Cervical spine evaluation and clearance in the intoxicated patient: A prospective Western Trauma Association Multi-Institutional Trial and Survey.

BACKGROUND: Intoxication often prevents clinical clearance of the cervical spine (Csp) after trauma leading to prolonged immobilization even with a normal computed tomography (CT) scan. We evaluated the accuracy of CT at detecting clinically significant Csp injury, and surveyed participants on related opinions and practice. METHODS: A prospective multicenter study (2013-2015) at 17 centers. All adult blunt trauma patients underwent structured clinical examination and imaging including a Csp CT, with follow-up thru discharge. alcohol- and drug-intoxicated patients (TOX+) were identified by serum and/or urine testing. Primary outcomes included the incidence and type of Csp injuries, the accuracy of CT scan, and the impact of TOX+ on the time to Csp clearance. A 36-item survey querying local protocols, practices, and opinions in the TOX+ population was administered. RESULTS: Ten thousand one hundred ninety-one patients were prospectively enrolled and underwent CT Csp during the initial trauma evaluation. The majority were men (67%), had vehicular trauma or falls (83%), with mean age of 48 years, and mean Injury Severity Score (ISS) of 11. The overall incidence of Csp injury was 10.6%. TOX+ comprised 30% of the cohort (19% EtOH only, 6% drug only, and 5% both). TOX+ were significantly younger (41 years vs. 51 years; p < 0.01) but with similar mean Injury Severity Score (11) and Glasgow Coma Scale score (13). The TOX+ cohort had a lower incidence of Csp injury versus nonintoxicated (8.4% vs. 11.5%; p < 0.01). In the TOX+ group, CT had a sensitivity of 94%, specificity of 99.5%, and negative predictive value (NPV) of 99.5% for all Csp injuries. For clinically significant injuries, the NPV was 99.9%, and there were no unstable Csp injuries missed by CT (NPV, 100%). When CT Csp was negative, TOX+ led to longer immobilization versus sober patients (mean, 8 hours vs. 2 hours; p < 0.01), and prolonged immobilization (>12 hrs) in 25%. The survey showed marked variations in protocols, definitions, and Csp clearance practices among participating centers, although 100% indicated willingness to change practice based on these data. CONCLUSION: For intoxicated patients undergoing Csp imaging, CT scan was highly accurate and reliable for identifying clinically significant spine injuries, and had a 100% NPV for identifying unstable injuries. CT-based clearance in TOX+ patients appears safe and may avoid unnecessary prolonged immobilization. There was wide disparity in practices, definitions, and opinions among the participating centers. LEVEL OF EVIDENCE: Diagnostic tests or criteria, level II.

Awareness and knowledge of HPV and cervical cancer in female students: A survey (with a cautionary note).

We conducted a survey to explore levels of awareness and knowledge of human papillomavirus (HPV) and cervical cancer in 170 female students and whether mode of data collection (online vs. paper) affected the results. 27% of women named HPV as a cause of cervical cancer with online respondents more likely to do so. 75% of women had heard of HPV. More online respondents had heard of HPV than paper respondents. 127 women reported having heard of HPV, with a mean knowledge score of 2.989 (standard deviation [SD] 1.599). Online respondents scored higher (3.57, SD 1.316) than paper respondents (2.688, SD 1.591). Knowledge and awareness of HPV and its link to cervical cancer appear to have increased which may be related to the HPV vaccination programme. However, there is still a considerable number of women with little to no knowledge of HPV. Online surveys may result in an inflated estimation of awareness and knowledge.

Insulin activates caspase-3 by a phosphatidylinositol 3'-kinase-dependent pathway.

Activation of the caspase proteases by c-Jun N-terminal kinase 1 (JNK1) has been proposed as a mechanism of apoptotic cell death. Here we report that insulin activates caspase-3 by a pathway requiring phosphatidylinositol 3'-kinase (PI3-kinase). JNK1 assays demonstrated that insulin treatment of myeloma cells induced 3-fold activation of JNK1. Inhibition of PI3-kinase with wortmannin and LY294002 blocked insulin-dependent activation of JNK1. Caspase assays demonstrated that insulin increased caspase-3 activity 3-fold and that inhibition of PI3-kinase blocked this effect. Cell death was doubled by insulin and was due to a 3-fold increase in apoptosis of cells in the G1/G0 phase of the cell cycle. Inhibition of PI3-kinase completely blocked this effect. Finally, inhibition of caspase-3 with benzyloxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone blocked cell death due to insulin. Taken together, these findings indicate that insulin activates caspase-3 by a PI3-kinase-dependent pathway resulting in increased apoptosis and cell death.

Phosphatidylinositol 3'-kinase, but not S6-kinase, is required for insulin-like growth factor-I and IL-4 to maintain expression of Bcl-2 and promote survival of myeloid progenitors.

Phosphatidylinositol 3'-kinase (PI 3-kinase) catalyzes the formation of 3' phosphoinositides and has been implicated in an intracellular signaling pathway that inhibits apoptosis in both neuronal and hemopoietic cells. Here, we investigated two potential downstream mediators of PI 3-kinase, the serine/threonine p70 S6-kinase (S6-kinase) and the antiapoptotic protein B cell lymphoma-2 (Bcl-2). Stimulation of factor-dependent cell progenitor (FDCP) cells with either IL-4 or insulin-like growth factor (IGF)-I induced a 10-fold increase in the activity of both PI 3-kinase and S6-kinase. Rapamycin blocked 90% of the S6-kinase activity but did not affect PI 3-kinase, whereas wortmannin and LY294002 inhibited the activity of both S6-kinase and PI 3-kinase. However, wortmannin and LY294002, but not rapamycin, blocked the ability of IL-4 and IGF-I to promote cell survival. We next established that IL-3, IL-4, and IGF-I increase expression of Bcl-2 by >3-fold. Pretreatment with inhibitors of PI 3-kinase, but not rapamycin, abrogated expression of Bcl-2 caused by IL-4 and IGF-I, but not by IL-3. None of the cytokines affected expression of the proapoptotic protein Bax, suggesting that all three cytokines were specific for Bcl-2. These data establish that inhibition of PI 3-kinase, but not S6-kinase, blocks the ability of IL-4 and IGF-I to increase expression of Bcl-2 and protect promyeloid cells from apoptosis. The requirement for PI 3-kinase to maintain Bcl-2 expression depends upon the ligand that activates the cell survival pathway.

Insulin growth factor-I inhibits apoptosis in hematopoietic progenitor cells. Implications in thymic aging.

A decline in plasma concentrations of both growth hormone and IGF-I occurs during aging of humans and rodents, and this is accompanied by involution of the thymus gland. Exogenous growth hormone induces the synthesis of IGF-I, which acts on bone marrow-derived hematopoietic progenitors of the myeloid and lymphoid lineages to promote their replication and survival. The increase in survival of these cells is caused by the ability of IGF-I to inhibit their apoptotic death. In contrast to the multipotential colony-stimulating-factor IL-3, inhibition of apoptosis by IGF-I requires the activation of the critical intracellular effector PI 3-kinase. These data establish that hematopoietic progenitors can use more than one intracellular signaling pathway in order to maintain their survival. The data also extend the original hypothesis that IGF-I shares with the colony-stimulating factors the properties of promoting DNA synthesis and inhibiting programmed cell death. Collectively, these data establish that hematopoietic progenitor cells are important targets for IGF-I, and this is likely to be important in understanding thymic aging.

IL-4 and insulin-like growth factor-I inhibit the decline in Bcl-2 and promote the survival of IL-3-deprived myeloid progenitors.

The proto-oncogene product Bcl-2 regulates cell survival in both the immune and central nervous systems. We withdrew growth factors from IL-3-dependent murine myeloid progenitor cells (factor dependent cell progenitors (FDCP)) and measured a time-dependent 80% reduction in endogenous expression of Bcl-2. This decline in Bcl-2 is directly associated with a fourfold increase in the apoptotic population after 12 h and an eightfold increase after 24 h. Since IL-4 and insulin-like growth factor-I (IGF-I) regulate myeloid cell growth, we used IL-3-deprived FDCP cells to determine whether IL-4 and IGF-I maintain Bcl-2 expression and prevent apoptosis. We demonstrate that IL-4, like IGF-I and IL-3, promotes survival of FDCP cells by reducing the apoptotic population. Flow cytometric measurement of intracellular Bcl-2 established that IL-4 and IGF-I maintain 10-fold higher levels of Bcl-2 than in IL-3-deprived cells. Similarly, Western analysis of Bcl-2 in lysates of IL-3-deprived myeloid progenitors confirmed that both IL-4 and IGF-I share with IL-3 the ability to maintain intact Bcl-2 protein. However, IL-4 and IGF-I do not change expression of the apoptotic inducer, Bax, although they maintain high levels of Bcl-2 that coimmunoprecipitate with Bax. Collectively, these data demonstrate that IL-4 and IGF-I, like IL-3, inhibit apoptosis in myeloid progenitors and maintain high levels of Bcl-2/Bax heterodimers, suggesting that Bcl-2 is a critical convergence point in the signaling pathways used by IL-4 and IGF-I.

Requirement for phosphatidylinositol 3'-kinase to protect hemopoietic progenitors against apoptosis depends upon the extracellular survival factor.

Hemopoietic growth factors promote survival of progenitor cells by preventing their apoptotic death. Recently, phosphatidylinositol 3'-kinase (PI 3-kinase) has been shown to be integral in the pathway by which insulin and nerve growth factor prevent apoptosis. In this work, we show that IL-3-dependent FDCP-1/Mac-1 murine hemopoietic progenitors express receptors for another growth factor, insulin-like growth factor-I (IGF-I), and that both IL-3 and IGF-I stimulate PI 3-kinase activity. We then demonstrate that IGF-I shares with IL-3 the properties of significantly promoting proliferation and enhancing survival of myeloid progenitor cells at concentrations as low as 3 ng/ml. IL-3 and IGF-I efficiently promote cell survival in the presence of inhibitors of either RNA synthesis (actinomycin D) or mitosis (mitomycin C), suggesting that both ligands promote survival by a process that is largely independent of RNA synthesis. To determine whether PI 3-kinase mediates IL-3- and IGF-I-induced inhibition of apoptosis, FDCP-1/Mac-1 cells were incubated with the PI 3-kinase inhibitor, wortmannin. While wortmannin inhibited both basal and IGF-I- and IL-3-induced PI 3-kinase enzyme activity, it did not affect the ability of IL-3 to protect FDCP-1/Mac-1 cells from apoptosis, even though it abrogated the IGF-I-induced inhibition of apoptosis. These data demonstrate that even though activation of PI 3-kinase is a pleiotropic feature of both IL-3 and IGF-I receptors in myeloid progenitors, prevention of apoptosis by IL-3 but not IGF-I is independent of PI 3-kinase. Survival of hemopoietic progenitors is therefore maintained by at least two different intracellular signaling pathways, one requiring PI 3-kinase and one that does not.

Growth hormone, growth factors and hematopoiesis.

Hypocellularity of primary lymphoid organs is a distinctive and reproducible characteristic of aged humans and animals. Similar changes have been reported in both hypophysectomized and dwarf rodents. In the bone marrow of these animals, there is an associated reduction in the number of erythroid, lymphoid and myeloid elements. Implantation of growth hormone (GH)-secreting GH3 pituitary cells or infusion of growth hormone into aged rodents dramatically improves cellularity of both the thymus gland and bone marrow. At present it is unknown whether these effects are due to direct effects of growth hormone on hematopoietic cells or if they are caused by the induction of insulin-like growth factor-1 (IGF-1) synthesis. We recently discovered that colony-stimulating factor-1 (CSF-1) and interleukin-3 (IL-3) induce expression and synthesis of the IGF-1 peptide in murine bone marrow cells. Transcripts for IGF-1 increase approximately 50-fold during differentiation over the negligible levels that are expressed in freshly isolated bone marrow cells. Two potential functions of macrophage-derived IGF-1 are to: (a) increase the proliferation of early or committed bone marrow progenitors and (b) reduce their rate of cell death. In support of the first possibility, IGF binding protein-3 significantly inhibits the proliferation of CSF-1-treated bone marrow cells and this inhibition can be reversed by addition of exogenous IGF-1. In support of the second possibility, we have induced apoptosis of both nonadherent bone marrow cells and a myeloid progenitor cell line by depriving these cells of CSFs. Preliminary results indicate that addition of IGF-1 to these cells reduces apoptotic cell death by 50%. These data establish that two different CSFs, CSF-1 and IL-3, induce abundant expression of IGF-1 as these cells differentiate into more mature hematopoietic cells. This model offers a novel approach for investigating the developmental expression of IGF-1 during defined differentiation pathways of hematopoietic cells. If IGF-1 is indeed proven to act as a survival factor for hematopoietic progenitors, these data would support the idea that the hypocellularity of primary lymphoid tissues in aged animals is related to the limited availability to these cells of either growth hormone or IGF-1.

The colony-stimulating factors induce expression of insulin-like growth factor-I messenger ribonucleic acid during hematopoiesis.

Murine bone marrow cells cultured in the presence of colony-stimulating factor-1 (CSF-1) showed coordinate induction of insulin-like growth factor-I (IGF-I) messenger RNA (mRNA) during the differentiation process, and these transcripts increased approximately 50- to 75-fold over the virtually negligible levels measured in freshly isolated bone marrow. In contrast, transcripts for the IGF-I receptor were evident in freshly isolated rat bone marrow cells and showed a 50% down-regulation during differentiation. Addition of a variety of single lineage and multilineage CSFs, including CSF-1, interleukin-3, granulocyte-macrophage-CSF, and granulocyte-CSF to mouse bone marrow cultures revealed that induction of IGF-I mRNA is a universal feature of differentiation with these CSFs, although IGF-I transcripts are at least 10- to 20-fold higher in CSF-1- and interleukin-3-differentiated lineages than in other cultures. The IGF-I induced by CSF-1 was biologically active because a natural ligand of IGF-I, IGF-binding protein-3, caused significant down-regulation of cellular proliferation, and this could be reversed by the addition of exogenous IGF-I. In addition, whereas IGF-I mRNA could be detected in resident peritoneal macrophages, these transcripts were increased 6-fold after a local injection of thioglycollate, a stimulus that induces macrophage proliferation and differentiation in vivo. These results show that CSFs induce expression of the growth factor IGF-I during differentiation of hematopoietic cells into multiple myeloid lineages and that this endogenously produced IGF-I is also a growth factor for hematopoietic cells. The induction of IGF-I mRNA during hematopoiesis should provide a new approach to understanding the expression of this gene during development and differentiation.

Competitive reverse transcriptase-polymerase chain reaction using a synthetic internal RNA standard to quantitate transcripts for leukocyte-derived hormones.

Leukocytes synthesize a variety of hormones that were once thought to be unique products of endocrine tissues. Understanding the regulation of leukocyte-derived hormone synthesis requires an accurate means for measuring steady-state expression of specific mRNA transcripts. Here we describe a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) technique to accurately quantitate macrophage-derived insulin-like growth factor-I (IGF-I) mRNA, and demonstrate the utility of this approach for measuring expression of leukocyte-derived hormone transcripts. A riboprobe was constructed to generate approximately 1 kb of synthetic competitor IGF-I RNA (exons 1 and 3-6) that differed from cellular IGF-I RNA by insertion of 122 bp of beta-actin RNA. One set of oligonucleotide primers could thus be used to simultaneously reverse transcribe and amplify both 144 bp of cellular (exons 3 and 4) and 266 bp of competitor IGF-I RNA. Densitometric scanning of the PAGE-separated PCR products revealed that the ratio of competitor to cellular amplified DNA bore a linear relationship (r2 > or = 0.98) to the amount of competitor RNA for both rat liver and splenocytes. However, rat liver contained 104 x 10(6) IGF-I molecules per microgram of total cellular RNA compared to only 2 x 10(6) IGF-I molecules for splenocytes.(ABSTRACT TRUNCATED AT 250 WORDS)