OBJECTIVE

To assess the impact of an algorithm defining resuscitation according to early goal-directed therapy, glycemic control, administration of stress doses of hydrocortisone, and use of recombinant human activated protein C (rhAPC) on measures of organ dysfunction and outcome in septic shock.

METHODS

Retrospective cohort study.

METHODS

Multidisciplinary ten-bed intensive care unit of a university hospital.

METHODS

Sixty patients were analyzed: 30 consecutive patients fulfilling criteria for diagnosis of septic shock, treated from September 2002 until December 2003 after implementation of a standard operating procedure (SOP) for severe sepsis and septic shock; and 30 patients with septic shock treated from January until August 2002 in the same unit, who served as controls.

RESULTS

Data for blood gas analysis, lactate, glucose, serum creatinine, bilirubin, white blood cells, platelets, and C-reactive protein were obtained from patient files on admission or at time of diagnosis of septic shock and at 7:00 a.m. on days 2 and 4; Sequential Organ Failure Assessment scores were calculated and 28-day survival was assessed. With implementation of the SOP, use of dobutamine (12/30 vs. 2/30), insulin (blood glucose <150 mg/dL, day 4: 26/28 vs. 13/25), hydrocortisone (30/30 vs. 13/30), and rhAPC (7/30 vs. 0/30) significantly increased, whereas volume for resuscitation and use of packed red blood cells were unaffected. Mortality was 53% in the historical control group and 27% after implementation of the SOP (p < .05).

CONCLUSIONS

The combined approach of early goal-directed therapy, intensive insulin therapy, hydrocortisone administration, and additional application of rhAPC in selected cases seems to favorably influence outcome. The implementation of a "sepsis bundle" can be facilitated by a standardized protocol while significantly reducing the time until the defined therapeutic measures are realized in daily practice.

Infusion of norephinephrine (NE) (1 - 3 mug/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of prostaglandin E-like substance (PGE) at a concentration of 2.81 +/- 0.65 ng/ml in terms of PGE2. Indomethacin (3 mug/ml) abolished the NE-induced release of PGE. Arachidonic acid (0.2 mug/ml) in the presence of indomethacin did not restore the NE-induced release of PGE. Hydrocortisone (10 - 30 mug/ml) and dexamethasone (2 - 5 mug/ml) also inhibited the NE-induced release of PGE. The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 mug/ml). Antigen-induced release of a prostaglandin-like substance (PGs) (43.1 +/- 3.8 ng/ml in terms of PGE2 and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completely abolished by indomethacin (5 mug/ml) or by hydrocortisone (100 mug/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 +/- 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 mug/ml) into the pulmonary artery reversed the hydrocortisone-induced blockade of the release of RCS and PGs. It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.

BACKGROUND

Exposure to intense physical and psychological stress during septic shock can result in posttraumatic stress disorder in survivors. Patients with chronic posttraumatic stress disorder often show sustained reductions in serum cortisol concentration. This investigation examines whether increasing serum cortisol levels with hydrocortisone treatment during septic shock reduces the incidence of posttraumatic stress disorder in survivors.

METHODS

Patients (n = 20) were recruited from a prospective, randomized double-blind study on the hemodynamic effects of hydrocortisone during septic shock. Eleven patients had received placebo and nine stress doses of hydrocortisone. Posttraumatic stress disorder was diagnosed 31 months (median) after intensive care unit discharge using SCID-IV (DSM-IV-criteria). Furthermore, the number of categories of traumatic memory from ICU treatment was determined in both groups at that time.

RESULTS

Only one of nine patients from the hydrocortisone group developed posttraumatic stress disorder, compared with seven of 11 patients in the placebo group (p =.02). There was no significant difference with regard to the number of categories of traumatic memory between the hydrocortisone and placebo groups.

CONCLUSIONS

The administration of hydrocortisone during septic shock in a dosage similar to the endogenous maximal production rate was associated with a lower incidence of posttraumatic stress disorder in long-term survivors, which seems to be independent of the number of categories of traumatic memory.

BACKGROUND

We recently demonstrated that 28-d bed rest in healthy volunteers results in a moderate loss of lean leg mass and strength.

OBJECTIVE

The objective of this study was to quantify changes in muscle protein kinetics, body composition, and strength during a clinical bed rest model reflecting both physical inactivity and the hormonal stress response to injury or illness.

METHODS

Muscle protein kinetics were calculated during a primed, continuous infusion (0.08 micromol/kg.min) of 13C6-phenylalanine on d 1 and 28 of bed rest.

METHODS

The setting for this study was the General Clinical Research Center at the University of Texas Medical Branch.

METHODS

Participants were healthy male volunteers (n = 6, 28 +/- 2 yr, 84 +/- 4 kg, 178 +/- 3 cm).

METHODS

During bed rest, hydrocortisone sodium succinate was administered iv (d 1 and 28) and orally (d 2-27) to reproduce plasma cortisol concentrations consistent with trauma or illness (approximately 22 microg/dl).

METHODS

We hypothesized that inactivity and hypercortisolemia would reduce lean muscle mass, leg extension strength, and muscle protein synthesis.

RESULTS

Volunteers experienced a 28.4 +/- 4.4% loss of leg extension strength (P = 0.012) and a 3-fold greater loss of lean leg mass (1.4 +/- 0.1 kg) (P = 0.004) compared with our previous bed rest-only model. Net protein catabolism was primarily due to a reduction in muscle protein synthesis [fractional synthesis rate, 0.081 +/- 0.004 (d 1) vs. 0.054 +/- 0.007%/h (d 28); P = 0.023]. There was no change in muscle protein breakdown.

CONCLUSIONS

Prolonged inactivity and hypercortisolemia represents a persistent catabolic stimulus that exacerbates strength and lean muscle loss via a chronic reduction in muscle protein synthesis.

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-1D has been developed in which more than 35% of the cells express beta-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete beta-casein unidirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine beta-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency (greater than 150-fold induction in some cases). This regulation occurred primarily at the transcriptional level and was dependent on the length of the 5' flanking region of the beta-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous beta-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.

The present studies were aimed at determining if the use of a cell culture medium that supports proliferation of human mammary epithelial cells of the luminal lineage would allow routine isolation of breast cancer cells from primary and metastatic tumor specimens. Results obtained with mammary epithelial cells derived from reduction mammoplasty specimens and primary breast carcinomas indicated that growth of cells on type I collagen-coated dishes in Ham's F-12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, cholera toxin, and 5% fetal bovine serum resulted in the growth and serial passage of cells that stained positively for the luminal cell marker cytokeratin 19. By contrast, growth of mammary epithelial cells in a growth factor-supplemented serum-free medium resulted in the emergence of mammary epithelial cell colonies that were uniformly negative for keratin 19. Filter isolation methods were used to isolate individual keratin-19-positive colonies from primary cultures derived from breast cancer specimens. All of the luminal mammary epithelial cells isolated from breast cancer tissues expressed characteristics of normal cells. Keratin-19-positive colonies isolated from several different tumors all grew rapidly for 30 to 60 days in culture and then senesced. Cells were isolated from one tumor that was known to have undergone a loss of heterozygosity at a specific locus in the p53 gene. All colonies isolated from this specimen contained both p53 alleles, which was consistent with their origin from normal luminal cells. Cells were also isolated from one tumor in which the c-erbB2 protein was drastically overexpressed in the neoplastic cells. Once again, keratin-19-positive colonies isolated from this tumor did not overexpress the c-erbB-2 protein. Experiments were then performed with cells derived from pleural effusions and metastatic lymph nodes. Results obtained with these specimens indicated that the growth conditions that support the growth of normal luminal mammary epithelial cells do not support the growth of neoplastic cells. However, the omission of cholera toxin, epidermal growth factor, and type I collagen substratum resulted in the isolation of two long-term cell lines. Both cell lines have population doubling times of approximately 100 h, are hyperdiploid, and stain positively for cytokeratin 19. Thus, culture conditions that support the growth of normal luminal mammary epithelial cells do not, in general, support the growth of breast cancer cells.

This report describes a form of disseminated necrotizing leukoencephalopathy that has been observed in four children with acute lymphoblastic leukemia, and one child with Burkitt's lymphoma terminating in a leukemic phase. In addition to systemic vincristine, cytosine arabinoside, cyclophosphamide, and steroids, these patients received courses of intrathecal methotrexate, cytosine arabinoside, and hydrocortisone, because of meningeal tumor cell infiltration. Whole brain radiation was also given either before or during intrathecal therapy. Three of the children had a progressive irreversible neurologic illness, which developed either at or shortly after the completion of combined triple intrathecal therapy, death ensuing approximately 2 months later. The neuropathologic lesions consisted of discrete multifocal necroses of coagulative type, apparently extending by confluence, and disseminated in the cerebral white matter in a random manner. In one case, extensive symmetrical demyelinating and necrotizing lesions involved the centrum ovale bilaterally. There was a remarkable absence of inflammatory cellular response and a relative paucity of macrophage reaction, with usually little or no tissue breakdown. In addition to demyelination and glial cell loss, there was striking axonal damage, with conspicuous axonal swelling both within and around the necrotizing lesions. The surrounding white matter showed focal status spongiosus and a moderate astrocytic response. Vascular fibrinoid necrosis was inconstant and, except in one case, rarely observed. The possible causal relationship of these lesions to combined triple intrathecal antimetabolite therapy and brain radiation is discussed.

Lactogenic hormones and extracellular matrix (ECM) act synergistically to regulate beta-casein expression in culture. We have developed a functional subpopulation of the mouse mammary epithelial cell strain COMMA-1D (designated CID 9), which expresses high level of beta-casein, forms alveolar-like structures when plated onto the EHS tumor-derived matrix, and secretes beta-casein unidirectionally into a lumen. We have further shown that ECM- and prolactin-dependent regulations of beta-casein occur mainly at the transcriptional level and that 5' sequences play an important role in these regulations. To address the question of the nature of the DNA sequence requirements for such regulation, we analyzed the bovine beta-casein gene promoter in these cells. We now have located a 160-bp transcriptional enhancer (BCE1) within the 5' flanking region of the beta-casein gene. Using functional assays, we show that BCE1 contains responsive elements for prolactin- and ECM-dependent regulation. BCE1 placed upstream of a truncated and inactive beta-casein promoter (the shortest extending from -89 to +42 bp with regard to the transcription start site) reconstitutes a promoter even more potent than the intact promoter, which contains BCE1 in its normal context more than 1.5 kb upstream. This small fusion promoter also reconstitutes the normal pattern of regulation, including a requirement for both prolactin and ECM and a synergistic action of prolactin and hydrocortisone. By replacing the milk promoter with a heterologous viral promoter, we show that BCE1 participates in the prolactin- and ECM-mediated regulation.

OBJECTIVE

To assess the effects of corticosteroids on mortality in patients with severe sepsis and septic shock.

METHODS

Randomised and quasi-randomised trials of corticosteroids versus placebo (or supportive treatment alone) retrieved from the Cochrane infectious diseases group's trials register, the Cochrane central register of controlled trials, Medline, Embase, and LILACS.

METHODS

Two pairs of reviewers agreed on eligibility of trials. One reviewer entered data on to the computer and four reviewers checked them. We obtained some missing data from authors of trials and assessed methodological quality of trials.

RESULTS

16/23 trials (n = 2063) were selected. Corticosteroids did not change 28 day mortality (15 trials, n = 2022; relative risk 0.92, 95% confidence interval 0.75 to 1.14) or hospital mortality (13 trials, n = 1418; 0.89, 0.71 to 1.11). There was significant heterogeneity. Subgroup analysis on long courses >> or = 5 days) with low dose (< or = 300 mg hydrocortisone or equivalent) corticosteroids showed no more heterogeneity. The relative risk for mortality was 0.80 at 28 days (five trials, n = 465; 0.67 to 0.95) and 0.83 at hospital discharge (five trials, n = 465, 0.71 to 0.97). Use of corticosteroids reduced mortality in intensive care units (four trials, n = 425, 0.83, 0.70 to 0.97), increased shock reversal at 7 days (four trials, n = 425; 1.60, 1.27 to 2.03) and 28 days (four trials, n = 425, 1.26, 1.04 to 1.52) without inducing side effects.

CONCLUSIONS

For all trials, regardless of duration of treatment and dose, use of corticosteroids did not significantly affect mortality. With long courses of low doses of corticosteroids, however, mortality at 28 days and hospital morality was reduced.

Defined culture conditions for routine clonal growth of normal human adult bronchial epithelial cells have been developed. Serum and feeder cell requirements were abrogated by: (a) optimizing the calcium concentration in nutrient medium, MCDB 151; (b) supplementing with purified factors (epidermal growth factor, 5 ng/ml; insulin, 5 micrograms/ml; transferrin, 10 microgram/ml; hydrocortisone, phosphoethanolamine and ethanolamine, each at 5 x 10(-7) M; and trace elements); and (c) coating the surface of the culture dish with a mixture of fibronectin, collagen, and bovine serum albumin. Endothelial cell growth supplement (100 micrograms/ml) and retinoic acid (3 x 10(-10) M) further enchanced growth, whereas cholera toxin was nonmitogenic and serum supplementation (greater than 2%) markedly reduced the growth rate. Using the defined system, dissociated cultures of bronchial epithelial cells, obtained from more than 15 donors, have been subcultured at clonal densities with a colony forming efficiency of 3 to 4%. In addition, high density cultures have been subcultured more than five times with four to six population doublings per passage. The features of this system permit pathobiologic investigations of bronchial epithelial cells, e.g., aging, differentiation, and carcinogenesis using conditions that isolate the results from the influence of serum, feeder cells, and other undefined factors.

The effect of glucocorticosteroids on the kinetics of mononuclear phagocytes, i.e., peripheral blood monocytes and peritoneal macrophages, was studied in normal mice, as well as in mice in which an inflammatory reaction was evoked in the peritoneal cavity. The administration of glucocorticosteroids resulted in a rapid decrease (within 3-6 hr) in the number of circulating monocytes, the duration being dependent on the nature and dose of the compound. The water-soluble dexamethasone sodium phosphate is only briefly active (less than 12 hr), but hydrocortisone acetate, which forms a subcutaneous depot, reduced the number of monocytes for more than 2 wk. In normal mice, hydrocortisone did not affect the number of macrophages already present in the peritoneal cavity, but the transit of mononuclear phagocytes from the circulation into the peritoneal cavity was arrested. During an inflammatory response in the peritoneal cavity, hydrocortisone suppresses both the increase in the number of monocytes in the peripheral blood and the increase in the number of peritoneal macrophages. This reduction of the inflammatory exudate appeared to be due to a diminished influx of mononuclear phagocytes from the peripheral blood. No lytic action of glucocorticosteroids on the mononuclear phagocytes could be demonstrated.

A method has been described for the long-term culture of human bone marrow cells in liquid medium. Hematopoiesis, as measured by the production of granulocytic-macrophage progenitor cells (CFUc), continued for at least 20 weeks and was dependent upon the presence of a marrow-derived adherent layer of cells. As in the case of murine marrow liquid cultures, the adherent layer consisted of mononuclear phagocytic cells, endothelial cells, and lipid-laden adipocytes, the latter being essential for long-term hematopoiesis. Optimal growth conditions included McCoy's medium supplemented with fetal bovine serum, horse serum, and hydrocortisone and incubation at 33 degrees C. Horse serum in conjunction with hydrocortisone appeared essential for the growth of adipocytes.

We have previously demonstrated that cultured human bronchial epithelial cells produce cytokines with potent proinflammatory properties on exposure to several stimuli in vitro, and we have hypothesized that these epithelial cell-derived factors may contribute to the pathogenesis of some inflammatory diseases of the bronchial mucosa, particularly asthma, by promoting the infiltration of granulocytes and T cells and their local activation. We provide, in this study, direct evidence of an increased expression of granulocyte-macrophage-colony-stimulating factor, interleukin-6, and interleukin-8 genes and proteins in bronchial epithelium from patients with symptomatic asthma. The up regulation of the production of these cytokines in bronchial epithelial cells of patients with asthma could be abolished in vitro by corticosteroids (hydrocortisone, 10(-7) mol/L), but the up regulation also spontaneously disappeared during a period of 6 days after the removal of the cells from the diseased tissue.

OBJECTIVE

To confirm the feasibility and estimate the efficacy of methotrexate (MTX), teniposide, carmustine, and methylprednisolone (MBVP) chemotherapy combined with radiotherapy (RT) for patients with non-AIDS-related primary CNS lymphoma (PCNSL) treated in a multicenter setting.

METHODS

Treatment consisted of two cycles of MBVP (MTX 3 g/m2 days 1 and 15, teniposide 100 mg/m2 days 2 and 3, carmustine 100 mg/m2 day 4, methylprednisolone 60 mg/m2 days 1 to 5, and two intrathecal injections of MTX 15 mg, cytarabine 40 mg, and hydrocortisone 25 mg) followed by 40 Gy of RT. Primary end points were response and safety of this regimen.

RESULTS

Twelve centers included 52 patients who were all analyzed on an intent-to-treat basis. Median follow-up of all patients was 27 months. One patient progressed and died before treatment, and five patients died during treatment. Four patients received RT after one cycle of chemotherapy, and 42 patients completed the entire treatment. Hematologic grade 3 and 4 toxicity was seen in 78% of patients for leukocytes and 24% of patients for platelets. The overall response rate of all 52 patients was 81%. Two patients who did not fulfill the criteria of objective response survived more than 1 year; one of them is still alive without disease. Eighteen patients died; 11 deaths were a result of tumor, five were probably treatment-related, one was caused by late leukoencephalopathy, and one was a result of intercurrent disease. Median estimated overall survival was 46 months.

CONCLUSIONS

MBVP followed by RT for PCNSL has a high response rate. However, the 10% toxic death rate during treatment in a multicenter setting underlines the need for highly specialized care.

Topical corticosteroids are widely prescribed by dermatologists caring for patients with atopic eczema. Patients' fears about using topical corticosteroids may have important implications for compliance with treatment. We carried out a questionnaire-based study of 200 dermatology outpatients with atopic eczema (age range 4 months-67.8 years) to assess the prevalence and source of topical corticosteroid phobia. We also questioned patients on their knowledge of the potencies of different topical corticosteroids. Overall, 72.5% of people worried about using topical corticosteroids on their own or their child's skin. Twenty-four per cent of people admitted to having been non-compliant with topical corticosteroid treatment because of these worries. The most frequent cause for concern was the perceived risk of skin thinning (34.5%). In addition, 9.5% of patients worried about systemic absorption leading to effects on growth and development. The most commonly used topical corticosteroid was hydrocortisone, yet 31% of patients who used this preparation classified it as either strong, very strong or did not know the potency. Only 62.5% of the 48 patients who had used both Dermovate (Glaxo) and hydrocortisone in the past were able to correctly grade Dermovate as being more potent than hydrocortisone. The most common source of patient information regarding topical corticosteroid safety was the general practitioner. Although skin thinning and systemic effects can develop very occasionally in people using topical corticosteroids, the concern expressed by people using them seems out of proportion in relation to the evidence of harm. This study highlights the need for provision of better information and education to patients and possibly general practitioners regarding the safety, potency and appropriate use of topical corticosteroids.

The effect of corticosteroid administration on the redistribution of sirculating lymphocytes was studied in the guinea-pig, since this species closely resembles man in its relative resistance to the lymphopenic effect of corticosteroids. A single intravenous injection of hydrocortisone (either 10 mg or 100 mg/kg) caused a profound but transient lymphocytopenia which was maximal at 4 hours following injection, with a returnto normal counts by 24 hours. There was a proportionately greater decrease in circulating T lymphocytes compared to B lymphocytes, although both populations were diminished. Chronic cortisone acetate treatment (100 mg/kg subcutaneously for 7 days) caused a similiar pattern of lymphocytopenia except that it was sustained during the period of chronically elevated plasma cortisol levels. The lymphocytes remaining inthe circulation during the period of lymphocytopenia responded normally in vitro to the mitogens phytohemagglutinin, concanavalin A, and pokeweek mitogen. There was very littleeffect of corticosteroid administration on the numbers, proportions, or mitogenic response of splenic lymphocytes. There was a dramatic increase in the bone marrow of proportions and absolute numbers of lymphocytes bearing surface T-and B-cell markers, as well as a marked increase in response of bone marrow lymphocytes to mitogenic stimulation during the period of maximal circulating lymphocytopenia caused by the administration of corticosteroids, especially chronic cortisone acetate. There was a preferential homing of reinfused -51Cr-labelled syngeneic peripheral blood lymphocytes to the bone marrow of corticosteroid-treated recipients. These studies demonstrate aredistribution of circulating lymphocytes to the bone marrow during corticosteroid treatment, resulting in an increase in immunocompetence of this compartment, while the peripheral blood lymphocyte compartment is quantitatively immunosuppressed due to a lymphocytopenia.

The effects of various drugs on chemotaxis of polymorphonuclear leukocytes (PMN's) in vitro and in vivo have been studied. Response of rabbit PMN's in vitro to the chemotactic factor of rabbit serum, consisting of an activated protein-protein complex of the fifth and sixth (and probably seventh) components of complement (C'), is suppressed by hydrocortisone, methyl prednisolone, and chloroquine. Drug concentrations causing 50% inhibition of chemotaxis in vitro were found to be: hydrocortisone, 2.9 x 10(-4)M; methly prednisolone, 1.2 x 10(-4)M; and chloroquine 8.5 x 10(-6)M. The hydrocortisone effect on PMN's appeared to be irreversible, since washing of the cells did not restore their chemotactic response. 2, 4-Dinitrophenol (DNP), vitamin A, and endotoxin did not inhibit chemotaxis. Hydrocortisone and chloroquine did block serum C' activity in vitro, but only at substantially higher concentrations. Using the reversed passive Arthus reaction in guinea pigs as a model for chemotaxis in vivo, systemic treatment of animals with hydrocortisone or chloroquine inhibited development of the vasculitis. Circulating antigen and C' were fixed in vascular structures, and serum C' was not perceptibly altered. Nevertheless, PMN infiltrates failed to occur. Local administration of hydrocortisone also prevented influx of PMN's in the Arthus reaction, in spite of the fact that immune reactants were found fixed in the vascular walls. Systemic treatment of guinea pigs with DNP did not diminish the intensity of the Arthus reactions. Phagocytosis of zymosan particles by rabbit PMN's was inhibited by hydrocortisone, methyl prednisolone, and chloroquine, but not by DNP or endotoxin. The concentrations of drugs inhibitory in phagocytosis were substantially higher than those required for inhibition of chemotaxis in vitro. These findings suggest that hydrocortisone and chloroquine inhibit the inflammatory process by preventing the response of leukocytes to chemotactic stimuli.

BACKGROUND

Endothelial dysfunction occurs in many diseases associated with increased cardiovascular risk. We examined the effects of pro-inflammatory cytokines on endothelial function.

RESULTS

Subjects lay with one hand placed on an angled support. The diameter of a vein was recorded by measuring the linear displacement of a probe placed on the skin overlying the vein when the pressure in a congesting cuff placed around the upper arm was deflated from 40 to 0 mm Hg. A length of the vein was isolated by two wedges. TNF-alpha (1 ng), IL-1beta (1 ng), or IL-6 (100 pg) were instilled for 1 hour, either individually or together. At the end of the hour, the wedges were removed and the vein reconnected with the circulation. Dose-response curves (bradykinin: 2, 4, and 8 pmol/min; arachidonic acid: 0.2, 2, and 20 nmol/min; and glyceryl trinitrate 1, 2, and 4 pmol/min) were constructed before and 1, 6, 24, and 48 hours after instillation. In another study, hydrocortisone (100 mg) was given 2 hours before the study. In a different study, subjects were given oral aspirin (75 mg or 1 g) 2 hours before the study. TNF-alpha and IL-1beta alone but not IL-6 attenuated the dilatation to bradykinin and arachidonic acid; the response was greatest at 1 hour with recovery occurring by 6 hours. Combination of IL-1beta and TNF-alpha prolonged the endothelial dysfunction, resulting in recovery at 24 hours. Hydrocortisone and high-dose aspirin prevented endothelial dysfunction.

CONCLUSIONS

The results demonstrate that pro-inflammatory cytokines induce transient and reversible endothelial dysfunction and indicate that cyclooxygenase activity may contribute to the genesis of the effect. If other vessels behave similarly, this may provide further insight into the mechanisms precipitating acute cardiovascular events after inflammatory disorders.

Bacteria that survive inside polymorphonuclear neutrophils (PMN) following phagocytosis are protected from the bactericidal action of most antibiotics. Two possible explanations are altered metabolism by intraleukocytic bacteria or failure of antibiotics to enter the phagosome. The oxygen consumption of intraleukocytic and extraleukocytic bacteria was measured as an index of bacterial metabolism. PMN respiration and bactericidal activity were suppressed with large doses of hydrocortisone and extraleukocytic bacterial oxygen consumption was abolished by the addition of lysostaphin. Intraleukocytic bacterial continued to consume oxygen suggesting that surviving ingested micro-organisms are metabolically active. Neither penicillin (which cannot kill intraleukocytic bacteria) nor rifampin (which can kill intraleukocytic bacteria) was bactericidal for staphylococci at 5 degrees C. Thus, rifampin is not uniquely able to kill "resting" bacteria.Intraleukocytic or extraleukocytic Staphylococcus aurens were incubated with [benzyl-(14)C]penicillin for 2 h at 37 degrees C. Live intraleukocytic bacteria bound only 13% as much penicillin as live bacteria incubated with killed PMN. To measure the penetration of antibiotics into PMN, [(14)C]rifampin and [(14)C]penicillin were measured in leukocyte pellets and in the supernatant fluid. The total water space in the pellets was quantitated using tritium water and the extracellular water space was measured using Na(235)SO(4). All penicillin associated with the cell pellet could be accounted for in extracellular water. Thus penicillin was completely excluded from the leukocytes. Rifampin was concentrated in the cell pellet 2.2 times when compared with the supernatant concentration. These studies suggest that a likely explanation for the survival of phagocytized bacteria in the presence of high concentrations of most antibiotics is the inability of the antibiotic to enter the phagocyte. Rifampin, which is highly lipid soluble, can enter leukocytes and kill intracellular bacteria.

In the present article, we report on two studies performed in young human populations which tested the cognitive impact of glucocorticoids (GC) in situations of decreased or increased ratio of mineralocorticoid (MR) and glucocorticoid (GR) receptor occupation. In the first study, we used a hormone replacement protocol in which we pharmacologically decreased cortisol levels by administration of metyrapone and then restored baseline cortisol levels by a subsequent hydrocortisone replacement treatment. Memory function was tested after each pharmacological manipulation. We observed that metyrapone treatment significantly impaired delayed recall, while hydrocortisone replacement restored performance at placebo level. In the second study, we took advantage of the circadian variation of circulating levels in cortisol and tested the impact of a bolus injection of 35 mg of hydrocortisone in the late afternoon, at a time of very low cortisol concentrations. In a previous study with young normal controls, we injected a similar dose of hydrocortisone in the morning, at the time of the circadian peak, and reported detrimental effects of GC on cognitive function. Here, when we injected a similar dose of hydrocortisone in the afternoon, at the time of the circadian trough, we observed positive effects of GC on memory function. The results of these two studies provide evidence that GC are necessary for learning and memory in human populations.

1 A simple double-isotope assay for phospholipase A2 activity of perfused organs is described; Guinea-pig lungs perfused through the pulmonary circulation exhibit a low background enzyme activity. This activity is blocked by dexamethasone, betamethasone and hydrocortisone, mepacrine, procaine or chlorpromazine. Aspirin and indomethacin are without effect. 3 Mechanical trauma, antigen challenge or injections of bradykinin, rabbit aorta contracting substance-releasing factor (RCS-RF) or histamine increase "basal" phospholipase activity. The effect of these agents, except that of bradykinin, is blocked by dexamethasone or mepacrine. 4 The blocking effect of steroids is cumulative and dose-dependent. They do not work in cell-free systems. Inhibition by mepacrine is rapid and is effective in cell-free lung homogenates. 5 It is suggested that agents which liberate prostaglandin endoperoxides and thromboxane A2 from perfused lungs do so by activating phospholipase A2.

Human neutrophils rapidly undergo apoptotic cell death. Because glucocorticoids are known to modulate an array of neutrophil functional activities as well as induce rapid apoptosis in susceptible lymphocyte populations, we have examined the effects of glucocorticoids on apoptosis in mature human neutrophils. In cultures of neutrophils maintained in vitro, the glucocorticoids, dexamethasone, 6 alpha-methylprednisolone, and hydrocortisone, inhibited the development of apoptotic morphology by 59% to 90% when assessed at 12, 24, and 48 hours. In contrast, corticosteroids lacking anti-inflammatory activity and progesterone failed to affect development of the morphologic features of apoptosis. The concentration of dexamethasone required to reduce apoptosis by 50% at 24 hours was approximately 5 x 10(-8) mol/L, a concentration that is achievable in plasma after dexamethasone treatment. Dexamethasone (10(-6) mol/L), but not progesterone, reduced the percentage of hypodiploid (apoptotic) nuclei by 40% to 90% over this time course. Similarly, dexamethasone reduced the DNA cleavage associated with apoptosis and prolonged the viability of neutrophils maintained in culture for 12 to 48 hours. Glucocorticoid-mediated modulation of neutrophil apoptosis was qualitatively similar, but lesser in magnitude, when compared with the effects of granulocyte colony-stimulating factor (100 ng/mL). Thus, glucocorticoids exert a protective effect on human neutrophil survival by delaying apoptosis.

High-dose corticosteroids have been reported to reduce symptoms of acute stress and post-traumatic stress in polytrauma patients and in animal studies. The underlying mechanism of action remains largely unclear. These issues were addressed in parallel in the clinical and preclinical studies below. In this preliminary study, 25 patients with acute stress symptoms were administered a single intravenous bolus of high-dose hydrocortisone (100-140 mg) or placebo within 6 h of a traumatic event in a prospective, randomized, double-blind, placebo-controlled pilot study. Early single high-dose hydrocortisone intervention attenuated the core symptoms of both the acute stress and of subsequent PTSD in patients. High-dose hydrocortisone treatment given in the first few hours after a traumatic experience was associated with significant favorable changes in the trajectory of exposure to trauma, as expressed by the reduced risk of the development of PTSD post-trauma. In parallel, a comparative study of morphological arborization in dentate gyrus and its modulating molecules was performed in stress-exposed animals treated with high-dose hydrocortisone. Steroid-treated stressed animals displayed significantly increased dendritic growth and spine density, with increased levels of brain-derived neurotrophic factor (BDNF) and obtunded postsynaptic density-95 (PSD-95) levels. The animal study provided insights into the potential mechanism of this intervention, as it identified relevant morphological and biochemical associations to the clinical observations. Thus, evidence from clinical and animal studies suggests that there is a "window of opportunity" in the early aftermath of trauma to help those who are vulnerable to the development of chronic PTSD.