulted in greater synthesis of FFA than that obtained by bST, and the effect was linear. The results do not support the hypothesis that bST acts through the actions of IGF-I on mammary cells. Rather, bST alters the delivery of nutrients to the mammary cells from other tissues and affects milk component synthesis through a mechanism that may not involve IGF-I.

Human olfactory neuroepithelial cells: tyrosine phosphorylation and process extension are increased by the combination of IL-1beta, IL-6, NGF, and bFGF.

Olfactory neuroepithelial cells (ONC) grown from biopsies of human donors are a novel cell culture system that may facilitate studies into normal and disease-related human neurobiology. We further characterized the expression of cell surface markers and intermediate filaments, and responses to neurotrophic factors by ONC. ONC are positive for cell surface markers N-CAM, PSA-N-CAM, neutral endopeptidase, N-aminopeptidase, NGF low-affinity receptor homologue (CD40), and transferrin receptor by flow cytometry for the intermediate filament proteins peripherin, vimentin, and NF-H by immunocytochemistry. Responses to neurotrophic factors measured were process outgrowth, cytoskeletal protein expression, and protein phosphorylation. Process outgrowth was increased by interleukin-beta 164-171 (IL-1beta) or by the combination of IL-1beta, interleukin-6 (IL-6), nerve growth factor (NGF), and basic fibroblast growth factor (bFGF). This combination of IL-1beta, IL-6, NGF, and bFGF (16NF) increased expression of two cytoskeletal proteins, NF-H protein and microtubule-associated protein tau. Application of the individual neurotrophic factors IL-1beta, IL-6, NGF, and bFGF increased protein phosphorylation, while 16NF produced an immediate increase in tyrosine phosphorylation of several proteins (MW of 40-80, 120, 150, and 190 kDa). The 16NF combination appears to act through a tyrosine-kinase-mediated pathway to induce process extension and increase NF-H expression. The ONC culture has the potential to be further explored to examine the relationship among process outgrowth, protein phosphorylation, and synergy between neurotrophin and cytokine receptor systems.

Differential regulation of APP secretion by apolipoprotein E3 and E4.

The apolipoprotein E isozyme, apolipoprotein E4, has been implicated as a risk factor for Alzheimer's disease. One reason for the increased risk may be that apolipoprotein E binds to the A beta peptide, but there may be other factors as well. We show that apolipoprotein E is a potent regulator of the secretion of amyloid precursor protein. In cultures of PC12 cells, nanomolar levels of apolipoprotein E3 induce a rapid decrease in the secretion of APP, being observable in 30 min. and stable over 24 hours. Apolipoprotein E4, in contrast, increases secretion of APP over a similar time course. Reciprocal changes occur in cellular amyloid precursor protein. Differential characteristics are also seen in apo E binding to the cells, where apo E4 binds over a slower time course than apo E3. These results suggest a novel mechanism by which apolipoprotein E may be influencing the metabolism of amyloid precursor protein.

Morphology of pig uterine subepithelial capillaries after topical and systemic oestrogen treatment.

The ultrastructure of subepithelial capillaries in pig endometrium was studied after intrauterine and systemic oestradiol treatment. Gilts were killed on day 13 or 19 (n = 4 per day per treatment) after surgical introduction of Silastic beads containing either oestradiol or cholesterol into the uterine lumina on day 10 after oestrus. An additional group of gilts was injected i.m. with 5 mg oestradiol valerate on days 11-15 of the oestrous cycle and killed on day 13 or day 19 (n = 4 per day). Light and electron microscope studies of endometrial samples revealed that cholesterol beads did not appear to affect subepithelial capillary structure. Both intrauterine and systemic treatment of gilts with oestradiol were associated with regional modification of the capillary wall involving attenuation and fenestration of the face directly underlying the uterine epithelium, accumulation of caveolae in the opposite face of the capillary, and the occurrence of discontinuous, multilayered capillary basal laminae. The similarity of these structural changes to those observed in capillaries at the time of embryonic attachment suggests that oestrogens of blastocyst origin may function to modify capillary morphology and possibly facilitate increased transcapillary traffic during the establishment of pregnancy.

Ornithine-delta-aminotransferase in lactating bovine mammary glands.

The occurrence and subcellular distribution of ornithine-delta-aminotransferase have been studied in lactating bovine mammary glands. The enzyme is localized in the mitochondria and has a unique thermal reaction profile that distinguishes it from putative liver and kidney isozymes. The enzyme concentration in the gland correlates well with a role in the conversion of ornithine into the proline precursor, L-delta 1-pyrroline-5-carboxylate. However, an unusually high Michaelis constant for the mitochondrial enzyme (8.4 mM) raises the question of enzyme efficiency in vivo such that this pathway needs to be considered in estimating barriers to protein secretion into milk.

Protein alterations in olfactory neuroblasts from Alzheimer donors.

Definitive diagnosis of Alzheimer's disease (AD) is made by pathologic examination of postmortem brain tissue in conjunction with a clinical history of dementia. To date, there are no good biological markers for a positive diagnosis of AD in the living patient. In an effort to identify biological markers useful both in the clinical and pathologic diagnosis of AD, we have investigated disease-specific protein alterations in cultured olfactory neurons. Olfactory neurons are readily accessible by biopsy, can be propagated in primary cell culture as olfactory neuroblasts (ONs), and exhibit several elements of AD brain pathophysiology making them powerful tools for the study of AD. Two-dimensional gel analysis of ON proteins from neuropsychologically evaluated AD donors revealed a set of five proteins (Mr 17-50 kD, pI 4.8-6.7) that were significantly altered in concentration when compared to cells from age-matched controls. Further characterization and microsequence analysis could lead to the identification of proteins that may have important diagnostic or therapeutic value in the treatment of AD.

Compared with crystalloid, colloid therapy slows progression of extrapulmonary tissue injury in septic sheep.

We tested the hypothesis that the type of fluid infused to chronically maintain intravascular volumes would modify both microvascular integrity and cellular structure in extrapulmonary organs in hyperdynamic sepsis. After cecal ligation and perforation, awake sheep were treated for 48 h with 10% pentastarch (n = 9), 10% pentafraction (Du Pont Critical Care; n = 8), or Ringer lactate (n = 8) titrated to maintain a constant left atrial pressure. After 48 h of fluid therapy, biopsy samples were taken from the left ventricle and gastrocnemius for electron microscopy. At this time, all groups demonstrated a similar hyperdynamic circulatory response, increased systemic O2 utilization and organ blood flows, measured by radioactive microsphere injection. However, greater capillary luminal areas with less endothelial swelling and less parenchymal injury were found in septic sheep treated with pentastarch vs. Ringer lactate infusion in both muscle types. Pentafraction showed few benefits in study end points over pentastarch. Thus, we conclude that chronic intravascular volume resuscitation of hyperdynamic sepsis with pentastarch ameliorated the progression of both microvascular and parenchymal injury. These findings indicate that microvascular surface area for tissue O2 exchange in sepsis may be better preserved with chronically infused colloid, resulting in less parenchymal injury.