Circulating and urinary adrenal corticosterone, progesterone, and estradiol in response to acute stress in female mice (Mus musculus).

In studies of stress, it can be difficult to obtain blood rapidly enough to avoid confounding steroid measures. Noninvasive urinary steroid measures may provide an alternative insofar as they reflect systemic steroids. In Experiment 1, we profiled urinary corticosterone, progesterone, and estradiol in ovariectomized female mice following 1 h on an elevated platform. This increased urinary corticosterone for 3 h and progesterone for 4 h. In Experiment 2, blood and urine samples were obtained at 0-6 h following stressor offset. Females showed increased serum corticosterone and progesterone immediately after stressor offset. Urinary corticosterone was increased at both 0 and 2 h post-stress, while an increase in progesterone 2-6 h after stressor offset was not significant. Estradiol was not influenced by this mild stressor. In Experiment 3, mice were exposed to a more severe 1 h stressor, a rat across a wire-mesh grid. In serum, both corticosterone and progesterone were elevated immediately after stressor offset and returned to baseline within 2 h. In urine, this severe stressor elevated corticosterone immediately and 2 h after stressor offset, and in progesterone 2 h after stressor offset. Estradiol in serum was not dynamic, but it was significantly elevated in urine 4 h after stressor offset. Urinary measures generally reflected systemic measures; however, with a different time course resulting in a longer return to baseline. We suggest that the relative value of serum or urinary steroid measures in mice depends upon the experimental design, and that estradiol may only respond when the stressor is severe.

Strategic planning for employee happiness: a business goal for human service organizations.

Employee happiness can impact substantially on an organization's performance. It can influence employee retention, absenteeism, and work performance. Because of this importance, such happiness is inseparable from the real business of the organization and should be considered a business goal. Implementation and development of the strategic plan associated with this goal becomes the responsibility of a highly placed project team that has as its mission ensuring employee satisfaction. The strategic plan includes procedures that allow management to listen effectively to employees, assessing and responding to their values and needs. In this paper we discuss the workforce and environmental characteristics that are involved planning for employee happiness and the steps in creating an organizational culture in which this can become a business goal.

Myocardial infarction and remodeling in mice: effect of reperfusion.

Anatomic and functional changes after either a permanent left anterior descending coronary artery occlusion (PO) or 2 h of occlusion followed by reperfusion (OR) in C57BL/6 mice were examined and compared with those in sham-operated mice. Both interventions generated infarcts comprising 30% of the left ventricle (LV) measured at 24 h and equivalent suppression of LV ejection velocity and filling velocity measured by Doppler ultrasound at 1 wk. Serial follow-up revealed that the ventricular ejection velocity and filling velocity returned to the levels of the sham-operated controls in the OR group at 2 wk and remained there; in contrast, PO animals continued to display suppression of both systolic and diastolic function. In contrast, ejection fractions of PO and OR animals were depressed equivalently (50% from sham-operated controls). Anatomic reconstruction of serial cross sections revealed that the percentage of the LV endocardial area overlying the ventricular scar (expansion ratio) was significantly larger in the PO group vs. the OR group (18 +/- 1.7% vs. 12 +/- 0.9%, P < 0.05). The septum that was never involved in the infarction had a significantly (P < 0.002) increased mass in PO animals (22.5 +/- 1.08 mg) vs. OR (17.8 +/- 1.10 mg) or sham control (14.8 +/- 0.99 mg) animals. Regression analysis demonstrated that the extent of septal hypertrophy correlated with LV expansion ratio. Thus late reperfusion appears to reduce the degree of infarct expansion even under circumstances in which it no longer can alter infarct size. We suggest that reperfusion promoted more effective ventricular repair, less infarct expansion, and significant recovery or preservation of ventricular function.

The degree of neointimal formation after stent placement in atherosclerotic rabbit iliac arteries is dependent on the underlying plaque.

The purpose of this study was to determine the effects of stent placement on the underlying arterial morphology and the relations of stent-vessel wall interactions with subsequent neointimal formation in an atherosclerotic artery. Seven New Zealand White rabbits with experimentally induced atherosclerosis underwent balloon angioplasty (n = 7) and stent placement after balloon angioplasty (n = 7) in the iliac arteries. Histologic analysis of the treated arteries was performed at 28 days to assess device interactions with the artery and the pattern of the neointimal response. The area within the external elastic lamina of the stented vessels was 66% greater than the arteries with balloon angioplasty alone (p = 0.001) which contributed to a significantly greater late lumen area (3.33 +/- 0.51 mm2 versus 1.33 +/- 0.20 mm2, p = 0.0028). Neointimal thickness was measured at 220 stent wire sites from 21 sections of stented arteries of which 139 (63%) had underlying plaque and 81 (37%) were adjacent to normal media. Rupture of the internal elastic lamina (IEL) occurred at only 9 (11%) of the 81 stent wire sites over normal media. The mean neointimal thickness was 0.16 +/- 0.01 mm lor all stent wire sites. The neointimal thickness was greater at the stent wire sites with underlying plaque (0.23 +/- 0.01 min) than at the stent wire sites adjacent to normal media (0.08 +/- 0.01 mm) or at sites with rupture of the internal elastic lamina (0.16 +/- 0.02 mm, p = 0.0001). The degree of neointimal formation within the stents strongly correlated with the area of the underlying atherosclerotic plaque (r = 0.76, p = 0.0007) and the extent of plaque or medial compression by the struts (r = 0.90, p = 0.006). The present study characterizes stent interactions in a model commonly employed to evaluate novel therapies for the prevention of restenosis. The neointimal response was influenced by the local arterial morphology and correlated with the extent of plaque or medial compression by the stent. These data may be useful for future studies in this model and understanding the mechanism of in-stent restenosis.

Regulation of cytokine gene expression in experimental autoimmune encephalomyelitis.

We previously reported that recovery of Lewis rats from experimental autoimmune encephalomyelitis (EAE) is associated with the appearance of suppressor T cells (Ts). These Ts secrete TGF-beta which down-regulates the production of inflammatory cytokines by the effector T cells that mediate this disease. In the present study, we immunized Lewis rats with myelin basic protein (MBP)+CFA, and evaluated purified T cells and MBP-activated spleen cells (SpC) during the paralytic phase (day 12) and after recovery (days 30-33) for TGF-beta and interferon (IFN)-gamma mRNA. We used reverse transcriptase-polymerase chain reaction (RT-PCR), quantitated on the basis of beta-actin mRNA. Abundant IFN-gamma mRNA was present in MBP-activated SpC obtained on day 12. In contrast, only trace IFN-gamma mRNA was detected in day 30 activated SpC, and no IFN-gamma mRNA was present in purified, nonactivated T cells obtained at either time. The level of IFN-gamma mRNA correlated with secretion of IFN-gamma as determined by ELISA on SpC culture supernatants, and with severity of adoptively transferred EAE by the activated SpC. Thus, it appears that IFN-gamma mRNA is both transcribed and translated in response to antigen activation, resulting in secretion of IFN-gamma by the disease-inducing Te. In contrast, when we used RT-PCR to investigate the expression of TGF-beta mRNA, we found the transcript present in isolated T cells and MBP-activated SpC obtained from rats at both days 12 and 30. The presence of TGF-beta mRNA at time points corresponding to both clinical EAE and recovery suggests post-transcriptional regulation of the production of this immunoregulatory cytokine.

Encephalitogenic T cells are present in Lewis rats protected from autoimmune encephalomyelitis by coimmunization with MBP73-84 and its analog.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) that is mediated by T helper 1 (Th1) CD4+ T cells. Lewis rats can be protected from actively induced EAE by coimmunization with the encephalitogenic myelin basic protein (MBP) epitope 73-84 and its single alanine-substituted analog 1028. Although analog 1028 cannot induce either active or passive EAE, it does elicit a Th1-like response that is cross-reactive with MBP73-84. Analog 1028 can effectively inhibit clinical EAE in a dose-dependent manner when rats are coimmunized with the encephalitogenic peptide MBP73-84 and 1028 in complete Freund adjuvant (CFA). Stimulation of cells from MBP73-84:1028-coimmunized protected rats proliferate and secrete interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in vitro in response to MBP73-84. Furthermore, coimmunized protected rats harbor a population of MBP73-84-reactive potentially encephalitogenic T cells, because splenocytes from these rats can be stimulated to transfer passive EAE to naive recipients. Thus, the protection of coimmunized rats by analog 1028 is not due to the inhibition of priming of MBP73-84-reactive T cells or alteration of the cytokine secretion profile of the MBP73-84-reactive cell population. Rather, MBP73-84-reactive potentially encephalitogenic T cells are primed in these protected animals.

A comparative study of experimental autoimmune encephalomyelitis in Lewis and DA rats.

We compared the T cell responses of Lewis (LEW) and DA rats to guinea pig myelin basic protein (MBP), the synthetic peptides corresponding to the epitopes that are encephalitogenic in the LEW strain (MBP73-86, MBP68-86, and MBP87-99), and bovine proteolipid protein (PLP). DA and LEW rats were susceptible to experimental autoimmune encephalomyelitis (EAE) induced with MBP or MBP68-86, but the peptide was less active in DA rats than in intact MBP molecule. MBP73-86 and MBP87-99 induced EAE in LEW rats but not in the DA strain. MBP89-169 was also encephalitogenic in DA rats. Encephalitogenic CDa+ T cell lines and clones derived from MBP-sensitized DA rats secreted IFN-gamma and TNF-alpha and proliferated to MBP and MBP89-169, but not to MBP68-88. However, T cells from MBP68-86-sensitized DA or LEW rats proliferated specifically in an I-A-restricted response to MBP68-86. T cells from MBP87-99-immunized LEW rats responded to MBP87-99 in the context of I-E, whereas the peptide-specific response of MBP87-99 immunized DA rats was I-A-restricted, although FACScan analysis indicated that DA rats express both I-A and I-E. DA rats were also highly susceptible to EAE induced with PLP; 0.6 nmol was Encephalitogenic for DA rats, but did not induce clinical EAE in LEW rats. Although both DA and LEW rats are highly susceptible to EAE, we demonstrate marked differences in the array of myelin epitopes capable of inducing the disease, as well as the MHC restriction of these epitopes, between the two rat strains.

Variable susceptibility of Lewis rats to experimental autoimmune encephalomyelitis.

This report is to alert other investigators that Lewis rats, which are widely employed in studies of autoimmune diseases, vary considerably with respect to susceptibility to experimental autoimmune encephalomyelitis (EAE), depending upon commercial source.

Selective alpha IIb beta 3 receptor blockage with peptide TP9201 prevents platelet uptake on Dacron vascular grafts without significant effect on bleeding time.

Synthetic vascular prostheses lack the uniquely low thrombogenicity provided by the endothelial cell lining of autogenous saphenous vein or artery grafts. The thrombogenic nature of the synthetic graft surface becomes a major determinant of early prosthetic graft patency. We demonstrate in a baboon ex vivo synthetic graft model that modification of the host's platelet interaction with the graft surface results in inhibition of platelet thrombus formation and thereby, a possible enhancement of early prosthetic graft patency. This was achieved by selective blockage of the platelet alpha IIb beta 3 receptor by the arginine-glycine-aspartic acid-containing synthetic peptide TP9201. Platelet thrombus formation on a Dacron graft indicated by accumulation of indium III-oxine-labeled autologous platelets was measured by gamma camera imaging. After 60 minutes of circulation, TP9201 at a bolus of 125 micrograms/kg; infusion of 3 micrograms/kg/min, bolus of 190 micrograms/kg; infusion of 5 micrograms/kg/min, bolus of 250 micrograms/kg; infusion of 6 micrograms/kg/min, and bolus of 500 micrograms/kg; infusion of 12 micrograms/kg/min decreased platelet uptake on the graft to 50%, 40%, 30%, and 10% of control uptake, respectively. Forelimb template bleeding times were not found to be significantly prolonged at doses that effectively inhibit ex vivo platelet aggregation. As a result of drug treatment, no changes in hemodynamic parameters or hematologic profile, including platelet number and clotting time, were observed. We demonstrate here that the arginine-glycine-aspartic acid-containing peptide TP9201, which competitively inhibits the alpha IIb beta 3 integrin-fibrinogen interaction, significantly decreased the accumulation of platelets on a Dacron vascular graft. Molecules like peptide TP9201, because of its unique activity profile, may represent a superior approach to the control of platelet accumulation on thrombogenic surfaces.

Transforming growth factor-beta 1 inhibits tumor necrosis factor-alpha/lymphotoxin production and adoptive transfer of disease by effector cells of autoimmune encephalomyelitis.

We previously reported that the CD4+ suppressor cells (Ts) that regulate recovery of Lewis rats from experimental autoimmune encephalomyelitis (EAE) produce transforming growth factor-beta (TGF-beta). We also reported that TGF-beta downregulates interferon-gamma (IFN-gamma), but not interleukin-2 (IL-2) production, by the CD4+ effector T cells (Te) that mediate EAE. We now report that TGF-beta also inhibits the production of tumor necrosis factor/lymphotoxin (TNF/LT) by EAE effector cells. When activated in vitro with myelin basic protein (MBP), Te produced TNF/LT, as measured using a WEHI 164 cytotoxicity assay. The specificity of cytokine action was demonstrated using neutralizing antibodies to TNF/LT. When added to the Te+MBP cultures, TGF-beta inhibited TNF/LT production in a dose-dependent fashion. Moreover, neutralizing anti-TGF-beta antibodies augmented TNF/LT production in the Te+MBP cultures. We also confirm that TGF-beta inhibits adoptive transfer of EAE. In contrast, murine IL-10 only partially inhibited TNF/LT and IFN-gamma production by Te. We conclude that TGF-beta production by Ts plays a major role in recovery from EAE in the Lewis rat by inhibiting TNF/LT and IFN-gamma production by the effector cells that mediate EAE.

T and B cell responses to myelin basic protein and encephalitogenic epitopes.

The major encephalitogenic epitope of myelin basic protein (MBP) for the Lewis rat includes residues 68-84, although a minor epitope has been localized to MBP residues 87-99. We synthesized MBP68-84 and MBP87-99, and immunized rats with these peptides or with MBP in complete Freund's adjuvant (CFA). MBP and MBP68-84 induced paralytic experimental autoimmune encephalomyelitis (EAE) at equimolar concentrations, whereas significantly higher dosages of MBP87-99 were required to elicit paralytic disease. Spleen cells (SpC) from MBP- or MBP68-84-immunized rats could be activated with either MBP or MBP68-84 to transfer EAE to recipients. Anti-MBP antibodies were detected by ELISA in rats immunized with MBP-CFA, and anti-MBP68-84 specific antibodies were present in serum obtained from MBP68-84-immunized animals. However, these antibodies were non-cross reactive. MBP87-99 elicited only a meager antibody response to the immunizing peptide, and cross reactivity with MBP was not observed. Thus, although MBP and each peptide exhibited encephalitogenic activity, and MBP and MBP68-84 were cross reactive at the T cell level, the absence of cross reactivity at the humoral level indicates that significant immunological differences exist between MBP and the synthetic determinants, which may reflect differences in epitope recognition by T and B lymphocytes.

CD4+ suppressor cells of autoimmune encephalomyelitis respond to T cell receptor-associated determinants on effector cells by interleukin-4 secretion.

We have previously demonstrated that CD4+ suppressor T cells (Ts) inhibit the secretion of interferon (IFN)-gamma, but not interleukin (IL)-2, by effector cells of experimental autoimmune encephalomyelitis (EAE). Moreover, CD4+ Ts appear to regulate IFN-gamma by secretion of transforming growth factor-beta. We now show that CD4+ Ts produce a lymphokine with IL-4 activity in response to a determinant associated with EAE effector cells. CD4+ Ts do not proliferate or secrete IFN-gamma, IL-2, or IL-4 in response to myelin basic protein, nor do CD4+ Ts proliferate or secrete IL-2 when co-cultured with irradiated EAE effector cells. Rather, CD4+ Ts secrete IL-4 when co-cultured with either irradiated effector spleen cells or irradiated encephalitogenic line cells. CD4+ Ts do not secrete IL-4 in response to OVA-primed spleen cells, suggesting that the suppressor cells recognize a determinant specific to encephalitogenic T cells. Furthermore, CD4+ Ts secrete IL-4 when cultured with synthetic T cell receptor (TcR) V beta 8, but not TcR V beta 14 peptide, in the presence of antigen-presenting cells. This response is major histocompatibility complex class II restricted as demonstrated by inhibition of the response with anti-class II monoclonal antibody. These results suggest that CD4+ Ts recognize a determinant associated with TcR on the surface of EAE effector cells and respond by secreting IL-4, in a manner analogous to the Th2 lymphocyte subtype.

Studies of V beta 8 T cell receptor peptide treatment in experimental autoimmune encephalomyelitis.

Lewis rats immunized with T cell receptor (TCR) variable region peptide V beta 8 in complete Freund's adjuvant (CFA) were protected against experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein in CFA, although variable protection was also observed in rats injected with control peptide in CFA, or CFA alone. However, this adjuvant-mediated protection could be avoided by immunizing with TCR peptide in incomplete adjuvant (IFA). Clinical, but not histologic EAE was suppressed in rats given V beta 8 peptide in IFA, whereas control animals injected with V beta 14 peptide in IFA, or IFA alone developed severe clinical EAE. Anti-V beta 8 antibodies were present in the sera of all V beta 8-treated rats. These findings lend support to the hypothesis that autoimmune disease can be suppressed by inducing an immune response against the TCR-idiotope of autoreactive T cells.

Effects of aspirin, dipyridamole, and dibutyryl cyclic adenosine monophosphate on platelet-derived growth factor A chain mRNA levels in human saphenous vein endothelial cells and smooth muscle cells.

Aspirin and dipyridamole have frequently failed to control intimal hyperplasia in vascular grafts in animal and clinical trials. These trials were based on the concept that the smooth muscle mitogen, platelet-derived growth factor (PDGF) released from platelets, was a major cause of intimal hyperplasia. Both endothelial and smooth muscle cells (ECs and SMCs), however, can also release PDGF-like SMC mitogens that might cause intimal hyperplasia. We therefore tested whether aspirin and dipyridamole alone or together can affect PDGF-A chain mRNA levels in cultured human saphenous vein ECs and SMCs. Cultures were exposed for 72 hours to 3 x 10(-5) mol/L aspirin and/or 5 x 10(-6) mol/L dipyridamole. Cellular RNA was then extracted, and PDGF-A chain mRNA signal levels were measured by a reverse transcription/polymerase chain reaction method. mRNA for glyceraldehyde-3-phosphate dehydrogenase was used as a constitutively expressed control RNA species. Signal strength on Southern blots of amplified polymerase chain reaction products was measured by densitometry. Neither aspirin nor dipyridamole alone or together reduced the ratio (PDGF-A chain signal/glyceraldehyde-3-phosphate dehydrogenase signal) below that of control cultures. PDGF-A chain expression was not a constitutive artifact of culture because dibutyryl cyclic AMP (5 x 10(-4) mol/L) reduced PDGF-A chain signal from a control index of 1.0 to 0.5 +/- 0.1 (mean +/- SE) (n = 3; p less than 0.05) in EC cultures and to 0.2 (mean) (n = 2) in SMC cultures. These data may explain why aspirin and dipyridamole fail to reduce intimal hyperplasia in some animal and clinical trials despite effective inhibition of platelet aggregation.

Increased in-vitro incubation time of endothelial cells on fibronectin-treated ePTFE increases cell retention in blood flow.

Endothelial cell (EC) seeding is postulated as a mechanism of improving patency of small calibre vascular grafts. However, the majority of seeded cells are lost within hours following restoration of blood flow. We postulated that incubating EC in-vitro on a graft will improve adherence and resistance to the sheer stresses of pulsatile blood flow. Fibronectin-treated ePTFE (5 cm x 4 mm ID) seeded with Indium-111-labelled autologous canine EC (1.5 x 10(5) cells/cm2) were incubated for four different time periods; 90 min, 24 h, 72 h and 6 days. Incubated grafts were subjected to blood flow of 75 ml/min for 6 h, in a canine ex-vivo arteriovenous shunt circuit. EC retention during perfusion was studied by measuring gamma activity emitted by the grafts. Cell morphology of non-perfused control groups and perfused groups was compared using scanning electron microscopy (SEM). SEM of control grafts showed progressive EC spreading on the ePTFE surface for up to 72 h incubation. Gamma activity was significantly higher at 6 h perfusion in grafts incubated for 72 h (82 +/- 4%) and 24 h (63 +/- 6%) vs. 90 min (34 +/- 13%, p less than 0.05), and between grafts incubated for 72 h vs. 6 days (55 +/- 7%, p less than 0.05). Perfused grafts incubated for 72 h showed unaltered EC morphology on SEM, few cells remained on 90 min incubated grafts. We conclude that incubating EC on fibronectin-treated ePTFE for 72 h in-vitro after seeding improves cell retention during blood flow.

Protection of endothelium in vessel segments excised for grafting.

Contracture and collapse often complicate dissection and excision of blood vessels for grafting. Subsequent forceful redilatation and leak checking with cold saline almost always lead to partial or total denudation of endothelium, which increases the risk of early occlusion by thrombi or late occlusion by fibrocellular initimal thickening. To prevent endothelial damage, we used three major modifications of the excision and extracorporeal maintenance procedures: (1) adventitial application of the smooth-muscle relaxant papaverine in situ to avoid contracture and to allow subsequent gentle, gradual redilatation at low pressure; (2) balanced perfusion and maintenance fluid containing 10% protein (serum of albumin); and (3) extracorporeal maintenance of the vessel at body temperature. In an experimental extracorporeal vascular perfusion system, we could preserve morphologically intact endothelium of rabbit carotid arteries for over 3 hours, as evaluated by scanning electron microscopy. Applying the same principles to canine cephalic and saphenous veins, we gradually dilated the vessels at pressures as low as 100 mm Hg and obtained a morphologically intact endothelium after 1 hour of extracorporeal maintenance.

Vascular organ culture: prevention of endothelial damage due to removal and reperfusion.

Endothelial cell damage is recognized as a complication in blood vessels which are excised and reperfused for grafting or vascular organ culture. We demonstrate that the damage consists of lifting, crenation and detachment of endothelial cells, partially due to contracture and forceful redilation of the vessel wall. We use a mechanical brace and smooth muscle cell relaxation with papaverine to maintain length and circumference of rabbit carotid arteries during removal and reperfusion. With this technique, a considerable degree of morphological preservation of the endothelial cell layer can be achieved in isolated and perfused vessel segments. Furthermore, this principle of endothelial cell preservation by prevention of contracture during vessel preparation may be applicable clinically.