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.

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.

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.

Somatotrophin increases thyroxine-5'-monodeiodinase activity in lactating mammary tissue of the cow.

The effect of administration of bovine somatotrophin (bST) on peripheral conversion of thyroxine (T4) to tri-iodothyronine (T3) was studied in non-pregnant lactating Holstein cows. Six cows were injected daily for 5 days with 40 mg recombinantly derived bST, while six control cows received excipient alone. Blood samples were collected hourly from 08.00 to 19.00 h on a single day the week before treatment and on days 4-5 of treatment. All other tissue samples were obtained at slaughter, 20-23 h after the last injection. Administration of bST increased milk production and caused a 9% increase in hepatic DNA. Consumption of feed did not differ between control and bST-treated cows. Treatment did not alter serum concentrations of T4 or T3, although concentrations of thyroid hormones in the serum increased from 08.00 to 19.00 h. Activity of thyroxine-5'-monodeiodinase (5'-D) in liver and kidney was similarly unaffected. However, activity of 5'-D in mammary tissue increased approximately twofold in response to bST administration. We suggest that an increase in mammary conversion of T4 to the more biologically potent thyroid hormone T3 plays a role in mediating the galactopoietic response of dairy cattle to bST.

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.

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.

Mucosal induction of systemic T cell tolerance by Fusobacterium nucleatum.

The present study was undertaken to determine if mucosal presentation of the periodontopathic bacterium Fusobacterium nucleatum could induce systemic tolerance. Two separate protocols of mucosal priming were carried out. In the first, mice were gastrically intubated on 2 consecutive days; this was repeated 5 days later. In the second protocol, mice were similarly primed but received another priming dose after a further 7 days. Positive control mice were similarly primed with sheep red blood cells (SRBC) while negative control animals were sham-primed with saline. Following mucosal priming, mice were systemically sensitized with the respective antigen and then subsequently challenged in the left hind footpad. The right footpad was challenged with saline and served as a negative control. Serum antibody levels were measured using enzyme-linked immunoabsorbent assay (ELISA) and haemagglutination assays. Mucosal priming with F. nucleatum was found to suppress the local delayed type hypersensitivity reaction as determined by footpad measurements. Sham-priming did not suppress the local response. On the other hand, the levels of serum antibodies were not influenced by mucosal priming. These results suggest that under the experimental conditions used, mucosal presentation of F. nucleatum can induce a degree of split tolerance in which T cell responses are suppressed while B cell responses remain intact. The implication of this finding to human periodontal disease is yet to be determined.

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.

In vitro DNA synthesis as indicator of mammary epithelial cell division: [14C]thymidine uptake versus flow cytometry cell cycle analysis.

Mammary and adipose explants from eight mid-lactation Holstein cows were co-cultured for 24 h in the presence or absence of liver explants, 1 microgram/ml pituitary bovine somatotrophin, or 100 ng/ml insulinlike growth factor-I. Liver explants in the media significantly depressed DNA and protein synthesis by mammary tissue as measured by [14C]-thymidine and amino acid incorporation. As measured by flow cytometry, the concentration of DNA in the G0G1 and G2M cells and the percentage of cells in the G0G1 population of mammary tissue was also significantly depressed by liver tissue. Changes in the percentage of cells in the S and G2M phases were not significant. Insulinlike growth factor-I in the presence of liver explants depressed protein synthesis, thymidine incorporation, and the concentration of DNA in the G0G1 and G2M cells compared to control but did not affect the percentage of cells in the G0G1, S, or G2M phases. Previously it was assumed that changes in [14C]thymidine incorporation indicated that changes in cell division were occurring. Flow cytometry revealed that changes in DNA content of mammary cells as a result of liver or hormonal stimulation were not due to changes in cell division. Indications are that differences in cellular DNA content result from changes in the rate of amplification of individual genes responsible for milk protein synthesis.

Comparative study of mammary gland development and differentiation between beef and dairy heifers.

Sixteen Hereford and 16 Holstein heifers were used to study the relationship of milk production potential to mammary development and differentiation. Heifers were slaughtered at 150, 180, and 260 days of first gestation and at 49 days of first lactation. Prolactin binding capacity of mammary tissue was 2.5 fold higher in dairy than beef heifers at day 260 of gestation (27.2 vs 11.0 fmols/mg protein). In both breeds, maximal growth hormone binding in liver coincided with the beginning of the rapid phase of mammary growth at 180 days. Mammary tissue from dairy heifers released more casein and alpha-lactalbumin during in vitro incubations than tissue from beef heifers. No differences were observed between breeds with respect to incorporation of [14C]acetate into lipids. Mass of dairy mammary tissue at 49 days of lactation was 3.3 times greater (16.4 vs 4.9 kg) and produced 5.7 times more milk (20.3 vs 3.5 kg/day) than its beef counterpart. The total DNA content and the RNA/DNA ratio of lactating dairy mammary tissue was approximately twice that of lactating beef mammary tissue. The data suggested that the higher milk production observed in dairy cattle is a result of a greater number of secretory cells and greater activity per cell.

Teat stimulation-induced release of prolactin and basal concentrations of prolactin and growth hormone in pregnant dairy and beef heifers.

The physiological bases for differences in milk production between breeds of cattle selected for beef or milk production are largely unknown. This study was conducted to determine concentrations of prolactin (PRL) and growth hormone (GH) in serum before and after teat stimulation in primiparous Hereford and Holstein heifers. Blood was collected from 6 beef and 9 dairy heifers at 115, 175, 230 and 250 d of gestation. Sampling times were -15, -10, -5, 0, 2, 4, 6, 8, 10, 12, 15, 20, 25 and 30 min relative to test stimulation. Mean areas under PRL response curves for beef and dairy heifers at 115, 175, 230 and 250 d of gestation were 427, 447, 556, 273 and 243, 189, 167, 343 ng/ml/30 min, respectively. Calculations of area (but not basal levels) excluded instances when no PRL response to test stimulation was obtained (22%). Neither stage of gestation nor breed affected PRL response. Basal PRL did not differ between breeds and was 1.8, 2.6, 2.4 and 9.2 ng/ml at 115, 175, 230 and 250 d of gestation. GH did not differ between breeds and was 6.6, 6.2, 5.5 and 7.4 ng/ml at 115, 175, 230 and 250 d. No difference between breeds was apparent with regard to PRL or GH secretion during first gestation.

Effect of bovine growth hormone on incorporation of [14C]acetate into lipids by co-cultures of bovine mammary, liver, and adipose tissue explants.

Incorporation of [14C]acetate into lipids was measured in 24 hr co-cultures of mammary, liver and adipose tissue from Holstein cows at 53, 210 and 318 d of lactation in the presence or absence of bovine growth hormone. Little (less than 1%) of the labeled lipids appeared in the media relative to that incorporated into the tissue. In mammary tissue, incorporation of [14C]acetate was highest into triglycerides (16,298 cpm/mg mammary tissue), followed by phospholipids (1,887 cpm), free fatty acids (1,252 cpm), diglycerides (708 cpm), free cholesterol (360 cpm) and monoglycerides (93 cpm). Bovine growth hormone did not increase incorporation of [14C]acetate when mammary or adipose tissue were incubated separately. However, in the presence of liver and adipose tissue, bovine growth hormone significantly increased the incorporation of [14C]acetate into triglycerides, diglycerides, free fatty acids and free cholesterol by mammary tissue. These results suggest that bovine growth hormone acts on mammary tissue indirectly through liver and adipose tissue to increase lipid synthesis. This mechanism may play a role in the action of bovine growth hormone in vivo to increase milk and milk fat production.

Bovine mammary teat and ductal epithelial cell cultures.

Bovine mammary epithelial cells from teat and ductal tissue were isolated at necropsy and were grown in culture. Cells were characterized by the presence of cytokeratin filaments, cell morphologic features, synthesis of milk proteins, esterase activity, DNA content, and growth patterns on polystyrene, fibronectin, laminin, collagen, and reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma. Cultured teat and ductal cells stained intensely for cytokeratin and had similar morphologic features. Both cell types synthesized alpha-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin, and lactoferrin to variable degrees. Cell type and culture conditions did not affect the DNA content of the cells, as indicated by similar amounts of DNA in G0G1 and G2M phases of the mitotic cycle in cultured cells and in cells from freshly isolated mammary explants. Cells cultured on polystyrene, fibronectin, laminin, and collagen formed pavement-like cell monolayers suitable for cytotoxicity and bacterial adherence studies. Cells cultured on the reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma formed three-dimensional structures closely resembling lactiferous ducts and alveoli, which could be used for studying lactogenesis and galactopoiesis. Freshly isolated cells and cultured cells were stored at -70 C or in liquid nitrogen. The latter storage method affected the cells less than did freezing at -70 C.

The Japanese management theory jungle.

Many competing hypotheses have been advanced to account for the apparent effectiveness of Japanese management practices. The present review of some of the leading theories attempts to classify and clarify the state of knowledge of Japanese management. Although each theory may be correct as a partial explanation of Japan's success, no single conceptualization has captured the complexity of Japan's managerial achievement. Further development of integrated, internally consistent models is needed.

General practitioners' use of non-steroidal anti-inflammatory drugs in Tayside and Fife regions.

The objectives of this study were to assess the prescribing of non-steroidal anti-inflammatory drugs (NSAIDs) by general practitioners and to determine their attitudes to problems caused by this class of drugs. The study consisted of two parts. The first was a questionnaire survey among general practitioners in Fife and Tayside, and the second was an analysis of NSAID prescribing over 12 months among the doctors in the Carnoustie Health Centre, using duplicate prescriptions. In the questionnaire survey 61% of the general practitioners responded. The three most preferred drugs were buprofen (56%), naproxen (20%) and mefenamic acid (7%); choice of drug was determined by efficacy and personal experience. Gastrointestinal side effects were most frequently encountered, although there was little consensus amongst respondents as to their management. The duplicate prescription study showed that 14% of patients (1607 individuals) received at least one NSAID prescription in the year of study. Ibuprofen (31%), naproxen (20%) and piroxicam (15%) were most frequently prescribed and up to 16% of the patients were co-prescribed a gastroprotective agent; ranitidine (75%) was the most commonly prescribed. Despite the introduction of newer NSAIDs, ibuprofen and naproxen are still the most commonly prescribed drugs. Furthermore, although gastrointestinal side effects are commonly encountered, there is some uncertainty about their management.

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.