Recent reports have shown that low birth weight infants have a higher incidence of adult hypertension. These observations have stimulated a number of studies designed to evaluate the mechanisms of this phenomenon. In this study, fetal growth retardation was induced by treating pregnant rats with dexamethasone. After birth, pups whose mothers were treated with dexamethasone had a lower body and kidney weight and a lower number of glomeruli than control pups. Immunohistochemistry on treated kidneys demonstrated a marked reduction in the number of cells undergoing mitosis in the cortical nephrogenic zone. In the treated group, body and kidney weight normalized by 60 d of age, but blood pressure was significantly higher compared with controls (130+/-4 versus 107+/-1 mm Hg). In addition, GFR was significantly lower, albuminuria was higher, urinary sodium excretion rate and fractional sodium excretion were lower, and sodium tissue content was higher. In contrast, when pregnant rats were treated with a natural glucocorticoid (hydrocortisone) which is metabolized by the placenta, fetal development and adult blood pressure were normal. In conclusion, we found that high levels of maternal glucocorticoids impair renal development and lead to arterial hypertension in offspring. Even though renal mass eventually normalizes, glomerular damage as well as sodium retention occur and these factors may contribute to the development of hypertension.

In vitro blood-brain barrier (BBB) models using primary rat brain microvessel endothelial cells (BMEC) are often hampered by a lack of culture purity and poor barrier properties. To address these problems, the translation inhibitor puromycin was used to purify rat BMEC cultures. BMEC purities of 99.8% were routinely attained using puromycin treatment, and this technique proved to be far superior to other purification methods of similar difficulty. In contrast to cultures without puromycin treatment, purity of puromycin-treated cultures was unaffected by initial seeding density. Next, rat BMEC monolayer transendothelial electrical resistance (TEER) was increased by glucocorticoid treatment with either corticosterone (CORT) or hydrocortisone (HC), and a corresponding decrease in monolayer permeability to small molecules was observed. Importantly, cultures treated with both puromycin and glucocorticoid attained significantly higher TEER values (CORT 168 +/- 13 Omega x cm2; HC 218 +/- 66 Omega x cm2) than those treated by the glucocorticoid alone (CORT 57 +/- 5 Omega x cm2; HC 70 +/- 2 Omega x cm2). Glucocorticoid induction resulted in BMEC morphological changes that accompanied the increases in TEER, and BMEC tight junctions exhibited improved integrity as visualized by the localization of tight junction proteins zonula occluden-1, occludin and claudin-5. The combined use of puromycin and glucocorticoid therefore provides an in vitro system that is well suited for molecular level BBB investigations.

Triamcinolone acetonide, triamcinolone hexacetonide, and a combination of betamethasone phosphate and acetate were given intra-articularly in different doses. Plasma levels of the steroids were measured and pharmacokinetic parameters were calculated. As a pharmacodynamic parameter for systemic steroid activity, plasma hydrocortisone levels were monitored for 3 weeks. Results indicate complete absorption from the site of injection over a period of 2 to 3 weeks. Because of its lower solubility, triamcinolone hexacetonide is absorbed slower than triamcinolone acetonide, thus maintaining synovial levels for a longer time and creating lower systemic corticoid levels. Endogenous hydrocortisone suppression correlated with exogenous steroid levels. Threshold concentrations for maximum suppression were determined.

OBJECTIVE

Despite their controversial role, corticosteroids (CS) are frequently administered to patients with H1N1 virus infection with severe respiratory failure secondary to viral pneumonia. We hypothesized that invasive pulmonary aspergillosis (IPA) is a frequent complication in critically ill patients with H1N1 virus infection and that CS may contribute to this complication.

METHODS

We retrospectively selected all adult patients with confirmed H1N1 virus infection admitted to the intensive care unit (ICU) of two tertiary care hospitals from September 2009 to March 2011. Differences in baseline factors, risk factors, and outcome parameters were studied between patients with and without IPA.

RESULTS

Of 40 critically ill patients with confirmed H1N1, 9 (23 %) developed IPA 3 days after ICU admission. Five patients had proven and four had probable IPA. Significantly more IPA patients received CS within 7 days before ICU admission (78 versus 23 %, p = 0.002). IPA patients also received significantly higher doses of CS before ICU admission [hydrocortisone equivalent 800 (360-2,635) versus 0 (0-0) mg, p = 0.005]. On multivariate analysis, use of CS before ICU admission was independently associated with IPA [odds ratio (OR) 14.4 (2.0-101.6), p = 0.007].

CONCLUSIONS

IPA was diagnosed in 23 % of critically ill patients with H1N1 virus infection after a median of 3 days after ICU admission. Our data suggest that use of CS 7 days before ICU admission is an independent risk factor for fungal superinfection. These findings may have consequences for clinical practice as they point out the need for increased awareness of IPA, especially in those critically ill H1N1 patients already receiving CS.

During the course of differentiation of preadipocytes into adipocytes, several differentiation-linked genes are activated synchronously with morphological changes. To follow this process we have used 3T3-F442A cells, known to undergo adipocyte conversion with high frequency. Accumulation of lipid droplets in the cytoplasm constitutes an easily visualized sign of the terminally differentiated phenotype. In this report we demonstrate that expression of the CCAAT/enhancer binding protein (C/EBP) is an important factor in determining the ability to accumulate lipid droplets in terminally differentiated adipocytes. In one experiment we can suppress C/EBP expression through administration of hydrocortisone to differentiating 3T3-F442A cells, which is accompanied by an inability of the cells to accumulate lipid. In another experiment a C/EBP antisense expression vector has been stably introduced into 3T3-F442A cells and as compared with control cells, a 62% decrease of C/EBP mRNA (p less than 0.01) is demonstrated. This decrease of C/EBP mRNA is accompanied by a change in cellular morphology characterized by a reduced ability to form lipid droplets. We can also demonstrate a correlation between the degree of reduction of C/EBP mRNA and the amount of lipid present in the cells. These findings strongly support the view that C/EBP is a necessary component of terminal adipocyte differentiation.

Genetic factors are known to strongly influence susceptibility to allergic inflammation. The Th2 cytokine IL-13 is a central mediator of allergy and asthma, and common single-nucleotide polymorphisms in IL13 are associated with allergic phenotypes in several ethnically diverse populations. In particular, IL13+2044GA is expected to result in the nonconservative replacement of arginine 130 (R130) with glutamine (Q). We examined the impact of IL13+2044GA on the functional properties of IL-13 by directly comparing the activity of WT IL-13 and IL-13 R130Q on primary human cells involved in the effector mechanisms of allergic inflammation. Our results show that IL-13 R130Q was significantly more active than WT IL-13 in inducing STAT6 phosphorylation and CD23 expression in monocytes and hydrocortisone-dependent IgE switching in B cells. Notably, IL-13 R130Q was neutralized less effectively than WT IL-13 by an IL-13R2 decoy. Decreased neutralization of the minor variant could contribute to its enhanced in vivo activity. Neither IL-13 variant was able to engage T cells, which suggests that increased allergic inflammation in carriers of IL13+2044A depends on enhanced IL-13-mediated Th2 effector functions rather than increased Th2 differentiation. Collectively, our data indicate that natural variation in the coding region of IL13 may be an important genetic determinant of susceptibility to allergy.

Glucocorticoids (GCs) are administered to human fetuses at risk of premature delivery and to infants with life-threatening respiratory and cardiac conditions. However, there are ongoing concerns about adverse effects of GC treatment on the developing human brain, although the precise molecular mechanisms underlying GC-induced brain injury are unclear. Here, we identified what we believe to be novel cross-antagonistic interactions of Sonic hedgehog (Shh) and GC signaling in proliferating mouse cerebellar granule neuron precursors (CGNPs). Chronic GC treatment (from P0 through P7) in mouse pups inhibited Shh-induced proliferation and upregulation of expression of N-myc, Gli1, and D-type cyclin protein in CGNPs. Conversely, acute GC treatment (on P7 only) caused transient apoptosis. Shh signaling antagonized these effects of GCs, in part by induction of 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2). Importantly, 11betaHSD2 antagonized the effects of the GCs corticosterone, hydrocortisone, and prednisolone, but not the synthetic GC dexamethasone. Our findings indicate that Shh signaling is protective in the setting of GC-induced mouse neonatal brain injury. Furthermore, they led us to propose that 11betaHSD2-sensitive GCs (e.g., hydrocortisone) should be used in preference to dexamethasone in neonatal human infants because of the potential for reduced neurotoxicity.

Classically, osteoarthritis (OA) has been considered a noninflammatory disease. However, the detection of selected inflammatory mediators in osteoarthritic fluid, in the absence of significant inflammatory cell infiltrate, is increasingly appreciated. We sought to identify the inflammatory component in human OA-affected cartilage that may be involved in cartilage damage/destruction. Using Western blot analysis and an antibody to the conserved region of nitric oxide synthase (NOS), we have observed up-regulation of NOS, one of the "key players" of inflammation, in chondrocytes of OA-affected patients. Remarkably, none of the cartilage samples examined from normal joints demonstrated detectable amounts of this NOS. Western blot analysis using the same alpha-NOS antibody indicated that this NOS from OA-affected cartilage (OA-NOS) was larger in size than (and distinct from) transfected human hepatocyte or murine inducible NOS (iNOS) (150 versus 133 kD) and similar in size to neuronal constitutive NOS (ncNOS). Antibodies specific for iNOS showed binding to murine and human iNOS but not to OA-NOS, endothelial constitutive NOS, or ncNOS. Antibodies specific for ncNOS bound to ncNOS and also to OA-NOS, but not to murine or human iNOS or endothelial constitutive NOS. Incubation of OA cartilage in serum-free medium resulted in spontaneous release, for up to 72 h, of substantial amounts of nitrite (up to approximately 80 microM/100 mg wet tissue), which could be inhibited by at least 80% with various inhibitors of iNOS, including inhibitors of protein synthesis and transcription factor NF-kappa B, but which (unlike murine macrophage iNOS) was not sensitive to hydrocortisone or TGF-beta. Exposure of OA-affected cartilage to interleukin 1 beta, tumor necrosis factor-alpha, and lipopolysaccharide resulted in approximately 20-50% augmentation of nitrite accumulation, which was also sensitive to cycloheximide and pyrrolidine dithiocarbamate. Hence, our data indicate that OA-NOS (based on immunoreactivity and molecular weight) is similar to ncNOS and that it releases nitric oxide, which may contribute to the inflammation and pathogenesis of cartilage destruction in OA.

A quantitative in vitro assay was employed to directly assess the effect of corticosteroids on the IgG and complement receptor function of human mononuclear phagocytic cells. In this system corticosteroids were solubilized with cholesterol-phospholipid sonicated dispersions before exposure to mononuclear cells. Solubilized corticosteroids at concentrations between 10(-4) and 10(-3) M inhibited both IgG and complement receptor activity in a dose-response fashion. Inhibition was dependent upon the time of interaction of the mononuclear cells with corticosteroids and was half-maximal by 15 min. The inhibitory effect at all concentrations of hydrocortisone was partially overcome by increasing the number of IgG molecules per erythrocyte. Hydrocortisone also inhibited the binding of erythrocytes coated with both IgG and C3, despite the fact that when both were on the erythrocyte surface a synergistic effect on binding to mononuclear cells was observed. At the steroid concentrations employed, the capacity of mononuclear cells to exclude trypan blue and to take up latex particles and neutral red was unaffected. Mineralocorticoids also inhibited receptor activity, but the sex hormones were less effective. These studies demonstrate an effect of steroid hormones on cell membrane receptor function, and they suggest that an inhibition of the recognition system for IgG and C3 in vivo may explain, in part, the effect of corticosteroids in man.

Increased afterload results in 'pathological' cardiac hypertrophy, the most important risk factor for the development of heart failure. Current in vitro models fall short in deciphering the mechanisms of hypertrophy induced by afterload enhancement. The aim of this study was to develop an experimental model that allows investigating the impact of afterload enhancement (AE) on work-performing heart muscles in vitro. Fibrin-based engineered heart tissue (EHT) was cast between two hollow elastic silicone posts in a 24-well cell culture format. After 2 weeks, the posts were reinforced with metal braces, which markedly increased afterload of the spontaneously beating EHTs. Serum-free, triiodothyronine-, and hydrocortisone-supplemented medium conditions were established to prevent undefined serum effects. Control EHTs were handled identically without reinforcement. Endothelin-1 (ET-1)- or phenylephrine (PE)-stimulated EHTs served as positive control for hypertrophy. Cardiomyocytes in EHTs enlarged by 28.4 % under AE and to a similar extent by ET-1- or PE-stimulation (40.6 or 23.6 %), as determined by dystrophin staining. Cardiomyocyte hypertrophy was accompanied by activation of the fetal gene program, increased glucose consumption, and increased mRNA levels and extracellular deposition of collagen-1. Importantly, afterload-enhanced EHTs exhibited reduced contractile force and impaired diastolic relaxation directly after release of the metal braces. These deleterious effects of afterload enhancement were preventable by endothelin-A, but not endothelin-B receptor blockade. Sustained afterload enhancement of EHTs alone is sufficient to induce pathological cardiac remodeling with reduced contractile function and increased glucose consumption. The model will be useful to investigate novel therapeutic approaches in a simple and fast manner.

To determine whether elevations of cortisol levels have more pronounced effects on glucose levels and insulin secretion in the evening (at the trough of the daily rhythm) or in the morning (at the peak of the rhythm), nine normal men each participated in four studies performed in random order. In all studies, endogenous cortisol levels were suppressed by metyrapone administration, and caloric intake was exclusively under the form of a constant glucose infusion. The daily cortisol elevation was restored by administration of hydrocortisone (or placebo) either at 0500 h or at 1700 h. In each study, plasma levels of glucose, insulin, C-peptide, and cortisol were measured at 20-min intervals for 32 h. The initial effect of the hydrocortisone-induced cortisol pulse was a short-term inhibition of insulin secretion without concomitant glucose changes and was similar in the evening and in the morning. At both times of day, starting 4-6 h after hydrocortisone ingestion, glucose levels increased and remained higher than under placebo for at least 12 h. This delayed hyperglycemic effect was minimal in the morning but much more pronounced in the evening, when it was associated with robust increases in serum insulin and insulin secretion and with a 30% decrease in insulin clearance. Thus, elevations of evening cortisol levels could contribute to alterations in glucose tolerance, insulin sensitivity, and insulin secretion.

OBJECTIVE

To investigate the effect of prolonged glucocorticoid treatment for patients with acute respiratory distress syndrome (ARDS).

METHODS

We conducted two sets of intention-to-treat analyses: (1) a primary analysis of individual patients' data (IPD) of four randomized controlled trials (RCTs) which investigated methylprednisolone treatment (n = 322) and (2) a trial-level meta-analysis incorporating four additional RCTs which investigated hydrocortisone treatment in early ARDS (n = 297). We standardized definitions to derive outcomes in all datasets. The primary outcome for the IPD analysis was time to achieving unassisted breathing (UAB) by study day 28. Secondary outcomes included mechanical ventilation (MV) and intensive care unit (ICU)-free days, hospital mortality, and time to hospital mortality by day 28.

RESULTS

By study day 28, compared to the placebo group, the methylprednisolone group had fewer patients who died before achieving UAB (12 vs. 29 %; p < 0.001) and more patients who achieved UAB (80 vs. 50 %; p < 0.001). In the methylprednisolone group, time to achieving UAB was shorter [hazard ratio 2.59, 95 % confidence interval (CI) 1.95-3.43; p < 0.001], and hospital mortality was decreased (20 vs. 33 %; p = 0.006), leading to increased MV (13.3 ± 11.8 vs. 7.6 ± 5.7; p < 0.001) and ICU-free days (10.8 ± 0.71 vs. 6.4 ± 0.85; p < 0.001). In those patients randomized before day 14 of ARDS onset, the trial-level meta-analysis indicated decreased hospital mortality (36 vs. 49 %; risk ratio 0.76, 95 % CI 0.59-0.98, I (2) = 17 %, p = 0.035; moderate certainty). Treatment was not associated with increased risk for infections (risk ratio 0.77, 95 % CI 0.56-1.08, I (2) = 26 %; p = 0.13; moderate certainty).

CONCLUSIONS

Prolonged methylprednisolone treatment accelerates the resolution of ARDS, improving a broad spectrum of interrelated clinical outcomes and decreasing hospital mortality and healthcare utilization.

The process by which various anions, including bilirubin and several dyes, drugs, hormones and their metabolites, are transferred from plasma into the liver cell is poorly understood. Two hepatic cytoplasmic proteins, Y and Z, that bind various organic anions in vivo and in vitro have been postulated to be involved in this process. The concentration of Y, the major organic anion-binding protein, increases in rat liver after administration of phenobarbital in association with enhanced organic anion transfer from plasma into liver as determined by initial plasma disappearance rate (K(1)) and hepatic dye content for sulfobromophthalein (BSP) and indocyanine green (ICG), as well as increased relative hepatic storage of BSP. Acute bile duct ligation failed to alter plasma disappearance or hepatic content of BSP in normal or phenobarbital-treated rats. Other drugs and chemicals which cause proliferation of hepatic smooth endoplasmic reticulum and enhancement of drug metabolism, such as allylisopropylacetamide, dieldrin, DDT, 3-methylcholanthrene, and benzpyrene increased Y and BSP K(1) and, where studied, hepatic BSP content. Alcohol feeding had no effect on Y, Z, or K(1) for BSP. Hypophysectomy and thyroidectomy increased Y but decreased K(1) and, where studied, hepatic content of BSP. Of several hormones studied, only thyroxine restored Y and K(1) to normal in hypophysectomized or thyroidectomized rats. Mice with congenital pituitary insufficiency also manifested increased Y which returned to normal after thyroxine administration. In hormone-deficient rats and mice, phenobarbital administration produced a further increase in Y suggesting that different mechanisms may be responsible for the change in Y resulting from drug administration and hormonal deprivation. Thyroxine, testosterone, or hydrocortisone did not alter BSP K(1) or Y in normal rats.Cholestasis produced by ethinyl estradiol administration or biliary obstruction reduced Y, Z, BSP K(1) and hepatic BSP content. These results support the hypothesis that Y and Z are involved in the transfer of BSP, ICG, and possibly other organic anions from plasma into the liver. The concentration of Y increased after administration of various drugs and chemicals as well as in thyroid deficiency. Thyroid hormone appears to be important in regulation of the intracellular concentration of Y. Because thyroid deficiency increased Y but decreased BSP K(1) and hepatic BSP content, other factors beside Y and Z influence hepatic organic anion uptake.

The genomic gene coding for the human beta 2-adrenergic receptor (beta 2AR) from A431 epidermoid cells has been isolated. Transfection of the gene into eukaryotic cells restores a fully active receptor/GTP-binding protein/adenylate cyclase complex with beta 2AR properties. Southern blot analyses with beta 2AR-specific probes show that a single beta 2AR gene is common to various human tissues and that its flanking sequences are highly conserved among humans and between man and rabbit, mouse, and hamster. Functional significance of these regions is supported by the presence of a promoter region (including mRNA cap sites, two "TATA boxes," a "CAAT box," and three G + C-rich regions that resemble binding sites for transcription factor Sp1) 200-300 base pairs 5' to the translation initiation codon. In the 3' flanking region, sequences homologous to glucocorticoid-response elements might be responsible for the increased expression of the beta 2AR gene observed after treatment of the transfected cells with hydrocortisone. In addition, 5' to the promoter region, an open reading frame encodes a 251-residue polypeptide that displays striking homologies with protein kinases and other nucleotide-binding proteins.

Cortisol's effects on lipid metabolism are controversial and may involve stimulation of both lipolysis and lipogenesis. This study was undertaken to define the role of physiological hypercortisolemia on systemic and regional lipolysis in humans. We investigated seven healthy young male volunteers after an overnight fast on two occasions by means of microdialysis and palmitate turnover in a placebo-controlled manner with a pancreatic pituitary clamp involving inhibition with somatostatin and substitution of growth hormone, glucagon, and insulin at basal levels. Hydrocortisone infusion increased circulating concentrations of cortisol (888 +/- 12 vs. 245 +/- 7 nmol/l). Interstitial glycerol concentrations rose in parallel in abdominal (327 +/- 35 vs. 156 +/- 30 micromol/l; P = 0.05) and femoral (178 +/- 28 vs. 91 +/- 22 micromol/l; P = 0.02) adipose tissue. Systemic [(3)H]palmitate turnover increased (165 +/- 17 vs. 92 +/- 24 micromol/min; P = 0.01). Levels of insulin, glucagon, and growth hormone were comparable. In conclusion, the present study unmistakably shows that cortisol in physiological concentrations is a potent stimulus of lipolysis and that this effect prevails equally in both femoral and abdominal adipose tissue.

OBJECTIVE

During the 2009 H1N1 influenza A virus pandemic, a minority of patients developed rapidly progressive pneumonia leading to acute lung injury (ALI)-acute respiratory distress syndrome (ARDS). A recent meta-analysis provides support for prolonged corticosteroid treatment in ALI-ARDS. We prospectively evaluated the response to oseltamivir and prolonged corticosteroid treatment in patients with ALI-ARDS and suspected H1N1 influenza.

METHODS

From June 24 through 12 July 2009, 13 patients with suspected H1N1 pneumonia and ALI-ARDS were admitted to the intensive care unit (ICU) of a tertiary care hospital. H1N1 influenza was confirmed with real-time reverse transcriptase-polymerase chain reaction assay in eight patients. Oseltamivir and corticosteroid treatment were initiated concomitantly at ICU admission; those with severe ARDS received methylprednisolone (1 mg/kg/day), and others received hydrocortisone (300 mg/day) for a duration of 21 +/- 6 days.

RESULTS

Patients with and without confirmed H1N1 influenza had similar disease severity at presentation and a comparable response to treatment. By day 7 of treatment, patients experienced a significant improvement in lung injury and multiple organ dysfunction scores (P < 0.001). Twelve patients (92%) improved lung function, were extubated, and discharged alive from the ICU. Hospital length of stay and mortality were 18.7 +/- 9.6 days and 15%, respectively. Survivors were discharged home without oxygen supplementation.

CONCLUSIONS

In ARDS patients, with and without confirmed H1N1 influenza, prolonged low-to-moderate dose corticosteroid treatment was well tolerated and associated with significant improvement in lung injury and multiple organ dysfunction scores and a low hospital mortality. These findings provide the rationale for developing a randomized trial.

BACKGROUND

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome that remains difficult to treat. Even with current standard HLH therapy, only approximately half of patients will experience complete resolution of disease, and early mortality remains a significant problem. Salvage therapies have been described only in limited case reports, and there are no large studies of second-line therapies.

METHODS

We reviewed the charts of 22 pediatric and adult patients who received alemtuzumab for the treatment of refractory HLH at our center or in consultation with our group.

RESULTS

Patients had received conventional therapies for a median of 8 weeks (range: 2-70) prior to alemtuzumab, and treatment immediately prior to alemtuzumab included dexamethasone (100%), etoposide (77%), cyclosporine (36%), intrathecal hydrocortisone ± methotrexate (23%), methylprednisolone (9%), and rituximab (14%). Patients received a median dose of 1 mg/kg alemtuzumab (range: 0.1-8.9 mg/kg) divided over a median of 4 days (range: 2-10). Fourteen patients experienced an overall partial response, defined as at least a 25% improvement in two or more quantifiable symptoms or laboratory markers of HLH 2 weeks following alemtuzumab (64%). Five additional patients had a 25% or greater improvement in a single quantifiable symptom or laboratory marker of HLH (23%). Seventy-seven percent of patients survived to undergo allogeneic hematopoietic cell transplantation. Patients experienced an acceptable spectrum of complications, including CMV and adenovirus viremia.

CONCLUSIONS

Alemtuzumab appears to be an effective salvage agent for refractory HLH, leading to improvement and survival to HCT in many patients. Prospective trials to define optimal dosing levels, schedules, and responses are needed.

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow's udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.

Epidemiological studies have shown not only a relationship between the intake of dietary lipids and an increased risk of developing metastatic prostate cancer, but also the type of lipid intake that influences the risk of metastatic prostate cancer. The Omega-6 poly-unsaturated fatty acid, Arachidonic acid, has been shown to enhance the proliferation of malignant prostate epithelial cells and increase the risk of advanced prostate cancer. However, its role in potentiating the migration of cancer cells is unknown. Here we show that arachidonic acid at concentrations <or=5 microM is a potent stimulator of malignant epithelial cellular invasion, which is able to restore invasion toward <em>hydrocortisone</em>-deprived adipocyte-free human bone marrow stroma completely. This observed invasion is mediated by the arachidonic acid metabolite prostaglandin E2 and is inhibited by the Omega-3 poly-unsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid at a ratio of 1 : 2 Omega-3 : Omega-6, and by the COX-2 inhibitor NS-398. These results identify a mechanism by which arachidonic acid may potentiate the risk of metastatic migration and secondary implantation in vivo, a risk which can be reduced with the uptake of Omega-3 poly-unsaturated fatty acids.

The effect of glucocorticoids on adipose tissue lipolysis in animals and humans is controversial. To determine whether a physiological increase in plasma cortisol, similar to that observed in diabetic ketoacidosis and other stress conditions, stimulates lipolysis, palmitate kinetics were measured in seven nondiabetic volunteers on two occasions with [1-14C]palmitate as a tracer. Subjects received a 6-h infusion of either 2 micrograms.kg-1.min-1 hydrocortisone or saline in random order. On both occasions, a pancreatic clamp (0.12 micrograms.kg-1.min-1 somatostatin, 0.05 mU.kg-1.min-1 insulin, and 3 ng.kg-1.min-1 growth hormone) was used to maintain plasma hormone concentrations at desired levels. Plasma cortisol concentrations increased to approximately 970 nM during cortisol infusion. Palmitate rate of appearance (Ra) and concentration increased by approximately 60% during cortisol infusion but did not change during saline infusion. Increments in palmitate Ra and concentration over the 6-h study were significantly greater during cortisol than saline infusion when compared by area-under-the-curve analysis (152 +/- 52 vs. -48 +/- 23 mumol.kg-1 and 12.2 +/- 4.1 vs. -4.9 +/- 4.1 mmol.min-1.L-1, respectively; P less than 0.02). Plasma glucose concentrations did not change significantly during cortisol (5.0 +/- 0.3 vs. 6.1 +/- 0.6 mM, NS) or saline (4.9 +/- 0.2 vs. 4.9 +/- 0.1 mM, NS) infusion. In nondiabetic volunteers, a 6-h cortisol infusion was associated with a 60% increase in palmitate Ra that did not occur with saline infusion. Thus, physiological hypercortisolemia may contribute to the increased rates of lipolysis observed in humans during stress.

OBJECTIVE

To investigate the relationship between adrenal gland function and pressor response to noradrenaline in septic shock.

METHODS

Basal cortisol level, noradrenaline--mean arterial pressure dose-response curve and cortisol response to intravenous corticotrophin bolus were obtained in nine patients fulfilling usual criteria for septic shock and in six normal volunteers. In patients with septic shock, dose-response curve to noradrenaline was determined a second time 60 min after a 50 mg intravenous hydrocortisone bolus.

RESULTS

As compared with controls, patients with septic shock had increased basal cortisol levels (mean+/-s.d.: 1564+/-818 vs 378+/-104 nmol l(-1) , P=0.002, 95% confidence interval for difference in means: [452, 1920]) and a blunted cortisol response to corticotrophin (403+/-461 vs 1132+/-195 nmol l(-1), P=0.008, [-1163, -2951). Five patients had impaired adrenal function reserve. As compared with controls, septic patients displayed a moderate and non significant decrease in pressor sensitivity to noradrenaline (P=0.112). As compared with patients with adequate adrenal response, patients with impaired adrenal function reserve showed a significant decrease in pressor sensitivity to noradrenaline (P=0.038). In septic patients, hydrocortisone improved pressor response to noradrenaline (P=0.032). This effect was more marked in patients with impaired adrenal function reserve so that, as compared with patients with adequate response, the difference was no longer significant (P=0.123).

CONCLUSIONS

In septic shock, impaired adrenal function reserve may partly be accounted for by the depressed pressor sensitivity to noradrenaline. The latter may be substantially improved by physiological doses of hydrocortisone.

Patients with rare defects in the gene encoding proopiomelanocortin (POMC) have extreme early-onset obesity, hyperphagia, hypopigmentation, and hypocortisolism, resulting from the lack of the proopiomelanocortin-derived peptides melanocyte-stimulating hormone and corticotropin. In such patients, adrenal insufficiency must be treated with hydrocortisone early in life. No effective pharmacologic treatments have been available for the hyperphagia and obesity that characterize the condition. In this investigator-initiated, open-label study, two patients with proopiomelanocortin deficiency were treated with setmelanotide, a new melanocortin-4 receptor agonist. The patients had a sustainable reduction in hunger and substantial weight loss (51.0 kg after 42 weeks in Patient 1 and 20.5 kg after 12 weeks in Patient 2).

The present review catalogues 1418 reported cases of drug-induced hypoglycemia. The main findings are that sulfonylureas (especially chlorpropamide and glyburide), either alone or with a second hypoglycemic or potentiating agent, still account for 63% of all cases; that alcohol, propranolol, and salicylate, either singly or with another hypoglycemic drug, are the next most frequent offenders (19% of the total); and that one older drug (quinine) and three new ones (pentamidine, ritodrine, and disopyramide) have caused an additional 7% of all episodes of severe hypoglycemia. The clinical factors that set the stage for drug-induced hypoglycemia are still restricted food intake, age, hepatic disease, and renal disease, both individually and even more so in combination. Drug-induced hypoglycemia continues to be so common that virtually every unconscious patient should be considered hypoglycemic until immediate estimation of the blood sugar level rules it in or out. If ruled in, the clinician should promptly start 10% intravenous glucose and plan to maintain it uninterruptedly for 1 or more days, with added glucagon, hydrocortisone, and diazoxide administration if necessary, until sustained hyperglycemia guarantees that all drug effects have worn off.

The response to a beta2-agonist (salbutamol) was assessed by measuring specific airways conductance (sGaw) in healthy volunteers following increasing doses of inhaled or intravenous salbutamol. Regular inhaled salbutamol in doses exceeding 200 microgram four times a day produced a progressive loss of airways responsiveness to both inhaled and intravenous salbutamol (beta-agonist resistance). In salbutamol-resistant subjects full bronchodilator sensitivity was restored 3-5 h after intravenous hydrocortisone.