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Supernova (SN) 2018oh (ASASSN-18bt) is the first spectroscopically-confirmed type Ia supernova (SN Ia) observed in the $Kepler$ field. The $Kepler$ data revealed an excess emission in its early light curve, allowing to place interesting constraints on its progenitor system (Dimitriadis et al. 2018, Shappee et al. 2018b). Here, we present extensive optical, ultraviolet, and near-infrared photometry, as well as dense sampling of optical spectra, for this object. SN 2018oh is relatively normal in its photometric evolution, with a rise time of 18.3$\pm$0.3 days and $\Delta$m$_{15}(B)=0.96\pm$0.03 mag, but it seems to have bluer $B - V$ colors. We construct the "uvoir" bolometric light curve having peak luminosity as 1.49$\times$10$^{43}$erg s$^{-1}$, from which we derive a nickel mass as 0.55$\pm$0.04M$_{\odot}$ by fitting radiation diffusion models powered by centrally located $^{56}$Ni. Note that the moment when nickel-powered luminosity starts to emerge is +3.85 days after the first light in the Kepler data, suggesting other origins of the early-time emission, e.g., mixing of $^{56}$Ni to outer layers of the ejecta or interaction between the ejecta and nearby circumstellar material or a non-degenerate companion star. The spectral evolution of SN 2018oh is similar to that of a normal SN Ia, but is characterized by prominent and persistent carbon absorption features. The C II features can be detected from the early phases to about 3 weeks after the maximum light, representing the latest detection of carbon ever recorded in a SN Ia. This indicates that a considerable amount of unburned carbon exists in the ejecta of SN 2018oh and may mix into deeper layers., Comment: 48 pages, 23 figures. This paper is part of a coordinated effort between groups. Accepted for publication in ApJ

Summary Reasons for performing study Antimicrobial stewardship within the veterinary profession is recognised by governing and professional bodies as being important; the attitudes and behaviour of veterinary surgeons merit investigation. Objectives To investigate levels of protected antimicrobial use and accuracy of antimicrobial dosing in a common clinical scenario in equine practice. Study design Retrospective cohort study. Methods Antimicrobial use was evaluated retrospectively in 113 cases subsequently referred to a single referral hospital for the treatment of limb wounds over a 20-month period. Antimicrobial classification (first-line, alternative or protected) was made according to guidelines produced by the British Equine Veterinary Association. These guidelines also served as the reference for recommended dose rates. Results Systemic antimicrobials were administered prior to referral in 94/113 (83.2%) horses, of which 8 (8.5%) received the protected third or fourth generation cephalosporins or fluoroquinolones. Forty-eight of 87 (55.2%) horses for which complete dosing data were available received antimicrobials at ≤90% of the recommended dose. Practitioners who held a postgraduate clinical qualification or worked in purely equine practice were no more or less likely to use protected antimicrobials (P = 0.06 and P = 0.64, respectively) or administer inadequate doses (P = 0.75 and P = 0.85, respectively). Veterinary surgeons with more experience were less likely to use protected antimicrobials (P

Rolipram is a type IV phosphodiesterase inhibitor that suppresses inflammation and TNF-alpha production. As anti-TNF-alpha therapy is effective in rheumatoid arthritis, we investigated the effect of rolipram on collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis. Rolipram was administered after the onset of clinical arthritis at doses of 0.5, 3, 5, or 10 mg/kg twice daily, with a dose-dependent therapeutic effect on clinical severity and joint erosion. Immunohistochemical analysis of joints of rolipram-treated mice revealed 67% reduction in TNF-alpha-expressing cells compared with control arthritic mice. In vitro studies using bone marrow-derived macrophages confirmed that rolipram directly suppressed TNF-alpha and IL-12 production following stimulation with IFN-gamma and LPS. The effect of rolipram on T cell activity was studied by measuring Th1/Th2 cytokine production by collagen-stimulated draining lymph node cells from arthritic mice treated in vivo with rolipram. Rolipram reduced IFN-gamma production and increased IL-10, indicating that rolipram down-regulated the ongoing Th1 response to type II collagen. Finally, the effect on CIA of combination therapy was studied using rolipram plus either anti-TNF-alpha or anti-CD4 mAbs. Rolipram plus anti-TNF-alpha was not therapeutically additive, whereas rolipram plus anti-CD4 mAb was clearly additive. This result indicates that the therapeutic effects of rolipram overlap with TNF-alpha blockade, but are complementary to anti-CD4 treatment. It is therefore proposed that a major mechanism of action of rolipram in CIA is suppression of TNF-alpha activity. These findings suggest that type IV phosphodiesterase inhibitors may be effective in pathologic conditions, such as RA, with overexpression of TNF-alpha.

We and others have shown that overexpression of SLP-76 augments TCR-stimulated IL-2 promoter activity in the Jurkat T cell line. In this report we investigate the signaling mechanisms through which SLP-76 mediates its effect on T cell activation. We show that overexpressed SLP-76 acts downstream of TCR-stimulated protein tyrosine kinases, but does not affect calcium signaling. Overexpression of SLP-76 does, however, augment TCR stimulation of both ERK (extracellular signal-regulated kinase) activity and a reporter construct driven by activating protein-1 binding sites. Structure/function analysis reveals that three distinct regions of SLP-76, each important for protein associations, are required for augmentation of TCR-induced nuclear factor-AT activity. These data suggest that SLP-76 functions as an adapter molecule that requires three unique domains to link proximal TCR signals in T cells.

Les maladies inflammatoires ont generalement une etiologie complexe et mettent en jeu une multitude de mediateurs; il semblerait donc approprie de les considerer en utilisant une approche polytherapeutique. Dans certaines conditions inflammatoires chroniques, nous croyons que la bradykinine (BK) et l'elastase des neutrophiles humains (ENH) interviennent de maniere cooperative. Nous avons deja synthetise des composes dotes d'une activite inhibitrice pour le recepteur B 2 de la BK et l'ENH. La presente etude decrit des composes simples, concus pour incorporer l'activite inhibitrice de l'ENH et l'activite antagoniste des recepteurs B 1 et B 2 de la BK. Un inhibiteur brevete (IENH, CP-955) a ete directement fixe via la formation d'une liaison amide a un antagoniste peptidique combine B 1 /B 2 de la BK (B-9430). Trois composes ont ete obtenus en utilisant divers sites de fixation dans le peptide antagoniste. Pour ces trois composes, la fixation a des recepteurs B 2 et B 1 clones d'humains a ete au moins 10 fois plus faible que la fixation a B-9430, tandis que dans le dosage fonctionnel in vitro du recepteur B 2 de l'ileon de cobaye, leur puissance a ete equivalente a celle de B-9430. Le compose I a induit une augmentation d'un facteur 4 de l'activite IENH comparativement a CP-955, alors que les composes Il et III ont ete inactifs. Ces resultats demontrent clairement qu'il est possible de retenir une activite ENH et une activite antagoniste des recepteurs B 1 /B 2 de la BK dans un heterodimere.

Previous studies have shown that the protein tyrosine phosphatase CD45 is required for initiation of signal transduction through several lymphoid receptors. In contrast, there is increasing evidence that another protein tyrosine phosphatase, hemopoietic cell phosphatase (known as HCP, SHP, PTP1C, SHPTP-1, or PTPN6), is a negative regulator of signaling in hemopoietic cells. To determine the effect of HCP on signal transduction through the TCR, HCP was expressed as a chimeric molecule with extracellular and transmembrane domains of the HLA-A2 molecule (A2/HCP) on wild-type Jurkat T cells and the CD45-deficient variant, J45.01. In this report, we show that expression of A2/HCP, unlike A2 chimeras containing the enzymatic regions of CD45, fails to rescue TCR-mediated signal transduction in J45.01. Furthermore, expression of A2/HCP on wild-type Jurkat T cells results in diminished inositol phosphate production following TCR ligation as well as markedly diminished nuclear factor of activated T cells promoter activity. Surprisingly, however, TCR-mediated tyrosine phosphorylation of phospholipase C gamma 1 remains intact in the Jurkat cells expressing the A2/HCP chimera. These experiments provide further evidence that HCP can serve a negative regulatory role in receptor-mediated signaling in immune cells. Additionally, our studies suggest that surface expression of HCP in T cells may provide a means to identify phosphotyrosine-containing proteins that are required for coupling signaling pathways initiated by ligation of the T cell Ag receptor.

SLP-76 (SH2 domain leukocyte protein of 76 kDa) is a recently identified substrate of the TCR-stimulated protein tyrosine kinases that functions in the signal transduction cascade linking the TCR with IL-2 gene expression. In this report, we demonstrate that engagement of the TCR results in tyrosine phosphorylation of SLP-76 in its amino-terminal acidic region. Two tyrosines (Y113 and Y128) fall within an identical five amino-acid motif and are shown to be phosphorylated upon TCR ligation. Although mutation of either Y113 and Y128 has a minimal effect on SLP-76 function, mutation of both residues decreases significantly the ability of SLP-76 to promote T cell activation. A third tyrosine within the amino-terminal region (Y145) appears to be the most important for optimal SLP-76 function, as altering it alone to phenylalanine has a potent impact on SLP-76 augmentation of NFAT promoter activity.

Ligation of the T-cell antigen receptor (TCR) results in the rapid activation of several protein tyrosine kinases, with the subsequent phosphorylation of numerous cellular proteins. We investigated the requirement for tyrosine phosphorylation of proteins which bind the Grb2 SH2 domain in TCR-mediated signal transduction by transfecting the Jurkat T-cell line with a cDNA encoding a chimeric protein designed to dephosphorylate these molecules. Stimulation of the TCR on cells expressing this engineered enzyme fails to result in sustained tyrosine phosphorylation of a 36-kDa protein likely to be the recently cloned pp36/Lnk. Interestingly, TCR ligation of the transfected cells also fails to induce soluble inositol phosphate production and intracellular calcium mobilization, although receptor-mediated tyrosine phosphorylation of phospholipase C gamma 1 still occurs. TCR-mediated Ras and mitogen-activated protein kinase activation remain intact in cells expressing the engineered phosphatase. These data demonstrate that tyrosine phosphorylation of a protein(s) which binds the SH2 domain of Grb2 correlates with phospholipase C gamma 1 activation and suggest that such a phosphoprotein(s) plays a critical role in coupling the TCR with the phosphatidylinositol second-messenger pathway.

Numerous recent studies have implicated the src homology 2 and 3 domain-containing protein, Grb2, in coupling protein tyrosine kinase signaling pathways with the Ras signaling pathway. Ligation of the T cell antigen receptor results in the activation of both a PTK, and Ras; therefore, we investigated whether Grb2 may serve a similar function in T cells. Here we report that a GST/Grb2 fusion protein associates with several tyrosine phosphoproteins from lysates of T cell antigen receptor-stimulated Jurkat T cells. Two of these proteins, pp36 and pp116, bind to the Grb2 fusion protein with high affinity. Through the use of mutated Grb2 fusion proteins, we demonstrate that pp116 binds the amino-terminal src homology 3 domain of Grb2, the same domain of Grb2 thought to be primarily responsible for its interaction with SOS. We demonstrate further that pp116 associates with Grb2 in vivo, and we provide evidence that in the Jurkat T cell line Grb2 may exist complexed with either pp116 or with SOS.

The earliest biochemical event known to be associated with T cell receptor (TCR) engagement is the activation of a protein tyrosine kinase thought to be a member of the src family. Expression of CD45, the major receptor tyrosine phosphatase present on T cells, is required for efficient coupling between the TCR and its signaling machinery. One model to explain the role of CD45 in regulating TCR signaling is that the phosphatase dephosphorylates the regulatory C-terminal tyrosine of lck. In the present report we confirm the finding of a trimolecular complex containing CD45, lck, and a 34-kDa protein (p34) in the Jurkat T cell line. Additionally, we extend this work with the observation that specific in vitro kinase activity associated with CD45 requires the expression of lck in Jurkat-derived clones as does the in vivo phosphorylation of p34. The association between CD45 and lck is shown not to require the expression of or activation of the TCR. Finally, we demonstrate that even in the absence of lck p34 associates with CD45, implying a direct association between this molecule and the phosphatase. These data suggest strongly that lck is the relevant protein tyrosine kinase found in CD45 immunoprecipitates in the Jurkat T cell line and that there is an additional association between CD45 and p34 which does not require the presence of the protein tyrosine kinase.

Studies have shown that severe daytime restriction of dietary protein improves the efficacy of L-dopa and reduces response fluctuations in some Parkinson's disease (PD) patients. This study investigated the nutritional adequacy of the daytime restricted-protein diet. Eleven free-living PD patients suffering from unpredictable response fluctuations to L-dopa were counseled to limit protein intake to approximately 10 g before 1700. Three sets of 6-d food records obtained during the 8-wk study showed that while on the test diet, mean intakes of most nutrients remained above the recommended nutrient intakes, although significant decreases occurred in protein, calcium, iron, phosphorus, riboflavin, and niacin intakes. The impact of the test diet on nutritional status as evaluated by changes in body weight and serum prealbumin was small. We conclude that healthy and highly motivated patients can maintain adequate intakes of most nutrients while restricting daytime protein intake. However, nutrient intakes might be compromised in patients whose regular diets are marginally adequate.

Murine respiratory mycoplasmosis resulting from Mycoplasma pulmonis infection in rats provides a useful model for the study of immunological and inflammatory mechanisms operative in the respiratory tract. We have previously shown that LEW rats develop more severe disease than do F344 rats. To further study the production of antibody responses in chronic respiratory disease due to M. pulmonis infection, we examined the distribution and development of M. pulmonis-specific antibody-forming cells (AFC) in different segments of the respiratory tracts of infected LEW and F344 rats. In these studies, the upper respiratory nodes were the initial site of antibody production after infection and remained the major site for recovery of AFC. Since infected LEW rats had equal or higher numbers of AFC than did infected F344 rats, these results suggest that the level of local antibody production alone is not responsible for the decreased susceptibility of F344 rats to murine respiratory mycoplasmosis. The differences in total antibody responses appear to be due to the greater numbers of cells recovered from the tissues of infected LEW rats compared with those recovered from F344 rats, suggesting that LEW rats may have greater production of chemotactic factors. Also, we demonstrate that nonspecific activation and/or recruitment of B cells occurs in the respiratory tracts of both LEW and F344 rats after infection with M. pulmonis.

Purification of the gastrin-releasing peptide (GRP) or bombesin receptor has proved elusive in part due to technical difficulties. In the present studies, the problem of oxidized radioligand was avoided by the use of 125I-GRP, which was verified to be not oxidized by high performance liquid chromatography. Specific 125I-GRP binding (at 0 degrees C) to intact human small cell lung carcinoma NCI-H345 cells which had been subjected to a dilute acid wash was 6 fmol/10(6) cells. Inhibition of GRP degradation by human H345 cell membranes through the use of phenanthroline or phosphoramidon permitted the development of binding assays for the GRP receptor in detergent-solubilized crude membrane preparations. The solubilized GRP receptor exhibited saturable, high affinity (KD = 1.3 nM), temperature-dependent specific binding averaging 402 +/- 65 fmol/mg protein (mean +/- S.E. for eight separate membrane preparations with 125I-GRP concentration = 3 nM), with a Bmax = 434 fmol/mg protein using a gel filtration binding assay. That the GRP receptor had been solubilized was demonstrated by its failure to pellet when centrifuged at 100,000 x g for 60 min, its passage through a 0.22-micron filter without loss of binding activity, and its elution in the void volume of a Sephadex G-50 gel filtration column, but within the inclusion volume of a Sephacryl S-200 column (Ve/V0 = 1.1). Isolation of the GRP receptor from human H345 cell-solubilized membranes was achieved by ligand affinity chromatography. A unique 70-kDa band on silver-stained reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis was reproducibly eluted from GRP14-27 affinity columns by an acidic high salt buffer, but binding activity was denatured by these conditions. The protein nature of the GRP receptor was demonstrated by its sensitivity to proteases after isolation. In addition, two unique bands of 65 and 70 kDa were eluted from the GRP14-27 affinity column with GRP14-27 in neutral buffer, and this eluate possessed specific 125I-GRP binding with a stoichiometry of approximately 1:1. Thus, reported here is the isolation of a functional membrane-associated, saturable, high affinity GRP receptor with temperature-dependent binding from the solubilized membranes of human H345 cells.

Cohorts of European corn borer, Ostrinia nubilalis (Hubner), from eggs infested on whorl-stage corn ( Zea mays L.), were intensively sampled through fourth instar in 1986 and through fifth instar in 1987. In both years, central plants in plots (4 by 2 m, 55 plants) were infested with European corn borer egg masses to simulate a first-generation infestation. In 1986, one egg mass was placed in each plot, whereas in 1987, plots received one, two, or four egg masses. In both years, >50% of the recovered larvae were on the infested plant. Mean dispersal distance increased linearly with time in both years. However, maximum dispersal distance was independent of time in both years. In 1987, egg mass density had a significant linear effect on maximum dispersal distance but no effect on mean dispersal distance. The radius encompassing 90% of the recovered larvae included the infested plant and two plants on either side within the same row. Mortality occurred mainly within the first 48 h: 75.7% in 1986 and 82.9% in 1987. A logistic regression showed that egg and larval survival were independent of egg mass density. The number of tunnels produced per larva found at 8 d after infestation (considered equivalent to scouting) was 0.592. Knowledge of European corn borer dispersal and survival could be used to improve sampling plans and the calculation of economic thresholds.

Surface expression of the CD45 tyrosine phosphatase is essential for the T cell antigen receptor (TCR) to couple optimally with its second messenger pathways. CD45 may be required to dephosphorylate a TCR-activated protein tyrosine kinase, which then transduces an activation signal from the TCR. A chimeric molecule that contained extracellular and transmembrane sequences from an allele of a major histocompatibility class I molecule and cytoplasmic sequences of CD45 restored TCR signaling in a CD45-deficient mutant T cell line. Thus, expression of the complex extracellular domain of CD45 is not required for the TCR to couple to its signaling machinery.

The advent of cylindric titanium implants and the prevalence of periodontal disease have necessitated a restoration that facilitates the performance of oral physiotherapy. The use of the conical crown as a metallic covering over natural teeth and implants--with a suprastructure that is retained through a specific degree of convergence without cement or screws--allows access for enhanced oral hygiene practices, while providing for esthetic and functional goals.

We set out to determine whether the increases in intracranial pressure (ICP) associated with CO2 insufflation had any metabolic effect on the central nervous system in a head injury when compared with gasless laparoscopy (GL). To test this hypothesis, we looked at both the ICP and jugular bulb venous saturation (JVS), with and without a coexisting cerebral mass lesion. Twenty-five kilogram male pigs had tracheostomy, epidural balloon, pulmonary arterial catheter, arterial line, and jugular bulb catheter placed. Intravenous Pentobarbital was used for anesthesia. Either CO2 laparoscopy (CL; n=7) or GL (n=7) were performed both with and without an epidural balloon inflated to a baseline ICP of 25. Data were analyzed using the Student's t test with a P value0.05 being significant. Cerebral perfusion pressure and most hemodynamic values did not differ. Both central venous pressure and peak inspiratory pressure were significantly elevated whenever CO2 insufflation took place, reflecting an increased intrathoracic pressure. When comparing both study groups, the partial pressure of CO2 did not differ. CL increases ICP significantly above the gasless group in our head injury model. This is most likely secondary to increased intrathoracic pressure. The question still remains whether these changes are clinically significant. We could not demonstrate significant metabolic effects secondary to laparoscopy. In patients suffering head injury, GL rather than CL might be safer to avoid ICP elevation. Additional studies looking at central nervous system metabolic and objective histopathologic effects should be undertaken with larger numbers of study animals.

The leukocyte-specific adapter protein SLP-76 is known to augment the transcriptional activity of nuclear factor of activated T cells and AP-1 following TCR ligation. A role for SLP-76 in additional receptor-mediated signaling events is less clear. To define the pattern of SLP-76 expression during murine hemopoiesis, we stained cells isolated from various tissues with a combination of surface markers followed by intracellular staining with a fluorochrome-labeled SLP-76-specific Ab. In the bone marrow, SLP-76 expression is largely restricted to cells of granulocyte and monocyte lineage. Heterogeneous SLP-76 expression is first detected in the CD44+ CD25- subset within the CD3- CD4- CD8- thymocyte population. Interestingly, SLP-76 expression increases as thymocyte maturation progresses within the CD4- CD8- compartment but decreases as cells mature to a CD4+ CD8+ phenotype. SLP-76 expression is then up-regulated following selection and concomitant with maturation to a CD4+ or CD8+ phenotype. In the periphery, SLP-76 is expressed in T lymphocytes with no detectable expression in the B cell compartment. Exposure to the superantigen staphylococcal enterotoxin B augments SLP-76 expression in the reactive T cell subset. Furthermore, in vitro stimulation with TCR-specific Abs augments the existing levels of SLP-76. These data reveal that SLP-76 expression is coordinately regulated with surface expression of a pre-TCR or mature TCR complex during thymocyte development and that TCR ligation elicits signals that result in increased expression of SLP-76.

Treatment of 3T3-L1 adipocytes with insulin (IC50 approximately 200 pM insulin) or insulin-like growth factor-1 (IC50 approximately 200 pM IGF-1) stimulates dephosphorylation of CCAAT/enhancer binding protein alpha (C/EBPalpha), a transcription factor involved in preadipocyte differentiation. As assessed by immunoblot analysis of one- and two-dimensional PAGE, insulin appears to dephosphorylate one site within p30C/EBPalpha and an additional site within p42C/EBPalpha. Consistent with insulin causing dephosphorylation of C/EBPalpha through activation of phosphatidylinositol 3-kinase, addition of phosphatidylinositol 3-kinase inhibitors (e.g. wortmannin) blocks insulin-stimulated dephosphorylation of C/EBPalpha. In the absence of insulin, wortmannin or LY294002 enhance C/EBPalpha phosphorylation. Similarly, blocking the activity of FKBP-rapamycin-associated protein with rapamycin increases phosphorylation of C/EBPalpha in the absence of insulin. Dephosphorylation of C/EBPalpha by insulin is partially blocked by rapamycin, consistent with a model in which activation of FKBP-rapamycin-associated protein by phosphatidylinositol 3-kinase results in dephosphorylation of C/EBPalpha. The dephosphorylation of C/EBPalpha by insulin, in conjunction with the insulin-dependent decline in C/EBPalpha mRNA and protein, has been hypothesized to play a role in repression of GLUT4 transcription by insulin. Consistent with this hypothesis, the decline of GLUT4 mRNA following exposure of adipocytes to insulin correlates with dephosphorylation of C/EBPalpha. However, the repression of C/EBPalpha mRNA and protein levels by insulin is blocked with an inhibitor of the mitogen-activated protein kinase pathway without blocking the repression of GLUT4 mRNA, thus dissociating the regulation of C/EBPalpha and GLUT4 mRNAs by insulin. A decline in C/EBPalpha mRNA and protein may not be required to suppress GLUT4 transcription because insulin also induces expression of the dominant-negative form of C/EBPbeta (liver inhibitory protein), which blocks transactivation by C/EBP transcription factors.

Stimulation of the T cell antigen receptor (TCR) activates signaling pathways involving protein kinases, phospholipase Cgamma1, and Ras. How these second messengers interact to initiate distal activation events is an area of intense scrutiny. In this report, we confirm that TCR ligation results in phosphorylation of Sos, a guanine nucleotide exchange factor for Ras. This requires expression of both the CD45 tyrosine phosphatase and the Lck protein tyrosine kinase and depends upon signaling via protein kinase C. In contrast to previous studies examining requirements for Sos phosphorylation following insulin and epidermal growth factor receptor engagement, we show that TCR-induced phosphorylation of Sos does not require activation of the mitogen-activated protein kinase/extracellular-signal regulated kinase (MEK/ERK) pathway. However, the basal phosphorylation of Sos in T cells is affected by either MEK or MEK-dependent kinases. Although Sos phosphorylation results in its dissociation from Grb2 following insulin stimulation in Chinese hamster ovary cells, TCR engagement on the Jurkat T cell line fails to elicit a similar effect. These data demonstrate that the kinases responsible for Sos phosphorylation differ following ligation of various cell surface receptors and that the consequences of Sos phosphorylation relies, at least in part, on sites of its phosphorylation.

Inflammatory disorders typically have a complex etiology and involve a multitude of inflammatory mediators, and hence, a polytherapeutic approach to these diseases would seem appropriate. In certain chronic inflammatory conditions, we believe that bradykinin (BK) and human neutrophil elastase (HNE) are cooperatively involved. We have previously synthesized compounds with inhibitory activity toward both the BK B2 receptor and HNE. The present study describes single compounds designed to incorporate HNE inhibitory activity and BK B1 and B2 antagonist activity. A proprietary HNE inhibitor (HNEI, CP-955) was directly linked via amide bond formation to a peptide-based combined BK B1/B2 antagonist (B-9430). Three compounds were made using different linking positions in the antagonist peptide. For all compounds, B1 and B2 receptor binding in human cloned receptors was at least 10-fold less than that of B-9430, whereas in the in vitro guinea pig ileum B2 receptor functional assay, the compounds had potencies equivalent to B-9430. Compound I was found to have a fourfold increase in HNEI activity compared with CP-955, whereas compounds II and III were inactive. These data clearly demonstrate that it is possible to retain BK B1/B2 receptor antagonist and HNE activity in a heterodimer.

Recently we described the molecular cloning of SLP-76, a hematopoietic cell-specific 76-kD protein that was first identified through its association with GST/Grb2 fusion proteins. The primary sequence of SLP-76 predicts a protein of 533 amino acids comprising an amino-terminal region with numerous potential tyrosine phosphorylation sites, a central region rich in proline residues, and a single carboxy-terminal SH2 domain. Here we demonstrate formally that Grb2 associates with unphosphorylated SLP-76 and map the Grb2 binding site on SLP-76 undergoes rapid tyrosine phosphorylation and associates with tyrosine phosphoproteins of 36, 62, and 130 kD. In vitro experiments show that the SH2 domain of SLP-76 associates with the 62- and 130-kD proteins and additionally with a serine/threonine kinase. Finally, we demonstrate that transient overexpression of SLP-76 results in dramatically enhanced TCR-mediated induction of nuclear factor of activated T cells (NFAT) and interleukin (IL) 2 promoter activity; and we provide evidence that a functional SLP-76 SH2 domain is required for this effect. Our data document the in vivo associations of SLP-76 with several proteins that potentially participate in T cell activation and implicate SLP-76 itself as an important molecule in TCR-mediated IL-2 production.

A significant number of elderly drivers present without discernible external cause for their road crash. We hypothesize that syncope may contribute to their crash etiology and prospectively evaluated drivers older than 60 years with unexplained crash, presenting to our trauma center during a 1-year period. Prehospital data and prior medical history were obtained on all, followed by a standard syncope work-up. Of 79 drivers ages 60-98, 58 (73%) were at fault in the crash. In 33 (57%), the crash etiology could not be determined. Thirteen had prior syncope history. Workup was negative in two patients, and six died or were transferred before evaluation. Twenty-five of 33 (76%) had positive workups for syncope: cardiac causes in 10; neurologic in seven; psychiatric in five, endocrine in two; one indeterminant. Ten of 18 who survived had license revocation for medical reasons. Syncopal episodes are a frequent contributor to unexplained crashes among elderly drivers and should be suspected whenever external causes are not apparent.

Activation of Ras by the exchange of bound GDP for GTP is predominantly catalyzed by the guanylnucleotide exchange factor SOS. Receptor tyrosine kinases increase Ras-GTP loading by targeting SOS to the plasma membrane location of Ras through the small adaptor protein Grb2. However, despite the continuous stimulation of receptor tyrosine kinase activity, Ras activation is transient and, in the case of insulin, begins returning to the GDP-bound state within 5 min. We report here that the cascade of serine kinases activated directly by Ras results in a mitogen-activated protein kinase kinase (MEK)-dependent phosphorylation of SOS and subsequent disassociation of the Grb2-SOS complex, thereby interrupting the ability of SOS to catalyze nucleotide exchange on Ras. These data demonstrate a molecular feedback mechanism accounting for the desensitization of Ras-GTP loading following insulin stimulation.

The ability of elderly citizens to drive safely has been the subject of ongoing debate. To identify the type of elderly driver who becomes involved in an injury-producing road crash, we profiled all drivers over 39 years of age admitted to our Level I Trauma Center over a 1-year period. Data were prospectively collected and drivers age 40-59 years were compared with those over 60 years. Eighty-four drivers ageor = 60 and 130 drivers age 40-59 were studied. Of the 84 elderly drivers, 67 were deemed at fault. Twenty-four of those crashes were due to driving errors, 12 due to syncopes, and in 20 no crash cause was determined. Fifty-three of the 67 at fault drivers had significant underlying medical problems, compared to 9 of 17 deemed not at fault. Only four were legally intoxicated. Of the 130 drivers in the comparison group, only 19 had significant underlying medical problems; in three syncope was suspected; 18 were legally intoxicated. We conclude from our data that underlying medical disorders occur frequently in elderly drivers and may contribute to their incidence of road crashes.

Numerous recent studies have implicated the src homology 2 and 3 domain-containing protein, Grb2, in coupling protein tyrosine kinase signaling pathways with the Ras signaling pathway. Ligation of the T cell antigen receptor results in the activation of both a PTK, and Ras; therefore, we investigated whether Grb2 may serve a similar function in T cells. Here we report that a GST/Grb2 fusion protein associates with several tyrosine phosphoproteins from lysates of T cell antigen receptor-stimulated Jurkat T cells. Two of these proteins, pp36 and pp116, bind to the Grb2 fusion protein with high affinity. Through the use of mutated Grb2 fusion proteins, we demonstrate that pp116 binds the amino-terminal src homology 3 domain of Grb2, the same domain of Grb2 thought to be primarily responsible for its interaction with SOS. We demonstrate further that pp116 associates with Grb2 in vivo, and we provide evidence that in the Jurkat T cell line Grb2 may exist complexed with either pp116 or with SOS.

CD45, the major transmembrane tyrosine phosphatase of lymphoid cells, is required for optimal signaling via a number of receptors. A model for how CD45 regulates signaling is that it controls phosphorylation of the COOH-terminal tyrosine of src family kinases. We have shown that CD45 physically associates with lck, one src kinase. Others have shown that CD45 also interacts with the CD4 and CD8 surface antigens expressed on many T cells. In this report we examine further the relationship between CD45 and lck in a CD4+ T cell line and in peripheral T cells. We show now that CD45 associates with lck independently of both CD4 and CD8. We show also the time course of an association between CD45 and a form of lck that migrates at an apparent higher molecular mass. Finally, we demonstrate that the interaction between CD45, lck, and a previously reported 32-34 kD protein is stable after stimulation of T cells.

Interactions between mycoplasmas and B cells consist primarily of the development of specific antibody and of nonspecific interactions with B lymphocytes or antibody. Antibody responses are important in the resistance to mycoplasmal disease in both humans and animals. However, the ability of mycoplasmas to survive in their host despite vigorous responses suggests that these play a limited role in the host's recovery from infection. Antibody also may prevent dissemination of mycoplasmal infections from mucosal sites and may account for the appearance of systemic mycoplasmal infections in immunocompromised patients. In some cases, antibody responses may contribute to disease pathogenesis through the development of hypersensitivity responses or the deposition of immune complexes. In addition, nonspecific interactions between mycoplasmas and B lymphocytes have been implicated in disease pathogenesis, possibly leading to autoimmune reactions, modulation of immunity, and/or promotion of lesion development. For example, several mycoplasmas, including Mycoplasma pneumoniae and Mycoplasma pulmonis, are able to activate B cells polyclonally in vitro and in vivo, but the mechanisms and consequences of these responses have yet to be defined. In addition to activating B lymphocytes, mycoplasmas are capable of producing chemotactic factors, Fc receptors, and immunoglobulin proteases that may also be involved in lesion development and/or survival of the organisms. Thus, both specific and nonspecific interactions of mycoplasmas with B cells can have important effects on disease progression, especially since many mycoplasmal infections are chronic and the cumulative effect of these interactions may be substantial.

The importance of delayed or secondary brain insults in the eventual outcome of closed-head trauma has been documented in experimental models. To understand this phenomenon in the clinical setting, we studied a series of head-injured patients in whom multiple cranial computed tomographic (CT) scans were obtained. Patients whose follow-up CT studies revealed new intracranial lesions or worsening, compared with admission findings, were considered to have delayed cerebral injury. One hundred forty-nine (44.5%) of 337 consecutively studied patients developed delayed brain injury. There were highly significant associations (P0.001) between the appearance of delayed cerebral insults and the severity of the initial brain injury, the need for cardiopulmonary resuscitation in the field, the presence of coagulopathy at admission, and subdural hematoma on the initial CT scan. In addition, delayed injury was associated (P0.001) with higher mortality, slowed recovery, and poorer outcome at 6 months. Delayed brain injury was not significantly associated with patient age, sex, injury mechanism, associated injury, the need for endotracheal intubation in the field, early talking, CT abnormality other than intracranial hematoma, or type of residual neurological deficits. We used multiple regression analysis to explore the relationship between severity of injury, delayed insults, and outcome. As expected, the severity of the initial brain trauma contributed significantly to neurological outcome. The presence of delayed cerebral injury makes the outcome dramatically worse for each category of initial injury severity. The relationship between initial and secondary brain injury is discussed.

We reviewed the records of 1538 mild head injury patients admitted during a 4 1/2-year period to the Southern New Jersey Regional Trauma Center. All patients had experienced brief loss of consciousness or amnesia, but had a normal or near normal neurologic examination on admission, with Glasgow Coma Scale (GCS) scores of 13-15 and no focal neurologic deficit. Routine urgent cranial CT scans were obtained on all patients, and correlations between skull fractures and intracranial lesions investigated. Two hundred sixty-five patients (17.2%) harbored 340 lesions on CT scans, of which 131 were fractures and 209 were intracranial abnormalities. Fifty-eight patients needed surgery for their intracranial lesions; 23 of them had no skull fractures. None of the 1339 patients without CT evidence of intracranial lesions deteriorated under observation. We conclude that clinical observation with or without skull x-ray films is inadequate to rule out potentially dangerous intracranial lesions in apparently mild head injuries. If there is a history of loss of consciousness or amnesia, an immediate CT scan is indicated. If the results of the CT scan are normal and there are no other indications for admission, these patients may be safely discharged.

Chronic infections play a significant role in the morbidity and mortality of patients with chronic airflow limitation. By stimulating airway inflammation, persistent infection has the potential to cause airway fibrosis. However, in patient this condition is most typically found in lungs damaged by other factors, such as smoking, abnormal secretions, or barotrauma. We report the characterization of Mycoplasma pulmonis infection-induced lung fibrosis in two immunocompetent rat strains with no preexisting lung disease. The fibrosis was predominantly in the airways, as demonstrated by the findings for infected animals of increased airway inflammation, airway fibrosis, and airway wall thickness, which correlated with the collagen content of the lungs. Also, the physiological alterations were the opposite of those found in interstitial fibrosis, with a positive correlation between lung compliance and collagen content. The airway fibrosis was noted earlier and to a greater extent in Lewis rats than in Fisher rats, and this result apparently was related to regulation of the inflammatory response. Airway wall thickness, airway inflammation, and airway fibrosis are commonly reported in tissue specimens from patients with chronic airway diseases and have been shown to correlate with airflow limitation in patients with chronic obstructive pulmonary disease. Thus, this model may be useful in furthering our understanding of the role of chronic infection and airway inflammation in airflow obstruction.

Mycoplasma pulmonis causes chronic murine respiratory mycoplasmosis, which is characterized by extensive peribronchial and perivascular infiltration of mononuclear cells, including B lymphocytes. B-lymphocyte recruitment into sites of inflammation is presently poorly understood but must involve directed chemotaxis of these cells in response to some external recruitment stimulus. In these studies, picogram amounts of M. pulmonis membrane protein were found to possess potent chemoattractant activity for resting rat B lymphocytes. This report is the first description of a bacterially derived chemoattractant for B lymphocytes and offers a unique opportunity to study regulation of B-lymphocyte recruitment to a site of chronic pulmonary inflammation. Furthermore, M. pulmonis membrane activation of fresh rat serum was found to produce a potent stimulus for recruitment of peritoneal and alveolar macrophages. M. pulmonis-mediated recruitment of lymphocytes and macrophages may play a significant role in the pathogenesis of murine respiratory mycoplasmosis, a role in which organisms on the bronchiolar epithelial surfaces may release proteins which can directly or indirectly promote chemotaxis of inflammatory cells from the circulation.

We reviewed the records of 253 patients with head injury who required serial computed tomographic (CT) scans; 123 (48.6%) developed delayed brain injury as evidenced by new or progressive lesions after a CT scan. An abnormality in the prothrombin time, partial thromboplastin time, or platelet count at admission was present in 55% of the patients who showed evidence of delayed injury, and only 9% of those whose subsequent CT scans were unchanged or improved from the time of admission (P less than 0.001). Among patients developing delayed injury, mean prothrombin time at admission was significantly longer (14.6 vs. 12.6 s, P less than 0.001) and partial thromboplastin time was significantly longer (36.9 vs. 29.2 s, P less than 0.001) than patients who did not have delayed injury. If coagulation studies at admission were normal, a patient with head injury had a 31% risk of developing delayed insults. This risk rose to almost 85% if at least one clotting test at admission was abnormal (P less than 0.001). We conclude that clotting studies at admission are of value in predicting the occurrence of delayed injury. If coagulopathy is discovered in the patient with head injury early follow-up CT scanning is advocated to discover progressive and new intracranial lesions that are likely to occur.

Central venous catheterization during resuscitation of trauma patients remains controversial. Such catheterizations performed at the UMDNJ-Robert Wood Johnson Medical School at Camden Cooper Hospital/University Medical Center (Camden, NJ) trauma center for the period January 1, 1988 to December 31, 1989 were retrospectively reviewed. Patients with underlying hemothorax, pneumothorax, or resuscitative thoracotomy were excluded. There were 269 catheters inserted using the Seldinger technique. Catheters were placed via the internal jugular or subclavian veins by attending traumatologists or emergency medicine and surgical house staff under direct attending supervision. Of 238 patients ages 6 to 90 years, 156 were men. Mean Trauma Score and Injury Severity Score were 9 and 30, respectively. Patients were divided into two groups: patients undergoing cardiopulmonary resuscitation or in shock (systolic bp less than 90) during resuscitation (Group 1: n - 161); and patients not in shock (Group 2: n - 77). Thirty patients (Group 1a) expired in the resuscitation area. The remainder (Group 1b) with 150 catheterizations had 12 complications (8%): pneumothorax (8), hemothorax (1), and malposition (3). Group 2, with 80 catheterizations, had 6 complications (7.5%): pneumothorax (4), hematoma (1), and malposition (1). The complication rate for patients surviving the resuscitation (230 catheterizations) was 7.8 per cent. There were no catheter-related deaths. In conclusion, emergency central venous catheterization during resuscitation of trauma patients carries a relatively low risk of serious complications when performed by experienced physicians.

A patient had a gunshot wound to the heart involving three cardiac chambers. Conventional echocardiography failed to identify the intracardiac injuries. The utility of transesophageal echocardiography in a patient with cardiac trauma is described.

The immediate transfusion of uncrossmatched type O blood in the initial resuscitation of the trauma victim remains controversial. To examine difficulties in crossmatching blood for later transfusions after use of uncrossmatched type O blood, we undertook a prospective 23-month study at a level I trauma center. One hundred thirty-five severely injured patients received uncrossmatched type O blood during the study period. Sixty-one patients (45%) died and 76 patients (56%) underwent emergent operation. There were no major transfusion reactions. Six patients had blood antigen-antibodies present on admission, and such antibodies developed in three patients during hospitalization. We conclude that uncrossmatched type O blood may be used safely in the exsanguinating patient, but blood antigen-antibodies, which may complicate later crossmatching, can develop after its use.

Outcome after traumatic brain injury, defined by the Glasgow Outcome Scale (GOS) and length of stay in acute rehabilitation, was measured in 59 patients admitted to an intensive rehabilitation program to examine the effects of severity of the initial brain injury, severity of multiple trauma, and length of stay in the acute care hospital. Severity of initial brain injury, best measured by length of coma, was the most significant predictor of GOS outcome. Length of acute hospitalization was a small, but significant, predictor of GOS. Severity of initial brain injury, length of acute hospitalization, and gender emerged as predictors of length of rehabilitation hospital stay. Although length of acute hospitalization is apparently affected by severity of brain injury, it adds significantly--more than severity of brain injury--to the prediction of length of rehabilitation.

To determine the value of serum amylase sampling as an indicator of intra-abdominal injury, the records of 940 consecutive victims of blunt trauma were retrospectively reviewed. The sensitivity, specificity, and predictive value were poor in the determination of intra-abdominal injury, whether accompanied by craniofacial injury or not. It was concluded that routine serum amylase determination is of no value in the clinical management of the patient suffering blunt injury.

Over a three month period, attempts to perform serial Pneumatic Cuff Venous Occlusion Plethysmography (VP) were carried out in 51 severely injured patients felt to be at risk for Pulmonary Embolus (PE). Studies could not be performed in 25 of these patients due to the nature of their injuries. Of the remaining 26 patients, two showed evidence of deep vein thrombosis (DVT). Venography confirmed the diagnosis in only one of these patients. Serial VP failed to predict PE in all five patients in whom it was diagnosed. This article concludes that serial VP is not an effective predictor of PE in these patients.

Of 27 patients admitted to our level I trauma center with acute disruption of the thoracic aorta, two patients died of exsanguination before aortic repair. One patient had massive leakage from the aneurysm after aortography and died during surgery. All patients suffered from multiple injuries. Eighty-three percent of the patients had major operations in addition to the aortic repair. "Clamp and sew" technique was used in 18 patients (75%), two of whom had multiple tears of the aortic arch. Heparin-coated shunts were used in five patients (20.8%), and a cardiopulmonary bypass was performed in one patient who had multiple tears. Three postoperative deaths were related to polytrauma, cardiogenic shock, and sepsis. Paraplegia developed in three patients, two of whom had multiple aortic lesions necessitating longer ischemia time during the repair. Only one patient had complete neurologic deficit at the 1-year follow-up. In our series, neither surgical procedure proved superior. We conclude that the "clamp and sew" technique for repair of the disrupted thoracic aorta may allow for a more favorable outcome.