Loss of BRCA1 tumor suppressor function is a critical event in breast tumorigenesis. We have previously identified the stress hormone hydrocortisone as a negative regulator of BRCA1 expression in nonmalignant mammary cells. Here, we have identified a direct role for the unliganded glucocorticoid receptor (GR) in BRCA1 upregulation in the absence of hydrocortisone. The positive regulatory effect of GR is lost upon the addition of hydrocortisone. We have shown that GR interacts with the BRCA1 promoter only in the absence of hydrocortisone, and that this interaction is mediated through the β-subunit of the ets transcription factor GA-binding protein (GABP) at the RIBS promoter element. GR and GABPβ interact in both coimmunoprecipitation and mammalian two-hybrid assays, and this interaction involves the N-terminal to central regions of both proteins. This work presents the first evidence of a ligand-independent role for GR as a positive regulator of gene expression, and loss of GR from the BRCA1 promoter in response to stress hormones leads to decreased BRCA1 expression. Because low levels of BRCA1 have been implicated in the development of sporadic breast cancer, this may represent a novel mechanism through which prolonged stress signaling increases breast cancer risk.

OBJECTIVE

Aldosterone is well recognized as the selective physiological ligand for mineralocorticoid receptor in epithelia. However, in-vitro studies have demonstrated that the affinity of aldosterone and glucocorticoids for mineralocorticoid receptor is similar. We hypothesized that glucocorticoids are involved in the development of renal injury through an mineralocorticoid receptor-dependent mechanism.

RESULTS

Uninephrectomized (UNX) rats were treated with 1% NaCl and divided into three groups: vehicle, bilateral adrenalectomy (ADX) + hydrocortisone (HYDRO; 5 mg/kg/day, s.c.), ADX + HYDRO + eplerenone (0.125% in chow). HYDRO-treated UNX-ADX rats showed increased blood pressure and urinary albumin-to-creatinine ratio with an increase in the expression of the mineralocorticoid receptor target genes, serum and glucocorticoid-regulated kinases-1 and Na+/H+ exchanger isoform-1, in renal tissues. HYDRO treatment induced morphological changes in the kidney, including glomerulosclerosis and podocyte injury. Treatment with eplerenone markedly decreased the gene expression and reduced the albuminuria and renal morphological changes. In contrast, dexamethasone (0.2 mg/kg per day, s.c.) + UNX + ADX induced hypertension and albuminuria in different groups of rats. Eplerenone failed to ameliorate these changes.

CONCLUSIONS

Our findings indicate that chronic glucocorticoid excess could activate mineralocorticoid receptor and, in turn, induce the development of renal injury.

The combination of thiamine, ascorbic acid, and hydrocortisone has recently emerged as a potential adjunctive therapy to antibiotics, infectious source control, and supportive care for patients with sepsis and septic shock. In the present manuscript, we provide a comprehensive review of the pathophysiologic basis and supporting research for each element of the thiamine, ascorbic acid, and hydrocortisone drug combination in sepsis. In addition, we describe potential areas of synergy between these therapies and discuss the strengths/weaknesses of the two studies to date which have evaluated the drug combination in patients with severe infection. Finally, we describe the current state of current clinical practice as it relates to the thiamine, ascorbic acid, and hydrocortisone combination and present an overview of the randomized, placebo-controlled, multi-center Ascorbic acid, Corticosteroids, and Thiamine in Sepsis (ACTS) trial and other planned/ongoing randomized clinical trials.

BACKGROUND

The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro.

METHODS

Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture.

RESULTS

The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n=60. Furthermore, they were capable of synthesizing beta-casein (CSN2), acetyl-CoA carboxylase-alpha (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line.

CONCLUSIONS

The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs).

BACKGROUND

Current treatment guidelines generally suggest using lower and weight-adjusted glucocorticoid replacement doses in patients with insufficiency of the hypothalamic-pituitary-adrenal (HPA) axis. Although data in patients with acromegaly revealed a positive association between glucocorticoid dose and mortality, no comparable results exist in patients with nonfunctioning pituitary adenomas (NFPA).

OBJECTIVE

Our objective was to assess whether higher glucocorticoid replacement doses are associated with increased mortality in patients with NFPA and HPA axis insufficiency.

METHODS

We included 105 patients receiving glucocorticoid replacement after pituitary surgery due to NFPA and concomitant HPA axis insufficiency. Patients were grouped according weight-adapted and absolute hydrocortisone (HC) replacement doses. Mortality was assessed using Kaplan-Meier methodology as well as multivariable Cox regression models.

METHODS

This was a retrospective analysis conducted at a tertiary referral center.

RESULTS

We evaluated overall mortality based on HC replacement doses.

RESULTS

Average age at inclusion was 58.9±14.8 yr, and mean follow-up was 12.7±9.4 yr. The groups did not differ according to age, follow-up time, pattern of hypopituitarism, and comorbidities. Kaplan-Meier survival probabilities differed significantly when comparing individuals with differing weight-adjusted HC dose (P=0.001) as well as absolute HC dose (5-19, 20-29, and ≥30 mg, P=0.009). Hazard ratios for mortality increased from 1 (0.05-0.24 mg/kg) to 2.62 (0.25-0.34 mg/kg) to 4.56 (≥0.35 mg/kg, P for trend=0.006) and from 1 (5-19 mg) to 2.03 (20-29 mg) to 4 (≥30 mg, P for trend=0.029), respectively.

CONCLUSIONS

Higher glucocorticoid replacement doses are associated with increased overall mortality in patients with NFPA and insufficiency of HPA axis. This further substantiates the importance of a balanced and adjusted glucocorticoid replacement therapy in these patients.

OBJECTIVE

To determine whether glycemic control is less feasible when hydrocortisone is given as a bolus compared with continuous application in septic shock patients.

METHODS

Observational prospective pilot study.

METHODS

Fourteen-bed surgical university hospital ICU.

METHODS

Sixteen consecutive patients with septic shock receiving a continuous infusion of 200 mg hydrocortisone/day and an infusion regime of insulin keeping blood glucose below 150 mg/dl.

METHODS

Blood glucose and insulin infusion were adjusted to steady state before intervention. At baseline, the continuous hydrocortisone infusion was replaced with a single bolus of 50 mg hydrocortisone. During a subsequent 6-h period, blood glucose was monitored hourly and insulin infusion was kept constant. Afterwards, hydrocortisone application and adjustment of blood glucose was resumed according to standard treatment.

RESULTS

Mean blood glucose in steady state at baseline immediately prior to intervention was 128 mg/dl (range 114-141 mg/dl; 95% confidence interval). After bolus injection of hydrocortisone, blood glucose increased significantly within 6 h with peak levels of 154 mg/dl (range 132-178 mg/dl; p<0.01). Blood glucose returned to baseline with restoration of continuous hydrocortisone infusion. There were marked inter-individual variations with peak glucose values up to 254 mg/dl, but no significant difference in intra-individual glucose variability before and after bolus injection of hydrocortisone.

CONCLUSIONS

Bolus injections of hydrocortisone may induce significant increases of blood glucose in patients with septic shock. The individual response is highly variable and we speculate that repetitive boluses would induce marked undulation of blood glucose. In terms of glycemic-control strategies, a continuous infusion of hydrocortisone seems to be preferable.

BACKGROUND

The diagnosis and management of hypotension in the very low birth weight (VLBW) is a controversial area.

OBJECTIVE

To establish if there is any consensus in the diagnostic criteria and therapeutic interventions in the hypotensive VLBW among neonatologists in Canada.

METHODS

A postal questionnaire was sent to neonatologists in all level II and III neonatal intensive care units throughout Canada.

RESULTS

In total, 120 questionnaires were sent. Ninety-five completed questionnaires were returned. Seventy-six percent of respondents work in units where at least 50 VLBWs and 43% where at least 100 VLBWs are cared for annually. Fifty-seven percent of the respondents have at least 10 years experience as practicing neonatologists. 25.8% rely on blood pressure values alone when intervening, the most common being a blood pressure less than gestational age in weeks. Ninety-seven percent of respondents commence therapy with a fluid bolus. Normal saline is the predominant volume administered (95%). Dopamine remains the pressor of choice. Great variation exists in starting doses and total amount administered. Similar variation exists with epinephrine, with tenfold differences in starting doses (0.01-0.1 mcg/kg/min) and tenfold differences in maximum dose (0.4-4 mc/kg/min) administered. Steroid doses used ranged from 0.1 mg/kg/dose of hydrocortisone to 5 mg/kg/dose. Bicarbonate is rarely used. Three predominant therapeutic regimes exist. These include (i) volume followed by dopamine then a steroid (32%), (ii) volume, dopamine, dobutamine (29%), (iii) volume, dopamine, epinephrine (22%).

CONCLUSIONS

This is the first large study of practices among neonatologists addressing hypotension in the VLBW infant. There is wide variation in practice, which is a reflection of the lack of good evidence currently available for this very common problem.

BACKGROUND

Elevated levels of proinflammatory cytokines, especially interleukin-6 (IL-6), can mediate the greater risk for cardiovascular disease in individuals with posttraumatic stress disorder (PTSD), particularly in those with comorbid major depressive disorder (MDD). However, IL-6 levels are not consistently elevated in either PTSD or MDD. Although PTSD is associated with supersensitivity to glucocorticoids; prior studies have not evaluated the effect of comorbid MDD.

METHODS

Serum IL-6 levels were measured hourly between 7:00 pm and 7:00 am in individuals with PTSD with comorbid MDD (PTSD + MDD) (n = 9) and compared with those with PTSD without MDD (PTSD - MDD) (n = 9) and nontraumatized healthy control subjects (n = 14). Group differences in serum IL-6, plasma adrenocorticotropic hormone (ACTH), and plasma cortisol response to 30 mg of intravenous hydrocortisone were evaluated using linear mixed models.

RESULTS

Only subjects with PTSD + MDD exhibited higher, overnight serum IL-6 levels compared with individuals with PTSD - MDD (p < .01) and healthy control subjects (p < .001). Peak overnight IL-6 levels positively correlated with severity of PTSD (r = .56, p < .01) and depressive symptoms (r = .54, p < .01). Hydrocortisone administration significantly reduced IL-6 levels in both PTSD groups; however, IL-6 levels in PTSD + MDD were higher than both PTSD - MDD (p < .05) and healthy control subjects (p < .01). Following hydrocortisone administration, there was a greater reduction in levels of ACTH in PTSD - MDD compared with control subjects (p < .01).

CONCLUSIONS

Sustained elevations of overnight IL-6 levels and relatively decreased sensitivity to hydrocortisone distinguish PTSD + MDD from PTSD - MDD. Novel strategies that decrease IL-6 levels offer a new direction in the prevention and treatment of PTSD and associated comorbid medical illnesses.

Some very preterm neonates admitted to the neonatal intensive care unit show circulatory and respiratory problems that improve after administration of steroids. It is unclear whether these symptoms could be caused by adrenal insufficiency. The objective of our study was to investigate the cortisol levels and the cortisol release from the adrenals after ACTH in very preterm infants with and without severe illness and to find whether a relation exists between adrenal function and outcome. An ACTH test (0.5 microg) was performed on d 4 in 21 very preterm infants (gestational age, 25.6-29.6 wk; birth weight, 485-1265 g). Baseline cortisol and 17-hydroxyprogesterone (17OHP) levels and the cortisol levels 30, 60, and 120 min after ACTH administration were measured. The Score for Neonatal Acute Physiology was used to measure illness severity. All infants showed an increase in cortisol levels after ACTH, but the cortisol levels were significantly lower in the ventilated more severely ill infants. After adjusting for birth weight and gestational age, the mean baseline cortisol levels and cortisol/17OHP ratios were significantly lower and the 17OHP levels significantly higher in the ventilated infants compared with the nonventilated infants. Patients with an adverse outcome had significantly lower baseline cortisol/17OHP ratios and 60-min cortisol levels during ACTH testing (p = 0.002 and p = 0.03, respectively). These data suggest an insufficient adrenal response to stress in sick ventilated very preterm infants with gestational ages younger than 30 wk compared with nonventilated less sick preterm infants. Further studies are required to investigate whether supplementation with physiologic doses of hydrocortisone may benefit the outcome.

Chemically modified tetracyclines [CMT-3 (IC50 approximately 6-13 microM = approximately 2.5-5 microg/ml) and CMT-8 (IC50 approximately 26 microM = 10 microg/ml), but not CMT-1, -2 or -5], which lack anti-microbial activity, inhibited nitrite production in LPS-stimulated macrophages. Unlike competitive inhibitors of L-arginine which inhibited the specific activity of inducible nitric oxide synthase (iNOS) in cell-free extracts, CMTs exerted no such direct effect on the enzyme. CMTs could, however, be shown to inhibit both iNOS mRNA accumulation and protein expression in LPS-stimulated cells. Tetracyclines (doxycycline and CMT-3) unlike hydrocortisone had no significant effect on murine macrophages transfected with iNOS promoter (tagged to a luciferase reporter gene) in the presence of LPS. However, doxycycline and CMT-3 augmented iNOS mRNA degradation, in LPS-stimulated murine macrophages. These studies show a novel mechanism of action of tetracyclines which harbours properties to increase iNOS mRNA degradation and decrease iNOS protein expression and nitric oxide production in macrophages. This property of tetracyclines may have beneficial effects in the treatment of various diseases where excess nitric oxide has been implicated in the pathophysiology of these diseases.

OBJECTIVE

To update the 2008 consensus statements for the diagnosis and management of critical illness-related corticosteroid insufficiency (CIRCI) in adult and pediatric patients.

METHODS

A multispecialty task force of 16 international experts in Critical Care Medicine, endocrinology, and guideline methods, all of them members of the Society of Critical Care Medicine and/or the European Society of Intensive Care Medicine.

METHODS

The recommendations were based on the summarized evidence from the 2008 document in addition to more recent findings from an updated systematic review of relevant studies from 2008 to 2017 and were formulated using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) methodology. The strength of each recommendation was classified as strong or conditional, and the quality of evidence was rated from high to very low based on factors including the individual study design, the risk of bias, the consistency of the results, and the directness and precision of the evidence. Recommendation approval required the agreement of at least 80% of the task force members.

RESULTS

The task force was unable to reach agreement on a single test that can reliably diagnose CIRCI, although delta cortisol (change in baseline cortisol at 60 min of <9 µg/dl) after cosyntropin (250 µg) administration and a random plasma cortisol of <10 µg/dl may be used by clinicians. We suggest against using plasma free cortisol or salivary cortisol level over plasma total cortisol (conditional, very low quality of evidence). For treatment of specific conditions, we suggest using intravenous (IV) hydrocortisone <400 mg/day for ≥3 days at full dose in patients with septic shock that is not responsive to fluid and moderate- to high-dose vasopressor therapy (conditional, low quality of evidence). We suggest not using corticosteroids in adult patients with sepsis without shock (conditional recommendation, moderate quality of evidence). We suggest the use of IV methylprednisolone 1 mg/kg/day in patients with early moderate to severe acute respiratory distress syndrome (PaO2/FiO2 < 200 and within 14 days of onset) (conditional, moderate quality of evidence). Corticosteroids are not suggested for patients with major trauma (conditional, low quality of evidence).

CONCLUSIONS

Evidence-based recommendations for the use of corticosteroids in critically ill patients with sepsis and septic shock, acute respiratory distress syndrome, and major trauma have been developed by a multispecialty task force.

BACKGROUND

Paediatric Cushing's disease (CD) is rare, but is associated with considerable morbidity and requires effective treatment. Control of hypercortisolaemia is recommended prior to definitive therapy by transsphenoidal pituitary surgery with selective adenomectomy. We describe a 6.2-year-old male with severe hypercortisolaemia and life-threatening complications of Cushing's disease. Control of cortisol with metyrapone and ketoconazole was ineffective, and due to his deteriorating condition, the decision was taken to proceed to bilateral adrenalectomy.

METHODS

Low-dose IV infusion of etomidate, with dose titration according to serum cortisol levels, was administered.

RESULTS

Etomidate infusion (3.0 mg/h i.v.) decreased serum cortisol from 1,250 to 250 nmol/l within 24 h. Combined etomidate and hydrocortisone therapy was maintained to provide stable serum cortisol levels within the desired range for 12 days prior to successful bilateral adrenalectomy.

CONCLUSIONS

In our experience, etomidate was effective and safe for short-term control of severe hypercortisolaemia in a severely ill child.

OBJECTIVE

A major problem in rat liver endothelial cell culture is the rapid loss of cells after 48 h. This study aimed to develop a protocol that allowed easy maintenance and proliferation of sinusoidal endothelial cells in serum-free culture for 5-6 days.

METHODS

Cells isolated from adult rat liver by collagenase digestion were purified by centrifugal elutriation and cultured on glutaraldehyde-crosslinked collagen.

RESULTS

At high plating densities cells could be maintained serum-free for 6 days in the presence of hydrocortisone and basic fibroblast growth factor; at lower plating densities medium had to be supplemented with additional growth-promoting factors. Conditioned medium of adult rat hepatocytes proved to be the most effective growth stimulus; it increased thymidine incorporation, DNA content and cell number per dish with a half-maximal effect at 20% (v/v). Cell proliferation was also observed with either vascular endothelial growth factor, phorbol ester or conditioned media from FAO or HEPG2 liver cell lines provided the cultures were additionally supplemented with 1% newborn calf serum. Vascular endothelial growth factor was detected in all conditioned media. In the absence of hepatocyte-conditioned medium, 1% serum helped to maintain cultures; it itself exerted a low proliferative effect. Higher serum concentrations (>5%), however, led to cell loss after 48 h. The numerous sieve plates of 6-h-old cells progressively disappeared during culture and were replaced by randomly distributed pores, which later grouped together at cell-cell borders. More than 90% of the cells endocytosed acetylated low-density lipoprotein.

CONCLUSIONS

The study shows that cultured hepatocytes secrete growth-promoting substances that stimulate in vitro endothelial cell proliferation in the absence of serum; this effect could be mimicked by the combined addition of vascular endothelial growth factor and 1% serum. The new media formulations should facilitate future research on liver endothelial cells in mono- or coculture.

Bone marrow stromal stem cells differentiate into adipocytes and osteoblasts. These two lineages are thought to be reciprocally related, in part due to the observation that the osteoblast-inducing factor, 1,25 dihydroxy vitamin D3 [1,25(OH)2D3], inhibited adipogenesis of rat femoral-derived stromal cell cultures. However, the literature is divided concerning the adipogenic effects of this steroid hormone. This work examined the effect of 1,25(OH)2D3 (10(-12)-10(-8) M) on murine femoral-derived bone marrow stromal cell differentiation in response to adipogenic agonists employing two different classes of nuclear hormone receptors: the glucocorticoid receptor (hydrocortisone) or peroxisome proliferator-activated receptors (thiazolidinediones). Experiments used the multipotent murine bone marrow stromal cell line, BMS2, and its subclones, as well as primary-derived murine bone marrow stromal cell cultures. In all systems examined, 1,25(OH)2D3 blocked adipogenesis induced by hydrocortisone, methylisobutylxanthine, and indomethacin based on flow cytometric analysis of lipid accumulation. This correlated with reduced messenger RNA levels of the late adipocyte gene markers, aP2 and adipsin. In the BMS2 subclone no. 24, the 1,25(OH)2D3 actions were concentration dependent. Whereas 1,25(OH)2D3 partially inhibited thiazolidinedione-induced adipogenesis in the parental BMS2 cell line, it had minimal effect on the thiazolidinedione-induced differentiation of the BMS2 subclone and primary cultures. These findings indicate that 1,25(OH)2D3, at nanomolar concentrations, completely inhibits murine bone marrow stromal cell differentiation in response to glucocorticoid-based adipogenic agonists but is a less effective adipogenic antagonist following induction with thiazolidinediones. This work supports the conclusion that 1,25(OH)2D3 inhibits murine femoral-derived bone marrow stromal cell adipogenesis.

The alphavbeta6 integrin is an exclusively epithelial integrin that is highly expressed during fetal development. In adult tissue, alphavbeta6 integrin is expressed during inflammation, carcinogenesis, and in wound healing. We previously reported that alphavbeta6 integrin is highly expressed in poorly healing human wounds and its over-expression is associated with chronic wounds in a mouse model. The objective of this study was to investigate the role of alphavbeta6 integrin in compromised wound healing induced by hydrocortisone treatment or aging by using young and old mice deficient in or overexpressing the beta6 integrin subunit in the epidermis. Untreated aged beta6 integrin-deficient (beta6-/-) animals showed a significant delay in wound healing when compared to their age-matched controls or younger beta6-/- mice. The most significant delay was observed at the stages where granulation tissue deposition was occurring. Hydrocortisone treatment significantly delayed wound healing in wild-type and beta6 integrin-deficient mice in comparison with the untreated controls. However, hydrocortisone treatment in beta6 integrin overexpressing animals did not cause a significant delay in wound healing. The results of this study suggest that alphavbeta6 integrin plays an important role in wound healing in animals compromised by either age or stress mimicked by hydrocortisone.

Research indicates that glucocorticoids affect hippocampal function and the form of cognition that the hippocampus is thought to subserve, explicit memory. However, some studies suggest that glucocorticoids affect the frontal lobe, attention and working memory. Thus, it is not clear whether glucocorticoids specifically target the hippocampus and explicit memory or if the effects are more ubiquitous. By simultaneously measuring event-related potentials and behavioral performance in tasks designed to tap particular cognitive and neural processes, the present study examined the effects of hydrocortisone on 24 healthy humans. In an intentional face recognition memory task where the stimuli were presented again after a brief delay (6-18 s) and a long delay (30 min), hydrocortisone altered the P600 component (an electrophysiological index of recognition memory and hippocampal activity) following the brief delay and impaired behavioral performance after the long delay. ERPs and behavioral performance were not affected in the attention and working memory tasks. These findings are consistent with reports indicating that glucocorticoids affect explicit memory and hippocampal function.

A unique population of human bone marrow-derived, adherent fibroblastlike cells differentiates to adipocyte morphology when grown in vitro in the presence of horse serum and hydrocortisone sodium hemisuccinate. Over the initial 8-weeks growth at 37 degrees C, 7% CO2, these cells accumulate Oil Red O-positive lipid and form colonies of over 100 cells, which persist in confluent cultures for over 30 weeks. Similar to cultures derived from mouse marrow, corticosteroid-induced adipocyte differentiation is associated with long-term granulopoiesis. Human marrow preadipocytes, as well as human, mouse and rat embryo fibroblast cell lines, failed to differentiate to adipocyte morphology in the presence of insulin. In contrast, the 3T3-L1 insulin-dependent preadipocyte cell line was not induced to differentiate in the presence of hydrocortisone. These studies demonstrate that human marrow preadipocytes are dependent upon corticosteroid for differentiation in vitro.

Growth of Chlamydia trachomatis B/TW-5/OT and L(2)/434/Bu strains in cultures of thioglycolate-activated mouse peritoneal macrophages was studied. Both strains grew to a limited extent in the macrophages, but lymphogranuloma venereum (LGV) grew better than trachoma. Growth was enhanced by centrifugation of the inoculum onto the macrophage cell layer and inhibited by pretreatment of macrophages for 2 h with 100 mug of concanavalin A per ml. No significant effect was observed by pretreatment of macrophages with diethylaminoethyl-dextran (30 mug/ml, 30 min), cytochalasin B (20 mug/ml, 1 h), and cyclophosphamide (200 mug/ml, overnight) or by treatment with hydrocortisone (1 and 10 mug/ml, overnight before inoculation and during a 2-day incubation after inoculation). Resistance to intracellular growth of the two organisms was not increased in macrophages obtained from mice immunized with the organisms compared with macrophages from nonimmunized mice unless they were pretreated in vitro with penicillin (100 U/ml, overnight). The yields of LGV organisms from the penicillin-pretreated macrophages of LGV-immunized mice were 100-fold less than from the pretreated macrophages of nonimmunized control mice. At the same time, the yields of LGV organisms from penicillin-pretreated macrophages of mice immunized with trachoma, gonococcus, and HeLa cells were not different from those obtained in pretreated macrophages of nonimmunized control mice.

In a previous longitudinal study of basal cortisol levels and cognitive function in humans, we showed that elderly humans with 4- to 7-yr cumulative exposure to high levels of cortisol present memory impairments, compared with elderly humans with moderate cortisol levels over years. Here, we measured whether memory performance in two groups of elderly humans separated on the basis of their cortisol history over a 5-yr period could be modulated by a hormone-replacement protocol in which we inhibited cortisol secretion by the administration of metyrapone and then restored baseline cortisol levels by infusion of hydrocortisone. We showed that in elderly subjects with a 5-yr history of moderate cortisol levels (n = 8), metyrapone treatment significantly impaired memory performance, a deficit that was reversed following hydrocortisone replacement. In the elderly subjects with a 5-yr history of high cortisol levels and current memory deficits (n = 9), metyrapone treatment did not have any significant effect on memory performance, but hydrocortisone treatment significantly decreased delayed memory. These results suggest that memory function in elderly humans can be intensely modulated by pharmacological manipulation of glucocorticoids, although the direction of these effects depends on the cortisol history of each individual.

Mus dunni endogenous virus (MDEV) is activated from cells of the Asian wild mouse M. dunni (also known as Mus terricolor) in response to treatment with either 5-iodo-2'-deoxyuridine or hydrocortisone. MDEV represents a new murine retrovirus interference group and thus appears to use a different receptor for entry into cells than do other murine retroviruses. Here we show that MDEV is also not in the gibbon ape leukemia virus or RD114 virus interference groups. A retroviral vector with an MDEV pseudotype was capable of efficiently infecting a wide variety of cells from different species, indicating that the MDEV receptor is widely expressed. We isolated a molecular clone of this virus which exhibited no hybridization to any cloned retrovirus examined, suggesting that MDEV has an unusual genome. One copy of a possible retrovirus element that weakly hybridized with MDEV was present in the genomes of laboratory strains of mice, while no such elements were present in other species examined. A virus activated by 5-iodo-2'-deoxyuridine from cells of a BALB/c mouse, however, was not related to MDEV by either hybridization or interference analyses.

Studies of normal cellular function as well as the understanding of cellular mechanisms of carcinogenesis and other diseases of the large intestine have been limited, particularly due to the lack of long-term culture of normal human large intestinal epithelial cells (NHLIEC). Using the epithelia from surgically resected human colon, we have dissociated a sufficient number of viable NHLIEC and maintained them in in vitro culture for up to 5 months. Normal-appearing human large intestinal mucosal fragments (1 mm2) were treated with 0.01 mg/ml trypsin, 0.2 mg/ml collagenase + 0.1 mM EGTA or 0.1 mg/ml trypsin + 0.1 mM EGTA in a Stomacher laboratory blender to isolate the cells. Compared with other methods, the use of the Stomacher blender combined with low concentrations of proteolytic enzymes yielded greater numbers of cells per gram of tissue, with up to 84% viable cells. Primary and serially passaged NHLIEC were cultured in CMRL-1066, MEM with 5% serum, and serum-free KGM. These media were all supplemented with insulin, hydrocortisone, epithelial growth factor, and bovine pituitary extract. CMRL-1066 was found to be the best medium for NHLIEC. Contaminating fibroblasts were selectively removed by briefly allowing the cells to adhere to the culture vessel and adding 25 U/ml collagenase to the culture media at the first subculture treatment. The epithelial nature and secretory function of the established cells were confirmed by morphological criteria (light microscopy, phase contrast microscopy and electron microscopy), immunoreactivity to cytokeratin, and positive mucin cytochemistry. We propose that using this methodology for the culture and maintenance of NHLIEC for an extended period of time would serve as a valuable model for a variety of investigations.

OBJECTIVE

Neurosurgical patients have a high risk of hyponatremia and associated complications. We critically evaluated the existing literature to identify the determinants for the development of hyponatremia and which management strategies provided the best outcomes.

METHODS

A multidisciplinary panel in the areas of neurosurgery, nephrology, critical care medicine, endocrinology, pharmacy, and nursing summarized and classified hyponatremia literature scientific studies published in English from 1950 through 2008. The panel's recommendations were used to create an evaluation and treatment protocol for hyponatremia in neurosurgical patients at the University of Florida.

RESULTS

Hyponatremia should be further investigated and treated when the serum sodium level is less than 131 mmol/L (class II). Evaluation of hyponatremia should include a combination of physical examination findings, basic laboratory studies, and invasive monitoring when available (class III). Obtaining levels of hormones such as antidiuretic hormone and natriuretic peptides is not supported by the literature (class III). Treatment of hyponatremia should be based on severity of symptoms (class III). The serum sodium level should not be corrected by more than 10 mmol/L/d (class III). Cerebral salt wasting should be treated with replacement of serum sodium and intravenous fluids (class III). Fludrocortisone may be considered in the treatment of hyponatremia in subarachnoid hemorrhage patients at risk of vasospasm (class I). Hydrocortisone may be used to prevent natriuresis in subarachnoid hemorrhage patients (class I). Hyponatremia in subarachnoid hemorrhage patients at risk of vasospasm should not be treated with fluid restriction (class II). Syndrome of inappropriate antidiuretic hormone may be treated with urea, diuretics, lithium, demeclocycline, and/or fluid restriction (class III).

CONCLUSIONS

The summarized literature on the evaluation and treatment of hyponatremia was used to develop practice management recommendations for hyponatremia in the neurosurgical population. However, the practice management recommendations relied heavily on expert opinion because of a paucity of class I evidence literature on hyponatremia.

Many techniques for the production of solid dispersions rely on elevated temperatures and prolonged material residence times, which can result in decomposition of temperature-sensitive components. In this study, hydrocortisone was used as a model temperature-sensitive active ingredient to study the effect of formulation and processing techniques as well as to characterize the benefits of KinetiSol Dispersing for the production of solid dispersions. Preformulation studies were conducted using differential scanning calorimetry and hot stage microscopy to identify optimum carriers for the production of amorphous solid dispersions. After identification, solid dispersions were prepared by hot melt extrusion and KinetiSol Dispersing, with material characterized by X-ray diffraction, dissolution and potency testing to evaluate physicochemical properties. Results from the preformulation studies showed that vinylacetate:vinylpyrrolidone (PVPVA) copolymer allowed for hydrocortisone dissolution within the carrier at temperatures as low as 160 degrees C, while hydroxypropyl methylcellulose required temperatures upward of 180 degrees C to facilitate solubilization. Low substituted hydroxypropyl cellulose, a high glass transition temperature control, showed that the material was unable to solubilize hydrocortisone. Manufacturing process control studies using hot melt extruded compositions of hydrocortisone and PVPVA showed that increased temperatures and residence times negatively impacted product potency due to decomposition. Using KinetiSol Dispersing to reduce residence time and to facilitate lower temperature processing, it was possible to produce solid dispersions with improved product potency. This study clearly demonstrated the importance of carrier selection to facilitate lower temperature processing, as well as the effect of residence time on product potency. Furthermore, KinetiSol Dispersing provided significant advantages over hot melt extrusion due to the reduced residence times and lower required processing temperatures. This allowed for the production of solid dispersions with enhanced product potency.